RU2793467C2 - Mesenchymal stromal cells and methods for obtaining mesenchymal stromal cells from the umbilical cord - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention relates to a process for obtaining a mesenchymal stromal cell. To implement this process, stromal Wharton's jelly or part thereof is first isolated from the umbilical cord. Next, enzymatic treatment of stromal Wharton's jelly or part thereof is carried out with collagenase enzyme in the presence of platelet lysate (PL), thereby releasing at least one cell from stromal Wharton's jelly or part thereof. After that, at least one cell is propagated in a growth medium containing PL.

EFFECT: improved yield of mesenchymal stem cells.

15 cl, 24 dwg, 7 tbl, 14 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to a method for obtaining stromal cells, in particular stromal precursor cells. Stromal progenitor cells are generally capable of giving rise to phenotypically and genotypically identical daughter cells (self-renewal) as well as differentiated cells of at least one type. Some stromal cells are capable of self-renewal, and such cells have received increasing attention in recent years. The stromal cells of the present invention are non-embryonic mesenchymal stromal cells. They can be obtained by isolation from the umbilical cord by the method of the invention, which is described in detail herein.

BACKGROUND OF THE INVENTION

Mesenchymal stromal cells (MSCs) are stromal cells of particular interest to the scientific community, in particular because of their multipotency and low immunogenicity (Pittenger et al., 1999, Science, vol. 284, p. 143-147). According to the current state of the art, mesenchymal stromal cells (MSCs) are traditionally usually obtained from the bone marrow, however, MSCs can also be isolated from various other animal sources such as adipose tissue, muscle, connective tissue, dental pulp, cord blood, placenta and amniotic fluid (Iudicone et al., 2014, J. Transl. Med., 12:28). Of the MSCs, bone marrow-derived adult MSCs (BM-MSCs) are the best studied (eg WO 2008/042174 A2). In the bone marrow, MSCs coexist with a variety of cell types, including more primitive and embryonic-like stem cells, mesenchymal stem cells, committed progenitor cells, and mature cells such as osteoblasts and fibroblasts, and various methods have been described for isolating, for example, MSCs from bone marrow (see, for example, Majore, Cell Commun. Signal., 2009, vol. 20; 7:6). Some efforts have also been made in the past to explore alternative sources of mesenchymal stromal cells. For example, the umbilical cord (UC) has been proposed as a suitable reservoir for various types of cells, some of which have been shown to have properties similar to those of stem cells. The umbilical cord is a structure in placental mammals that connects the placenta to the developing embryo, thereby providing nourishment to the fetus. It is available after birth without any invasive procedures or ethical concerns (Hass et al., 2011, Cell. Commun. Signal., vol. 9:12). The umbilical cord contains Wharton's jelly, a gelatinous, loose mucous connective tissue rich in stromal cells, which was named after the anatomist Thomas Wharton. Wharton's jelly contains cells dispersed in an amorphous substance composed of proteoglycans, including hyaluronic acid and other types of glycans (see, for example, WO 2004/072273 A1). It is considered to be a primitive mesenchymal tissue of predominantly extraembryonic origin (Weiss, 1983, Histology, cell and tissue biology, New York, Elsevier Biomedical, p. 997-998), and has been proposed as a promising source of MSCs (Wang et al., 2004, Stem Cells, vol. 22: pp. 1330-1337).

It is generally accepted that umbilical cord stromal cells (PCs) are heterogeneous in their structure, biological properties, and localization in PCs. Several methods have been described for obtaining cells from Wharton's jelly (WJ) of PC, and cells with MSC-like properties found in and originating from different regions of PC have been described. The human umbilical cord contains three vessels, which are usually arranged in a left-handed (counterclockwise) spiral. In a publication by Mitchell et al. (2003, Stem Cells, vol. 21, p. 50-60) describes a method for removing umbilical cord vessels before harvesting the remaining tissue. This remaining tissue, which contains both part of the BC and the amniotic epithelium, is then minced to obtain small tissue fragments, which are transferred to tissue culture plates; however, enzymatic cleavage of the umbilical cord or remaining tissue is not provided. Rather, the tissue fragments are subsequently used as primary explants from which cells migrate outside of the tissue samples onto the culture substrate. Another publication (Romanov et al., 2003, Stem Cells, vol. 21, p. 105-110) describes that fibroblast-like MSCs can be isolated from the umbilical cord vasculature by splitting the umbilical vein with collagenase, however, this results in a mixed population vascular endothelial and subendothelial cells. US 5,919,702 A (Purchio et al.) describes a method of isolation involving longitudinal cutting of a segment of the umbilical cord about 2.5 cm long, removal of the blood vessels and membrane, and collection of the BC in a sterile container, where it is crushed and cultured. For example, it is described that cells can be isolated by placing a 2-3 mm ³ section of BC on a glass slide at the bottom of a petri dish, covering it with another glass slide, and culturing it for 10-12 days to allow some cells ("prechondrocytes" to ) migrate outward in the culture dish. The use of prechondrocytes to obtain cartilage is also mentioned.

Isolation of cells derived from the umbilical matrix has also been described for tissue of animal origin. For example, Lange-Consiglio et al., (2011, Open Tissue Engineer. Regen. Med. J., vol. 4, p. 120-133) describe the isolation of "cells derived from the umbilical cord matrix" from "intervascular" and "perivascular" areas of the horse's umbilical cord.

Human umbilical cord weight at term of about 40 g (Raio et al., 1999, Eur. J. Obstet. Gynecol. Reprod. Biol., vol. 83; p. 131-135; Conconi et al., 2011 , Open Tissue Engineer, Regen. Med. J., vol. 4, pp. 6-20), imposes a natural upper limit on the starting material available for isolation of cells with MSC-like properties. According to US 2004/0136967 A1 and Seshareddy et al. (2008, Meth. Cell Biol., vol. 86, p. 101-119), it is difficult to obtain large numbers of desired cells from the umbilical cord. As a consequence, the authors recommend the extraction of a relatively large number of cells and describe conditions that are optimized for the isolation of a relatively large number of cells; for example, allowing the isolation of large numbers of cells. They propose a relatively harsh enzymatic treatment of the umbilical cord. Various zones of Wharton's jelly have been described, but the exclusion of specific zones of origin of the extracted cells, for example, the perivascular region, is not specifically recommended, in accordance with the general recommendation to use large numbers of cells. After isolation, the cells are cultured. An alternative approach is described in WO 2004/072273 A and Sarugaser et al. (Stem Cells, 2005, 23:220-229); these publications describe the isolation of cells with MSC-like properties exclusively from the perivascular region of the umbilical cord. Since this area is anatomically relatively limited, the amount of starting material is relatively low compared to methods in which the extraction is started from the whole umbilical cord. In addition, due to the close proximity to the blood vessels, contamination by blood cells cannot be ruled out; consequently, negative selection for CD45 expression has been described to avoid contamination with unwanted cells in culture. In addition, WO 2017/058003 A1 describes methods for isolating MSCs from the umbilical cord, dental pulp, foreskin, cornea, or other sources; the method for isolating mesenchymal stromal cells from the umbilical cord is preferably carried out under sterile conditions, and the umbilical cord is preferably treated with collagenase for relatively long periods of time. According to Conconi et al., 2011, Open Tissue Engineer. Reg. Med. J., vol. 4, p. 6-20, the most appropriate protocols for enhancing in vitro propagation and storage of MSCs have not yet been developed. Thus, there remains a need for methods of obtaining populations consisting of cells that have substantially uniform properties and that are capable of reliably propagating ex vivo; as well as in reliable ways to ensure them.

Problem to be solved

Thus, the aim of the present invention is to eliminate the shortcomings inherent in the state of the art. Specific objectives include providing a reliable method for producing mesenchymal stromal cells (MSCs), and reproducibly providing good yields of mesenchymal stromal cells (MSCs) and MSC populations having excellent self-renewal properties. It is desirable that the populations be relatively homogeneous and free of undesirable cells such as apoptotic cells, hematopoietic lineage cells, and so on. Thus, in other words, it is desirable to produce mesenchymal stromal cells in a manner that has advantages in terms of reliability and/or yield over currently known and practiced methods. Various shortcomings of the prior art determine the following goals for improvement, which were directed to the efforts of the authors of the invention, and these goals were achieved due to the contribution described and claimed in this document.

SUMMARY OF THE INVENTION

The present invention relates to a method for producing mesenchymal stromal cells (MSCs), corresponding mesenchymal stromal cells (MSCs), and other related aspects, all of which are described herein. The main aspects and embodiments of the invention are described below.

In a first aspect, the present invention relates to a method for producing a mesenchymal stromal cell. The method includes (a) isolating the stromal Wharton's jelly, or a portion thereof, from the umbilical cord, and (b) enzymatically treating the stromal Wharton's jelly, or a portion thereof, thereby releasing at least one cell from the stromal Wharton's jelly, or a portion thereof. Preferably, the cell obtained after step (b) is multipotent.

In addition to steps (a) and (b), it is highly preferred to include at least one additional step in the process according to the invention. In particular, it is preferable to include a step of increasing the number of cells, allowing you to take advantage of the cell division potential obtained in step (b). In such preferred embodiments, step (b) is followed by step (c); where step (c) includes propagation of at least one cell obtained in step (b). Preferably, the cells obtained after step (c) are multipotent.

The present inventors have unexpectedly found that cells derived from the umbilical cord stroma have particular advantages. Advantages include homogeneity of cell populations and properties similar to those of stem cells. In particular, these advantages can be used in the absence of umbilical cord vessels. Thus, it is preferable if step (a) isolating stromal Wharton's jelly, or a portion thereof, from the umbilical cord comprises the step of physically removing the vessels and perivascular region from the umbilical cord. Vessels and the perivascular region can be removed prior to step (a) or during step (a). One technically suitable method of removing the vessels and perivascular region, which is preferred by the present invention, is to separate the vessels and perivascular region from the umbilical cord. The inventors have found that the separation of the vessels, for example, by pulling the vessels, also leads to the separation of the perivascular region.

In addition, the inventors have found that it is preferable to segment the umbilical cord before said step of physically removing the vessels and the perivascular region. Segmentation is usually done by physical means, such as scissors or a scalpel. Preferably, the segmentation is performed perpendicular to the umbilical cord. More preferably, each of the perpendicularly cut segments has a length of from about 1 to about 4 cm, preferably from about 2 to about 3 cm, and more preferably about 2.5 cm. ) umbilical cord up to step (b). In particular, it is preferable to remove the vessels or, more precisely, the segments of the vessels after - preferably perpendicular - segmentation of the umbilical cord.

Preferably, the enzymatic treatment in step (b) is a treatment with a collagenase enzyme. The inventors have also found that the degree of collagenase activity has a certain value. In a preferred embodiment, the collagenase is present in the enzymatic treatment step (b) at a relatively low specific activity, as detailed below.

In the method of the present invention, the rapid isolation of stromal cells from the umbilical cord is preferred. In a preferred embodiment, steps (a) and (b) are carried out together for a total of less than 6 hours, more preferably less than 5 hours, more preferably less than 4 hours, and most preferably less than 3 hours. Obviously, the short combined duration of steps (a) and (b) also means that step (b) itself is short. In general, the short duration of step (b), that is, the rapid release, in particular the short duration of the enzymatic treatment, may contribute to the mildness of the conditions in step (b).

Surprisingly, the inventors also found that the presence of platelet lysate in the enzymatic digestion process has certain advantages. First, the inventors have found that the presence of human platelet lysate does not inhibit or reduce, or block, the enzymatic activity of the collagenase enzyme, at least not to the extent that would adversely affect the desired enzymatic release of cells from surrounding tissue. , for example, from the stromal matrix. It is known that blood plasma contains numerous proteases and thus there is a certain prejudice against adding a catalytic protein (such as collagenase) in the presence of blood plasma or its components, or vice versa, one would expect that a catalytic protein (such as collagenase) may degrade over time and therefore its activity will decrease; surprisingly, the present inventors have found that collagenase digestion is effective even in the presence of platelet lysate. Secondly, the inventors found that cells obtained by digestion in the presence of platelet lysate in the method of the present invention have excellent proliferative properties (see, for example, examples 2B and 2C). Thus, preferably, in step (b) Wharton's stromal jelly, or a portion thereof, is subjected to an enzymatic treatment in the presence of a mammalian platelet lysate (TL), more preferably a human platelet lysate (hTL).

In addition, the platelet lysate is preferably present in step (c). Preferably, in step (c), at least one cell is propagated in a growth medium containing platelet lysate (TL), preferably human platelet lysate (hTL). Preferably, the at least one cell is propagated in a growth medium free of any added serum, in particular free of fetal bovine serum (FBS).

In a particularly preferred embodiment, at least one cell is propagated in a particular growth medium, particularly suitable for the method of the invention, optionally containing additives. Such a growth medium may contain a minimal chemically defined medium and/or a platelet lysate.

The method according to the invention leads to the production of cells with specific properties, which are also described in this document. Thus, it is particularly preferred if the cell obtained by the method according to the first aspect of the invention is the cell described in the second aspect of the invention below.

In a second aspect, the present invention relates to a mesenchymal stromal cell (MSC). The MSC of the invention may also be referred to as the "cell of the invention" or simply "cell". The MSC of the invention is characterized in that it (a) expresses the APCDD1 gene and (b) does not express the 3G5 gene. Preferably, the cell is an isolated cell. More preferably, the cell is derived from umbilical cord stromal Wharton's jelly. Preferably, the cell is a primate cell. Thus, the cage is preferably not from a horse, that is, it is not a horse cage. Also, the cell is preferably non-rodent (eg mouse or rat), ie non-rodent (eg mouse or rat). Most preferably, the cell is a human cell.

In a preferred embodiment, the cell is a human cell that is derived from stromal Wharton's jelly and that expresses the APCDD1 gene.

In a preferred embodiment, the cell also expresses the PPARG gene. In another, and non-mutually exclusive, preferred embodiment, the cell also expresses the FLVCR2 gene. In the case of a human cell, preferably the human cell expresses at least one, preferably at least any combination of two, and most preferably all combinations, of the human leukocyte antigens HLA-A, HLA-B and HLA-C. In addition, in the case of a human cell, it is preferable if the cell does not exhibit the human leukocyte antigens HLA-DP and/or HLA-DQ. Optionally, HLA-DR is also not exposed. In one embodiment, the cell does not display any of the human leukocyte antigens HLA-DP, HLA-DR and HLA-DQ. Alternatively, HLA-DR is exhibited at low levels.

The cell of the present invention is generally smaller than some of the MSCs that have been isolated by various prior art methods. For example, the cell of the present invention may have a smaller volume and/or a smaller surface area. Various methods are available to determine the volume or surface area of a cell, such as flow cytometry to determine the cell volume of non-adherent cells and microscopic cell surface determination for adherent cells. Such methods are described later in this document.

Preferably, the cell does not exhibit any telomerase activity and/or does not express any telomerase. Preferably, the cell is multipotent. More preferably, the cell is capable of differentiating into different cell types. It is particularly preferred if the cell has at least all of the following differentiation potentials: (a) adipogenic; (b) chondrogenic; (c) osteogenic.

In a preferred embodiment, the cell is a mesenchymal stem cell.

The cell according to the second aspect of the invention is a cell obtainable by the method according to the first aspect of the invention. The preferred embodiments of the first aspect of the invention may be combined with the preferred embodiments of the second aspect of the invention in each, and in any combination, unless the context clearly dictates otherwise. Alternatively, the cell of the present invention may thus be described as a cell obtainable by the method of the first aspect of the invention.

In a third aspect, the invention relates to a population of cells. The population contains at least one cell according to the second aspect of the invention. More preferably, the cell population contains a plurality of cells, of which at least 80% (by number of cells), preferably at least 90% (by number of cells), more preferably at least 95%, for example, at least 96%, at least 97%, at least 98%, at least 99% (in each case by number of cells) are cells according to the second aspect. All preferred embodiments of the second aspect also apply to the third aspect of the invention. Preferably at least 70%, preferably at least 80%, more preferably at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least at least 99%, most preferably 100%, of the total number of cells are cells according to the second aspect, and optionally, one or more of its preferred embodiments, alone or in combination. The cell population is positive for APCDD1 gene expression.

Preferably, the population is an isolated population of cells.

Preferably, the cell population is also characterized in that the volume and/or surface area of the cells in the cell population are similar, or alternatively, in that a high percentage of cells have a small volume and/or surface area, as detailed below. Volume generally correlates with forward scatter (FSC) values determined by flow cytometry.

Preferably, the cell population is also characterized in that at least 70% of the cells (by number of cells), or more, are viable. This is a significant advantage over prior art methods in which the percentage of viable cells is lower. More preferably, at least 75% of the cells (by number of cells) are viable. More preferably, at least 80% of the cells (by number of cells) are viable. In one embodiment, viability is determined immediately after cell isolation, that is, prior to propagation.

In a preferred embodiment, the cell population does not contain any perivascular cells. The perivascular cell marker is 3G5. Thus, this embodiment can alternatively be described such that the cell population does not contain any cells expressing the 3G5 marker and/or any cells displaying 3G5 on its surface.

In a preferred embodiment, the cell population does not contain any epithelial cells. The epithelial cell marker is the epithelial cell adhesion molecule (EpCAM). Thus, this embodiment can alternatively be described such that the cell population does not contain any cell expressing the EpCAM marker and/or any cell exhibiting EpCAM on its surface.

In a preferred embodiment, the cell population does not contain any endothelial cells. The endothelial cell marker is CD31. Thus, this embodiment may alternatively be described such that the cell population does not contain any cells expressing the CD31 marker and/or displaying CD31 on its surface.

Preferably, the cell population is obtained by the method according to the first aspect of the invention, in particular, including the step (c), that is, the step of reproduction.

Alternatively, the cell population of the present invention may thus be described as the cell population obtainable by the method of the first aspect of the invention comprising step (c).

DETAILED DESCRIPTION OF THE INVENTION

In the following detailed description, specific and/or preferred embodiments of individual features of the invention are disclosed. The present invention also provides as particularly preferred embodiments those embodiments which result from the combination of two or more of the specific and/or preferred embodiments described for two or more features of the present invention.

The person skilled in the art understands that the invention described herein may be subject to variations and modifications not specifically described in this document. Thus, one skilled in the art will appreciate that the present invention includes all such variations and modifications. The invention also includes all of the elements, compounds, features, steps, methods, or compositions mentioned or referred to in this specification, alone or collectively, and any and all combinations, or any two or more, of said elements, compounds, features, steps, methods or compositions. Thus, unless specifically stated otherwise herein, or the context dictates otherwise, reference to a single element, compound, feature, step, method, or composition should be understood to include one or more (i.e., more than one, e.g., two or more, three or more, or all) of these elements, compounds, features, steps, methods, or compositions.

The present invention is not limited in scope to the specific embodiments described herein, which are provided herein for purposes of illustration and by way of example. Functionally or otherwise equivalent elements, compounds, features, steps, methods, or compositions are within the scope of the present invention.

Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions, presentations, and so on) above or below is incorporated by reference in its entirety. Nothing herein should be taken as an admission that the present invention is not entitled to backdating the specific ideas set forth.

This document should be read with a desire to understand and with the intention of understanding the technical side of the invention. All terms used herein are to be understood as having the meaning and scope which they ordinarily have in their respective field, unless, in specific instances in this document, one or more words are given special meaning by explicit definition or otherwise. To the extent that definitions given herein differ in part or all from definitions in some or all of the literature, the definitions in this document shall take precedence.

Unless specifically stated otherwise, or otherwise implied by the context, each embodiment, aspect, and example disclosed herein is to be considered applicable to, and compatible with, any other embodiment, aspect, or example described herein.

Unless specifically stated otherwise, all technical and scientific terms used herein have the meaning commonly given to them by those skilled in the art (e.g., genetics, molecular biology, gene expression, cell biology, cell culture, stem cells, stromal cells, neonatology, regenerative medicine, anatomy, histology, immunology, immunohistochemistry, protein chemistry and biochemistry). Educational literature and review articles tend to describe the meaning of terms commonly used by those skilled in the art.

The expression "and/or", e.g. "X and/or Y", should be understood to mean either "X and Y" or "X or Y" and should be taken as specifically meaning "and", "or" and both meanings ("and" or "or").

As used herein, unless specifically indicated otherwise, the terms "about", "about", and "practically" all mean "about" or "almost", and in the context of a numerical value or range given herein, preferably means ± 10%, more preferably ±5%, of the numerical value or range specified or claimed. Terms such as "substantially none", "substantially absent" and so on, when applied to characteristics of a cell, mean that the corresponding characteristic cannot be detected on the cell by analytical methods commonly used in the relevant field according to the prior art, and/or that virtually all cells in a population described in this way lack the corresponding characteristic.

Unless specifically stated otherwise, the term "comprise", or variations thereof such as "comprises" or "comprising", is used in the context of this document to indicate that additional components may optionally be present in addition to the components listed, preceded by the term "including". However, as a specific embodiment of the present invention, it is contemplated that the term "comprising" implies the likelihood that no additional components are present, i.e., for the purposes of this embodiment, the term "comprising" should be understood to mean "consisting of".

Unless specifically stated otherwise, all indications of relative amounts in the present invention are based on the ratio of mass (w/w). An indication of the relative amounts of a component characterized by a general term should refer to the total amount of all specific variants or representatives covered by the specified general term. If it is specifically stated that a particular component defined by a generic term is present in a specific relative amount, and if that component is further characterized as a specific variant or member covered by the generic term, this means that no other variants or members covered by the specific term are additionally are present such that the total relative amount of the components covered by the general term does not exceed the indicated relative amount; more preferably, any other variants or representatives covered by the general term are absent in their entirety.

The term "allogeneic" is used herein to describe an element obtained from another individual of the same species. Two or more individuals are said to be allogeneic to each other when their genes at one or more loci are not identical.

The term "area", or "surface area" or "cellular surface area", in relation to the cell means the surface area of the cell when viewed from above. In the context of the present invention, the 2-dimensional cell surface area is determined for adherent cells in culture, that is, cells attached to the culture plate. For reproducibility of the determination, it is preferable to assume that the cells are in a state of low confluence, since it is believed that in a state of low confluence, cells do not significantly inhibit each other due to contact inhibition. Methods for determining surface area are provided later in this document.

The expressions "average surface area" and "average area" mean the average of the surface areas of two or more individual cells, the first step is to determine the area of each individual cell, as described above, and then in the subsequent second step, the average area is determined as an average the arithmetic value of the areas of individual cells determined at the first stage. As a rule, to determine the average area, the first and second stages are carried out for at least 35 cells; in other words, the mean is the mean of at least 35 cells.

The term "autologous" is used herein to describe an element obtained from the same subject. By way of example, "autologous cell" means a cell obtained from the same subject. Autologous cell transplantation is sometimes considered preferable because it overcomes an immunological barrier that would otherwise lead to rejection.

A cell is a biological object consisting of a cytoplasm enclosed in a membrane. As used herein, the term "cell" preferably refers to a eukaryotic cell. The term "cell" may refer to a stem cell, or a cell lacking the properties of a stem cell, such as a terminally differentiated cell, or even a dead cell. Typically, a cell is a cell derived from a multicellular organism, or from part of it. Preferably, the cell is isolated from said multicellular organism, i.e., is physically unrelated and/or nutritionally independent of the multicellular organism from which it originates. The stromal cell is the preferred cell of the invention.

The terms "cell population" and "cell population" are used interchangeably and refer to a plurality of cells. In the most limited embodiment, the cell population includes two cells, but more often many cells. The term encompasses both populations of substantially identical cells, such as cell populations derived from a single clonogenic cell, and heterogeneous cell populations, that is, cells having a heterogeneous origin and/or that differ from each other in at least one functional or structural characteristic. . The term encompasses populations of only suspension cells, populations of only adherent cells, and mixed populations of suspension and adherent cells. The cell population may contain both stem cells and cells lacking the properties of stem cells. In another, but not mutually exclusive, example, a cell population includes both living cells and dead cells.

Unless the context dictates otherwise, the term "CD", usually used with a number (eg, "CD34"), in this document means "cluster of differentiation".

The term "clonogenicity" refers to the ability of a cell to reproduce, thereby giving rise to a plurality of daughter cells, which, as a rule, are genotypically identical. A "clonogenic" cell may also be referred to as a "colony forming unit", or "CFU".

As used herein, the term "collagenase" refers to an enzyme (E.C. 3.4.24.7) capable of breaking peptide bonds in collagen. As a rule, collagenase can contribute to the destruction of extracellular structures. The term does not impose any specific restrictions on the type or origin of collagenase, unless specifically indicated otherwise. The collagenase may be recombinant or derived from a natural source. Many commercially available collagenases have enzymatic activities other than E.C. 3.4.24.7. Such commercially available collagenases are suitable in the context of the present invention (see examples).

The term "confluency" is used herein to describe the proportion of the bottom surface of the culture vessel that is covered with cells, typically adherent cells. Confluence is typically reported as "% confluence".

The terms "% confluence" or "percent confluence" means the percentage of the lower surface of the culture vessel that is covered with cells, usually adhering cells, that is, the total area of cells relative to the surface of the culture vessel. For example, 50% confluency means that almost half of the surface is covered with cells, and there is still room for cell growth. 100% confluency means that the surface is completely covered with cells and there is no more room for cells to grow in a monolayer. In the context of the present invention, cells are typically subcultured to achieve 100% confluence to optimally maintain their proliferative phenotype.

The term "low confluency" means a percentage of confluence of 50% or less, typically 10-50%. The terms "subconfluent" and "subconfluent" mean a percentage of confluence greater than 50% but not greater than 80%.

The term "culture vessel" or "cell culture vessel" generally means a vessel suitable for culturing mammalian cells. As a rule, culture vessels are vials, plates and bags (in this case, packages usually consist of several plates placed one on top of the other). Particularly suitable culture vessels in the context of the present invention are culture vessels that allow cells to attach to the bottom of the culture vessel; such vessels are vials, tablets and bags with a flat bottom surface.

When referring to a culture vessel, the terms "surface" or "bottom surface" are used interchangeably and should refer to the inner bottom surface of the culture vessel, i.e., the surface to which adherent cells normally attach when they grow in the culture vessel. Typically, said surface is the inner bottom of the culture vessel and is limited solely by the side walls of the culture vessel.

The term "density" or "cell density" means the number of cells per two-dimensional surface, for example, in particular, the number of cells per cm ² , usually per cm ² of the bottom surface of the cell culture vessel, unless the context clearly indicates otherwise. Typically, the term is used to indicate the number of cells seeded in a culture vessel, for example, at the beginning of a particular passage. The total bottom surface of said culture vessel and the total number of cells seeded in that culture vessel determine the cell density per cm ² of said culture vessel at the start of said passage. In practice, the number of cells used as inoculum at the beginning of passage (I) is preferably determined by counting the cells before the start of said passage; I thus depends on the cell density and the bottom surface of the culture vessel. Unless specifically stated otherwise, "cell density" means the average density of cells over the entire lower surface of the culture vessel; however, according to the present invention, it is preferable if the cells are distributed evenly over the entire lower surface of the culture vessel.

The term "diameter", or "cell diameter", as applied to a cell, means the longest diameter of a cell when viewed from above. In the context of this document, the cell diameter is determined when the adherent cell is in culture, that is, the cell is attached to the culture plate. It is important that the cells are in a state of subconfluence. Subconfluence is considered to be preferable for reproducibility of the assay, since subconfluent cells are not considered to substantially inhibit each other through contact inhibition. To determine the diameter, one or more cells are photographed or otherwise visually observed or recorded, typically with a microscope, from a position perpendicular (i.e., 90°) to the surface of the culture plate to which the cell or cells are attached. The boundaries of the region are determined manually on a graphic image (ie, photograph) of the cell. Preferably, the culture vessel is not agitated prior to recording the graphic image. In the case of a bipolar cell, the diameter is the distance between the two poles of the cell. In the case of multipolar, for example, triangular, cells, the diameter is the distance between those two poles of the cell that are most distant from each other. In each case, "distance" means the shortest straight line connecting the respective two poles.

The term "average diameter" means the average of the diameters of two or more individual cells, in which case the diameter of each individual cell is determined in the first step, as described above, and then in the subsequent step the average diameter is determined as the arithmetic mean of the cell diameters determined in the first step .

As used herein, the terms "propagates" or "propagates" generally means the proliferation of a cell or population of cells. As a rule, in the context of the present invention, reproduction occurs in vitro or ex vivo. Propagation is generally an activity or process that occurs after cell isolation. As a rule, in the process of reproduction, the total number of cells increases. An increase in the number of cells can be described with "cumulative population doubling(s)" or with "number of passages ("P"), or with the total number of cells at the corresponding stage, and so on.

As used herein, the terms "express", "expressed", as well as "expression", "expression of a gene", and the like, refer to the use of information contained in a gene in the process of synthesizing a functional gene product. Gene expression includes at least transcription and optionally includes one or more additional features, optionally selected from an open list including RNA editing, translation and post-translational modification. When determining gene expression, the presence of an expression product is determined, such as unedited or edited RNA, or even an encoded protein. The above terms, in combination with a specific gene or locus, should specifically refer to the expression of genetic information from the gene or locus; for example, if it is indicated that APCDD1 is expressed, this should mean that the APCDD1 gene is expressed.

Used in this document, the term "extraction" ("extract") means isolation from the biological environment. Depending on the context, the term can mean "extract" (verb) or a physical component that has been extracted (eg, a cell or cell population obtained by extraction from its natural environment). For example, when cells are extracted from the umbilical cord, the cells are isolated from their original location in the umbilical cord and/or separated from other anatomical regions of the umbilical cord. Preferably, the extracted cell is a cell outside the living organism and/or physically separated from blood vessels and/or physically separated from other cells of the same organ or tissue, and/or physically separated from non-cellular anatomical regions of the same organ or tissue.

As used herein, the term “flow cytometry” means laser or impedance biophysical technology suitable for cell counting, cell sorting, cell property analysis, and biomarker detection (specifically, e.g., detection of cell surface molecules such as cluster of differentiation (CD) molecules) . Flow cytometry requires cells to be in suspension; for analysis of adherent cells, they must be separated from the substrate, eg the culture vessel to which they are attached, eg by enzymatic treatment, eg with trypsin, whereby they become cells in suspension. Cells in suspension, that is, cells in a fluid stream, are passed through an electronic detection device (flow cytometry device). The flow cytometry device makes it possible to analyze a cell, for example, based on the specific light scattering by each cell. A commercial flow cytometry instrument such as the FACSAria III flow cytometer (BD Biosciences) may be used. Data from flow cytometers can be used to plot in one dimension, to plot a histogram, or two-dimensional scatter plots, or even plots in three dimensions. Plots can be plotted using a selected scale, such as a linear or logarithmic scale. Regions on these plots can be sequentially subdivided based on fluorescence intensity, creating a series of extract subsets called "gates".

As used herein, the term "fluorescence-activated cell sorting", or interchangeably "FACS", refers to a specialized type of flow cytometry. FACS is a method for sorting a heterogeneous mixture of biological cells into two or more populations based on the specific light scattering and/or fluorescence characteristics of each cell. The type of fluorophore used as a label for FACS is not particularly limited; in some embodiments, fluorophores are attached to an antibody that recognizes a target feature, such as a cell surface protein (specifically, for example, detects cell surface molecules such as cluster of differentiation (CD) molecules). Alternatively, the fluorophore may be attached to a chemical moiety with affinity for a cell membrane or other cellular structure. Each fluorophore has a characteristic peak at a specific excitation and emission wavelength that is detected by a device suitable for FACS. A commercial instrument such as the FACSAria III flow cytometer (BD Biosciences) may be used.

As used herein, the term "heterologous" describes something that is made up of many different elements. For example, a cell transferred from one individual to another individual is a heterologous transplant. A heterologous gene is a gene originating from a source other than the specific subject.

As used herein, the terms "immortal" and "immortalized" refer to cells that can divide without being restricted by the Hayflick limit (the Hayflick limit is the point at which cells can no longer divide due to DNA damage or telomere shortening). Immortal cells typically express the telomerase-lengthening enzyme, but this is not a strict requirement.

Used in this document, the term "inoculum" means the total number of cells from which a particular cell culture (passage) begins in a particular culture vessel.

"Isolated" refers to a material that is substantially or substantially free of the impurities normally associated with it in its natural state. For example, as used herein, the term "isolated cell" means a cell that has been cleared of its cellular and extracellular environment, such as the tissue that surrounds it in its natural state, such as a mesenchymal stromal cell that has been removed from umbilical cord tissue, usually located near cells. Alternatively, as used herein, "isolated cell", and/or the like, refers to the in vitro isolation and/or purification of a cell from its natural cellular environment and from association with other tissue components in which the cell normally resides. Unless the context clearly dictates otherwise, an "isolated cell" need not be a single cell separated from any other cells; on the contrary, even a population of two or more cells, for example, several cells, can be called "isolated" if said cell population is isolated in the sense that it is substantially or substantially free from the components that normally accompany such a population of cells in its natural state. For example, a population of mesenchymal stromal cells may be termed "isolated" if it has been extracted from the original tissue, such as the umbilical cord. As used herein, the terms "isolated" and "isolated" as used herein describe an activity to produce an "isolated" material, such as, for example, an isolated cell.

As used herein, the term "committed" refers to the progeny of a cell that is capable of mitotic division but is generally incapable of an unlimited number of mitotic divisions. Eventually, the progeny of the committed cell must differentiate.

As used herein, the term "medium" means an aqueous mixture suitable for culturing cells, in particular mammalian cells. An aqueous mixture is usually a solution, although the term also covers suspensions and colloidal mixtures. The medium is usually a liquid, although some media may also be temporarily frozen, for example for storage purposes; in any case, the medium is used for cell culture when it is liquid. The medium can be either a "minimal medium", which is an aqueous mixture of a specific composition, or a "growth medium", which is an aqueous mixture containing a minimal medium and at least one additive, and which, as a rule, has the ability to maintain cell viability. ex vivo and, optionally, cell expansion, also ex vivo. Unless otherwise indicated, it is clear from the context whether the medium is a minimal medium or a growth medium. The minimum environment is preferably an environment with a defined chemical composition. The growth medium preferably comprises a minimal medium (typically 85% to 99.5% by volume) plus at least one supplement of human, animal or plant origin, such as serum, cell lysate or, in particular, in the context of the present invention, platelet lysate.

As used herein, the term "mesenchymal" refers to a cell that belongs to the mesenchymal cell type, such as an adipose cell, a cell of connective tissue, bone, cartilage, blood, lymph vessels and blood vessels, as well as a stromal cell or stem cell capable of differentiate into mesenchymal cells. Without any limitation to the present invention, mesenchymal cells in vivo are sometimes loosely grouped and found in a gelatinous interstitial substance. Typically, the mesenchymal cell originates from the mesoderm (germine mesoderm or extra-embryonic mesoderm). In some embodiments, the mesenchymal cell is a stromal cell (mesenchymal stromal cell) or a stem cell (mesenchymal stem cell), or a cell derived from one of these cells.

As used herein, the term "mesenchymal stromal cell", abbreviated "MSC", means a cell of mesenchymal origin, that is, derived from the stroma, in particular, for example, from mesodermal connective tissue. The tissue origin of the "mesenchymal stromal cell" is not further restricted; however, in preferred aspects of the present invention, the "mesenchymal stromal cell" is derived from the umbilical cord, more specifically from the umbilical cord stromal Wharton's jelly, as described in detail herein. The mesenchymal stromal cell typically expresses the surface markers CD105 and CD73, and usually also CD90. Preferably, the "mesenchymal stromal cell" of the present invention is characterized by the expression of CD105 (known as endoglin), CD73 (known as ecto-5'-nucleotidase) and CD90 (also known as Thy-1). Mesenchymal stromal cells hardly express hematopoietic antigens such as CD45, CD34, CD14, CD19 or CD3. Typically, MSCs express class I MHC molecules in vitro, but not class II molecules, unless stimulated, for example, with interferon, in tissue culture. Thus, the typical surface marker phenotype of MSCs is CD105+, CD73+, CD90+, CD45-, CD34- CD14-, CD19-, CD3-, HLA DR-. While this profile of surface markers makes it possible to uniquely identify MSCs, it is complex. A typical feature of MSCs is the ability to differentiate in vitro into various cell types, typically including osteoblasts, adipocytes, and chondroblasts. As a rule, MSCs are attached to plastic in vitro. The term "mesenchymal stromal cell" does not necessarily imply that the so-called cell is a stem cell; however, as described herein, the MSC of the present invention, or obtained by the present invention, may have properties similar to those of stem cells, in preferred embodiments.

The term “mesenchymal lineage progenitor cell” means a mesenchymal stromal cell that is capable of self-renewal while retaining multipotency and the ability to differentiate into a range of cell types, or of mesenchymal origin, such as osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons, and possibly also in cells of non-mesenchymal origin, such as neuronal cells and epithelial cells. Thus, the term "mesenchymal lineage progenitor cell" means a cell that is capable of differentiating into different cell types, although the cell is multipotent (as opposed to totipotent). Without wishing to be bound by a particular theory, it is understood that a "mesenchymal lineage progenitor cell" is a mesodermal lineage cell. However, unless otherwise indicated or the context dictates otherwise, the term "mesenchymal lineage progenitor cell" does not impose any limitation on the tissue origin of the respective cell. Thus, in general, cells referred to as "mesenchymal lineage progenitor cells" are not limited to a particular tissue origin and can be derived from, for example, bone marrow, umbilical cord, adult peripheral blood, adipose tissue, cancellous bone, and dental pulp. . In preferred aspects of the present invention, the "mesenchymal lineage progenitor cell" is derived from the umbilical cord, more specifically from umbilical cord Wharton's jelly, as described in detail herein. The term "mesenchymal lineage progenitor cell" encompasses both parental cells and their undifferentiated progeny. The term "mesenchymal lineage progenitor cell" also encompasses mesenchymal progenitor cells, multipotent stromal cells, mesenchymal stem cells and their undifferentiated progeny. In the present invention, it is highly preferred if the "mesenchymal lineage progenitor cell" is a stem cell as described herein, with the limitation that the stem cell is not totipotent.

As used herein, the terms "multi" and "plural" mean the plural, that is, any number of two or more.

As used herein, the term "multipotent" refers to a cell that is capable of giving rise to any of several types of mature cells. The term "multipotent" encompasses progenitor cells and all progeny of such cells that are still multipotent. The term "multipotent" refers to a progenitor cell having the ability to differentiate into more than one discrete cell type. Without wishing to be bound by a particular theory, it is understood that a multipotent stem cell is at the top of the lineage hierarchy and can give rise to several types of differentiated cells. Without limitation, multipotent cells can be found in the umbilical cord, adipose tissue, heart cells, bone marrow, and dental pulp. In some embodiments, multipotent cells are pluripotent.

As used herein, the term "perivascular" or "perivascular zone" or "perivascular region" refers to a physical region or region adjacent to a vessel, such as a blood vessel. The "perivascular zone", or "perivascular region", is indicated by the number 5 in figure 1. In particular, the perivascular zone/area of the umbilical cord (5) is any zone/area around each blood vessel of the umbilical cord. The perivascular zone/region can be defined as the space extending up to 3 mm from the outer walls of each umbilical cord vessel (WO 2004/072273 A1). As an example, the perivascular zone/region is described in Takechi et al., 1993 (Placenta vol. 14; p. 235-245).

The term "platelet lysate" means a preparation derived from blood platelets (also called platelets). Platelets can be obtained from a donor, preferably a human donor, either by isolating whole blood units from a pool and pooling, or by collecting platelets in apheresis: blood is collected from a donor and passed through a platelet-removing device. After their isolation, platelets, eg (re)suspended in plasma, are typically lysed, eg using one or more freeze/thaw cycles. Platelet lysate is available from many commercial suppliers, e.g. Macropharma (Mouvaux, France), from clinical blood banks, e.g. Polyclinic blood bank in Modena (Italy), but can also be obtained directly from blood platelets, as described, for example, in Naaijkens et al., Cell Tissue Res., 2012, vol. 348, p. 119-130 and WO 2013/042095A1.

As used herein, the term "pluripotent" refers to a cell that has the potential to differentiate into any of the three germ layers (endoderm, mesoderm, ectoderm) but not into germ line cells. As a rule, a pluripotent cell can differentiate into all types of adult cells, with the exception of germline cells. In one embodiment, a pluripotent cell is a cell capable of differentiating into any cell type of a mammalian body - without being bound by a particular theory, this may be about 260 cell types. A pluripotent cell may be self-renewing, and may remain dormant or inactive within the tissue. The pluripotent cell preferably has the following characteristics: (i) the ability to proliferate in an undifferentiated state, (ii) the maintenance of a normal karyotype during long-term culture, and (iii) the ability to differentiate in any of the three germ layers (endoderm, mesoderm, ectoderm) even after long-term culture. cultivation.

The term "passage number", abbreviated as "P", followed by a number (eg, "P1") means the number of times the cell culture has been subcultured. Thus, the term "passage number" is a useful term to describe the status in the process of cell propagation.

The term "population doubling", abbreviated as "PC", is used herein to describe that the number of cells has doubled, for example, in the time interval from an early time point t ₀ to a later time point t _0+x . In this case, x denotes the time interval during which the number of cells doubled. For example, t ₀ may represent the time point of the original selection, or the time point at which a certain passage was started. Thus, the term "population doubling" is a term that refers to the multiplication of cells. The term generally refers to doubling the number of cells in vitro. Population doubling (PP) is calculated by the formula:

UE=log(N/N ₀ )/log2

where N ₀ is the number of seeded cells and

N is the number of collected cells

(Purpura et al., 2004, Stem Cells. Vol. 22, p. 39-50).

The term "cumulative population doubling" is used herein to indicate the total number of times the number of cells in a cell population has doubled in vitro since a particular time point, eg, since their initial isolation. Thus, the term "cumulative population doubling" is also a term referring to cell propagation, typically in vitro. Accordingly, the total UE is determined for each passage. The value of the total UE is calculated by the formula:

n=log(N/N ₀ )/log2+X

where n=value of cumulative SP,

N ₀ =number of seeded cells (that is, at the beginning of the passage),

N=number of collected cells (i.e., at the end of the indicated passage),

X=the value of the cumulative NC at the beginning of the specified passage.

N ₀ is determined by counting the cells before the start of the passage; N ₀ depends on the cell density and the bottom surface of the culture vessel. N is determined by counting the cells after separation from the substrate, for example, after treatment with trypsin, at the end of the passage.

The cumulative UE is an additive quantity (that is, X in the formula above); for example, when cells passed through 5 cumulative PNs during the first P1 passage, and 5 more cumulative PNs during the subsequent second P2 passage, then the cells passed through 10 cumulative PNs during P1 plus P2. Freshly isolated cells have a cumulative SP=0 by definition. Thus, at the beginning P0 X=0. Because it is sometimes difficult to determine N ₀ to P0 (and therefore determine n for P0), the number of cumulative population doublings is optionally only determined from P1 onwards; however, in this case it is specifically stated in this document.

You can specify the cumulative FP as a value rounded to the nearest whole number. In the case of using certain culture conditions, it is possible to arithmetically convert the number of passages to the value of the cumulative EA, and vice versa.

The term "progenitor cell" is used herein to refer to a cell, which may be a stem cell and/or a descendant of a stem cell. The progenitors are usually descendants of stem cells, they are only more limited in their potential for differentiation or self-renewal. Progenitor cells may be, for example, multipotent or unipotent.

The terms "secrete" or "release" as used herein in relation to a cell generally refer to any material that escapes the cell into the external environment. Said material has typically been produced and/or modified by the cell, although not necessarily. For example, some cells secrete proteins and/or regulatory molecules such as hormones, prostaglandins and neurotransmitters and/or microvesicles and/or exosomes into their respective environment. For example, a prostaglandin produced by a cell can be released into the environment of the cell. These terms used herein are not limited to either an in vivo secretion/in vivo environment situation or an in vitro secretion/in vitro environment situation, unless the context clearly dictates otherwise.

The term "cell volume" or simply "volume" as applied to a cell means the cell volume as determined by flow cytometry, in particular in a FACS assay, more specifically by forward scatter (FSC) determination. In the context of the present invention, cell volume is determined when the cell, for example, a detached cell such as a trypsinized cell (from an adherent culture) or a collagenase-released cell (after isolation from a tissue), is in suspension, i.e., the cell is not attached. culture plate or tissue. The definition of SSC and, optionally, cell volume is described below in this document.

The term "average volume" or "average cell volume" means the average volume of two or more individual cells, wherein the first step determines the volume of each individual cell by flow cytometry as described herein, and then determines the average in a subsequent second step. volume in the form of the arithmetic mean of the cell volumes of individual cells determined in the first stage. As a rule, to determine the average cell volume, the first and second stages are carried out for at least 1000 cells; in other words, the average is the average of at least 1000 cells.

As used herein, the term "stem cell" means a eukaryotic cell capable of giving rise to phenotypically and genotypically identical daughter cells ("self-renewal"), as well as cells of at least one definitive cell type (e.g., terminally differentiated cells). The term "stem cell" means a cell, which may be totipotent, pluripotent or multipotent, as well as a progenitor and/or progenitor cell resulting from its differentiation, and/or a committed descendant cell. Generally, the term "stem cell" can refer to an adult stem cell or an embryonic stem cell. The term "stem cell" may optionally refer to an induced stem cell, such as an induced pluripotent stem cell, but more preferably refers to a cell that has not been induced.

The terms "stemness" or "stem cell-like property/stem cell-like properties" are used interchangeably to refer to a property of a cell, in particular, the ability to self-renew and the ability to form differentiated cells. More precisely, cells having properties similar to those of stem cells can give rise to daughter cells identical to the mother cell (self-renewal) as well as produce progeny with more limited potential, such as differentiated cells. Cells with properties similar to those of stem cells are characterized by a certain degree of potency, that is, the ability to produce differentiated progeny. Some cells with properties similar to those of stem cells may be capable of producing differentiated cells of several types (for example, "multipotent" or "pluripotent"), while others may be capable of producing differentiated cells of one type ("unipotent") . Although cells with stem cell-like properties are capable of self-renewal, most somatic cells, including those with stem cell-like properties, when cultured in vitro are able to proliferate for a finite number of cumulative population doublings before replication arrest, or senescence. In preferred embodiments, the mesenchymal stromal cells of the present invention are mesenchymal stromal cells with stem cell-like properties.

Typically, a "stroma" is a portion of a human or animal tissue or organ that performs a mechanical tissue support function, such as providing connective, structural support, and/or (preferably and) functional tissue support. Thus, in a living organism, the stroma provides the connective and functional support framework of a biological tissue or organ (as opposed to the parenchyma, which is the functional aspect of the tissue). As used herein, the term "stroma" refers to connective tissue cells in any organ, such as, but not limited to, the umbilical cord, uterine lining (endometrium), prostate, bone marrow, lymph nodes, and ovaries. In the respective organ, the stroma supports the function of the parenchymal cells of that organ. Thus, the stroma is made up of all parts that do not perform specific organ functions, such as connective tissue, blood vessels, nerves, ducts, and so on. Thus, the umbilical cord stroma is the matrix that makes up the umbilical cord, with the exception of the vessels, the perivascular region, and the amniotic epithelium. The term "stroma" may refer to the stroma of an entire organ or to a specific anatomical region of it. In particular, the term "stroma" may refer to the stroma of the entire umbilical cord or to a particular anatomical region thereof, that is, the stroma contained in a particular section of the umbilical cord.

As used herein, the term "stromal" refers to material present in or derived from the stroma. The term is not limited to the localization of "stromal" material (eg, a stromal cell) within the stroma, but also refers to material (eg, a cell) that was present in the stroma in an organ of a living human or animal, but was subsequently removed or isolated from the organ. , so that it is no longer present in the organ of a living person or animal. In other words, the term "stromal" refers to the specific in vivo origin of the material (eg, cell), whether or not the material (eg, cell) has been extracted or isolated. Material (eg, a cell) that has been extracted or isolated from the stroma may also be referred to herein as "stromal-derived", "stromal-derived", or the like. In some preferred embodiments of the present invention, stromal material (eg, a stromal cell) is, or has been, removed or isolated such that it is not, or no longer, present in an organ of a living human or animal.

Thus, a "stromal cell" is a biological cell that resides in or is derived from the stroma, that is, extracted or isolated, such that it is not, or no longer, present in an organ of a living human or animal. The stromal cell is alive unless otherwise indicated or otherwise implied by the context. In many organs, fibroblasts and pericytes are among the most common types of stromal cells. As an illustrative example, a stromal cell can be a cell located in the stroma (eg, umbilical cord stroma), or a cell that has been isolated (eg, extracted) - and optionally expanded - from the stroma (eg, umbilical cord stroma). Stromal cells are generally non-cancerous cells.

The term "syngeneic" is used herein to describe an element derived from individuals or cells having identical genotypes, such as identical twins or animals of the same inbred line, or cells thereof.

As used herein, the term "totipotent" refers to a cell that is capable of dividing and producing all of the differentiated cells in the body. As a rule, a totipotent cell is capable of giving rise to a whole embryo. In mammals, as a rule, only the zygote and subsequent blastomeres are totipotent.

The term "umbilical cord", or "umbilical cord", means the channel between the newborn and the placenta in placental animals. During prenatal development, the umbilical cord is physiologically and genetically part of the fetus. Although the umbilical cord is present throughout embryogenesis, connecting the placenta to the developing embryo or fetus, in this specification the term refers specifically to the umbilical cord of a child who has already been born. In humans, the umbilical cord usually contains three blood vessels, that is, two arteries (umbilical arteries) and one vein (umbilical vein). Three blood vessels, as a rule, are immersed in jelly whartons. Neither the placenta, or parts thereof, nor the newborn, or parts thereof, are part of the umbilical cord; this term refers exclusively to the channel.

The term "unipotent" refers to a cell with properties similar to those of stem cells, which can produce only one type of differentiated progeny. Such a unipotent cell may also be referred to as a progenitor cell.

Used in this document, the term "viable" in relation to the cell means that the cell is a living cell. Living cells are usually metabolically active. Although several different stains and fluorochromes can be used to stain non-viable cells, 7-amino-actinomycin D (7-AAD) staining is preferred herein. 7-AAD is a non-membrane-permeable dye that generally does not penetrate into viable cells. It binds to double-stranded DNA by intercalation between base pairs in G-C rich regions. 7-AAD can be excited at a wavelength of 488 nm, for example with an argon laser, and emits fluorescence at a maximum wavelength of 647 nm. Using 7-AAC, cell viability can be determined by flow cytometry, in particular FACS (fluorescence-activated cell sorting).

As used herein, the term "vessel" (not to be confused with "culture vessel" - see above) generally refers to a tube or conduit (e.g., vein or artery) that encloses and transports, or circulates, body fluid (e.g., blood). Unless the context clearly dictates otherwise, the term "vessel" is also used herein to refer specifically to vessels in the umbilical cord. In humans and many other mammals, the umbilical cord typically contains three blood vessels, that is, one vein that carries oxygenated, nutrient-rich blood to the fetus in vivo, and two arteries that carry oxygen-deprived, nutrient-depleted blood in vivo; although rare cases have been reported in which only two vessels (one vein and one artery) were present in the umbilical cord.

The term "Wharton's jelly", abbreviated as "BC", means the gelatinous substance of the placenta of animals, which contains cells. Typically, Wharton's jelly contains stromal cells, more specifically mesenchymal stromal cells. In addition, Wharton jelly typically contains mucopolysaccharides such as hyaluronic acid and chondroitin sulfate, as well as water. Although Wharton's jelly has also been described in the vitreous fluid of the eyeball, it is strongly recommended herein to consider Wharton's jelly as part of the umbilical cord of a newborn placental animal, preferably a mammal such as a human. In this case, Wharton jelly can also be called the gelatinous substance of the umbilical cord. In other words, it is strongly recommended that the Wharton jelly described herein originate from the umbilical cord, even more preferably originate from the human umbilical cord. As described below, the umbilical cord typically contains jelly in the perivascular region (5), in the intervascular region (4), and in the subamniotic region (1b).

The terms "periovascular Wharton's jelly" or "OSVS" or "VSVS" are synonymous and mean the Wharton's jelly of a specific anatomical region of the umbilical cord, namely, the area of Wharton's jelly, anatomically located around the vessels of the umbilical cord (5). In the case of the human umbilical cord, which is a preferred embodiment, the SSVR is located in a region extending from the outer surface of the vessel at a distance of 3.0 mm or less (for a review, see Davies et al., Stem Cells Translational Medicine, 2017, vol. 6, pp. 1620-1630). Thus, when the area located within 3.0 mm from the outer side of the vessels of the human umbilical cord is completely removed, the perivascular zone and, therefore, all the perivascular Wharton jelly located therein, are completely removed, respectively. For illustration, see also figure 1. The abbreviations "OSVS" and "VSVS" are also used in this document as synonyms and mean cells derived from the perivascular zone, including "perivascular Wharton's jelly", respectively, for example, in the captions to the figure. Perivascular Wharton's jelly (PRWS)-derived mesenchymal stromal cells may also be referred to as "perivascular-derived mesenchymal stromal cells".

The term "subamniotic stroma" means the area (1b) immediately adjacent to the amniotic epithelium (1a) of the umbilical cord. In the case of the human umbilical cord, which is a preferred embodiment, the subamniotic stroma is located in a region extending from the inner surface of the amniotic epithelium inwards by 3.0 mm or less.

The term "subamniotic warton jelly" means warton jelly in the subamniotic stroma (1b) immediately adjacent to the amniotic epithelium (1a) of the umbilical cord. In the case of the human umbilical cord, which is the preferred embodiment, the Wharton's subamniotic jelly is located in a region extending from the inner surface of the amniotic epithelium inwards by 3.0 mm or less.

As used herein, the term Whartonian stromal jelly, abbreviated as SHS, means Whartonian jelly of the umbilical cord, excluding the perivascular region (4). Thus, the term Whartonian stromal jelly does not include the perivascular region (4) and therefore does not include cells from the perivascular region (4). While there has been debate in the art as to whether or not the perivascular region contains mesenchymal stromal cells, such debate is not relevant to the present invention, at least because the present invention does not contemplate isolating mesenchymal stromal cells from the perivascular region, but at least for the reason that the perivascular region is not the source of the cells of the present invention. Unless specifically stated otherwise, the subamniotic stroma (1b) is contained within the stroma referred to herein. Unless specifically noted otherwise, subamniotic Wharton's jelly is contained within Wharton's jelly, referred to herein as "Wharton's stromal jelly". Subamniotic Wharton's jelly is contained in the subamniotic stroma. The abbreviation "SBC" is also used herein to refer to cells derived from "stromal Wharton's jelly", for example, in figure captions. Thus, with reference to figure 1, the stroma (and thus the source of Wharton's jelly) in the broadest definition includes all those areas of the umbilical cord that are not part of any of the following: (1a) amniotic (or epithelial) layer; (2) umbilical vein; (3) umbilical arteries; (5) perivascular region; (7): cord blood. In other words, in this broadest definition, the stroma (and thus the source of Wharton's jelly) includes (6) Wharton's jelly in the umbilical lumen plus (1b) the subamniotic (or mesenchymal) layer); plus (4) intervascular area. Unless specifically stated otherwise, subamniotic Whartonian jelly (1b) is preferably included in the Whartonian jelly referred to herein as "Whartonian stromal jelly". Preferably, (4) the intervascular region is included in the Whartonian jelly, referred to herein as "Whartonian stromal jelly". Thus, the stroma (and thus the source of Wharton's jelly) in its broadest definition includes all those areas of the umbilical cord that are schematically labeled 1b, 4 and 6 in figure 1. In the event that the anatomical definitions given in this document in relation to the umbilical cord differ in part or in whole from the definitions in some or all of the literature, the definitions given herein shall take precedence.

It has been suggested in the literature (Davies et al., Stem Cells Translational Medicine, 2017, vol. 6, p. 1620-1630) that, from a pragmatic point of view, only (1a) the lining of the umbilical cord and (5) the perivascular Whartonian jelly can be clearly dissected from other areas or zones. In one embodiment of the present invention, this clear cut is provided, that is, the separation of the stromal Wharton's jelly from (1a) the lining of the umbilical cord and (5) the perivascular zone containing the perivascular Wharton's jelly.

The present invention is based on several facts which are interrelated and thus have led the inventors to develop various aspects of the invention, all of which will now be described in detail. Thus, all aspects described in this specification may be combined with each other, even if not specifically stated, unless the context clearly dictates otherwise. All aspects of the present invention are based, in particular, on the fact that Whartonian stromal jelly contains cells with unique properties, in particular in a competent state for reproduction, and that a certain method of isolation allows these potentially multipotent stromal cells to be reliably obtained, so that they can be maintained and propagated in culture.

No human embryo is, or has been, destroyed in the context of the present invention. In particular, no method or cell or cell population of the present invention requires the death of a human embryo.

Method for obtaining mesenchymal stromal cells

In a first aspect, the invention relates to a method for producing a mesenchymal stromal cell. The method includes (a) isolating the stromal Wharton's jelly, or a portion thereof, and (b) enzymatically treating the stromal Wharton's jelly, or a portion thereof, thereby releasing at least one cell from the stromal Wharton's jelly, or a portion thereof.

In particular, with reference to Figure 1, which is a cross section of a (human) umbilical cord and a schematic representation of sections (anatomical regions) of a (human) umbilical cord, the method can be described as a method for obtaining a mesenchymal stromal cell, comprising (a) isolating stromal Wharton's jelly ( 1b, 4, 6), or a portion thereof, and (b) enzymatically treating said stromal Wharton jelly, or portion thereof, thereby releasing at least one cell from the stromal Wharton jelly, or portion thereof.

The cell obtained after step (b) may be pluripotent or, more commonly, multipotent. Optionally, but preferably, additional steps are included, in particular a propagation step, which is detailed below.

In the method of the present invention, the jelly is isolated from the umbilical cord. This is a paradigm shift regarding the general trend in the art that bone marrow (BM) derived mesenchymal stromal cells (MSCs) are the best studied population of adult MSCs and are considered the gold standard for MSC-based applications (e.g., Batsali et al. ., Blood, 2013 vol. 122, p. 1212). However, bone marrow aspiration is typically an invasive and painful procedure. The great advantage of the method of the present invention over isolation from bone marrow (BM) is that the umbilical cord is easily accessible at birth of the mammal, while the surgery required to obtain bone marrow is typically invasive and painful. As a consequence, patients, donors, and medical professionals are reluctant to use bone marrow-derived MSCs and are likely to be even less willing to do so if reliable alternatives are available.

Thus, the advantage of the method of the present invention is that the method is an ex vivo method. Preferably, an invasive procedure on a living human or animal is completely excluded. Thus, the method of the present invention is preferably not a method of treating a human or animal with surgery or therapy. As a result, the problems associated with pain, surgery and ethical aspects are successfully eliminated, in comparison with the method of isolation from the bone marrow described in the literature.

The next key difference from some early studies and reports is that, according to the prior art, MSCs can be efficiently isolated from the connective tissue surrounding umbilical cord vessels (e.g., Batsali et al., Blood, 2013 vol. 122, p. 1212) , while in accordance with the present invention, MSCs are isolated from an area that does not include the perivascular zone, and therefore the perivascular Wharton's jelly. Thus, the authors of the present invention have found that a certain anatomical region of the umbilical cord must be targeted to achieve the benefits described in this document. More specifically, the method of the present invention is characterized by a specific anatomical region of origin of cells and/or a specific division into sections and segmentation of the umbilical cord, and/or a specific isolation step, and/or, optionally, a specific expansion step, all of which are detailed below and exemplified. . It is important that the preferred and/or optional embodiments and features of the particular anatomical region of origin of the cells, the particular sectioning and segmentation of the umbilical cord, the particular isolation step, and optionally the particular propagation step can be combined with each other according to the present invention in each, and in any combination, unless the context clearly implies that a particular combination is not possible or desirable according to the present invention.

In the method of the present invention, the rapid isolation of stromal cells from the umbilical cord is preferred. In a preferred embodiment, steps (a) and (b) are carried out for a total of less than 6 hours, more preferably less than 5 hours, more preferably less than 4 hours, and most preferably less than 3 hours. Less than 3 hours includes time intervals from 0.5 to 2.5 hours and from 1 to 2 hours. Obviously, the short combined duration of steps (a) and (b) also means that the time interval for step (a) is itself short.

Unless specifically stated otherwise, all reagents, ingredients, culture vessels, containers, and materials that come into direct or indirect contact with cells are preferably sterile, and more preferably GMP rated.

The following is a detailed description of the method and its embodiments.

Fabric source

The first step of the method according to the invention is step (a) consisting in the isolation of stromal Wharton's jelly, or part thereof, from the umbilical cord. Step (a) is preferably a mechanical step, that is, no chemical or enzymatic isolation agents are usually used. Thus, in a preferred embodiment, in step (a), no ingredients with enzymatic activity are added during step (a). As further described below, however, enzymatic treatment is important for the subsequent step (b).

The term "portion" when used in conjunction with the term "stromal Wharton's jelly" is not particularly limited and generally means that, in some embodiments, less than 100% of the total amount of stromal Wharton's jelly can be used in the method of the invention. For example, in some embodiments, 10-99%, eg, 20-90%, 30-80%, 40-60%, of the total stromal Wharton's jelly may be used. The reasons for using less than 100% in some embodiments may be multiple and may be due to experimental limitations for recovering 100% umbilical cord stromal Wharton's jelly, or it may be intentional, such as when it is desired to use a portion of the stromal Wharton's jelly for comparative experiments. However, it is preferable to try to use as much as possible, ie 80% or more, of the stromal Wharton's jelly, preferably 90% or more. Values expressed in %, unless otherwise indicated, refer to the % of the total volume of stromal Wharton's jelly contained in one umbilical cord.

Thus, in accordance with the present invention, cells are isolated from the umbilical cord. The umbilical cord is derived from a placental animal, however, preferably the umbilical cord is derived from a mammal. In placental mammals, the umbilical cord is the structure that connects the placenta to the developing fetus and, immediately after birth, to the newborn. Neither the placenta nor the newborn is part of the umbilical cord; The umbilical cord is just a connecting tube between the placenta and the newborn.

The umbilical cord is received after birth. Preferably, the umbilical cord is obtained 24 hours or less after birth, such as 12 hours or less after birth, 6 hours or less after birth, 3 hours or less after birth, such as 2 hours or less after birth, and preferably 1 hour or less. less after birth. Thus, the umbilical cord can be stored, preferably for a maximum of 24 hours after birth, preferably at low temperatures of 0-10°C, preferably 2-8°C, until cells are isolated. However, the umbilical cord is not frozen prior to cell isolation. Optionally, the umbilical cord is washed with sterile water or sterile buffered aqueous solution before cell isolation.

The umbilical cord is preferably stored and processed under sterile conditions prior to and during cell isolation. Optionally, the umbilical cord can be further superficially sterilized by rapidly treating the surface of the umbilical cord, for example, with an aqueous (70% v/v) ethanol solution or betadine, followed by rinsing with sterile water.

Preferably, the cells are isolated from the human umbilical cord. The human umbilical cord (PC) at term birth has a mass of about 40 g and a mean diameter of about 1.5 cm (Raio et al., 1999, Eur. J. Obstet. Gynecol. Reprod. Biol., vol. 83; p. 131-135). Thus, preferably, the umbilical cord used in the present invention is a human umbilical cord. The human umbilical cord used in the method of the present invention preferably has a length of about 40-65 cm, such as 44-51 cm.

As a rule, the umbilical cord is obtained after the birth of a healthy baby from a healthy mother, preferably a female mother. Preferably, said mother has abstained from smoking, drinking alcohol and taking psychotropic pharmaceuticals for at least one month prior to delivery.

When it is said that Wharton's stromal jelly, or part thereof, is isolated from the umbilical cord, this preferably means that no more than one umbilical cord is used as starting material. However, it is also possible to use more than one umbilical cord as starting material. In one embodiment, the method of the present invention uses a single umbilical cord as the starting material. In this embodiment, all starting material is autologous, i.e., no allogeneic umbilical cord material is part of the starting material, and therefore all cells obtained by the method of the present invention are obtained from a single donor each time the method is used.

It is possible to use the entire umbilical cord as starting material, but it is also possible to use part of one umbilical cord as starting material. When one whole umbilical cord is used as the starting material in the method of the present invention, the method can be said to be used at "full scale". Accordingly, the method in the preferred aspect of the present invention, including steps (a), (b) and optionally (c), is called a "full-scale" cycle, when one whole umbilical cord is used as starting material. In this context, the word "whole" does not exclude that minor parts of the umbilical cord, for example the two ends of the umbilical cord, are separated or cut off and thus not used as starting material; it also does not preclude that the umbilical cord is cut into sections and/or segmented in the manner described herein; rather, it means that virtually the entire umbilical cord is used as starting material. In the case of a human umbilical cord at term, the whole umbilical cord typically has a mass of 35 to 40 grams, or more accurately, about 40 grams. In one embodiment, a full-scale cycle starts with 35-45 grams of umbilical cord from one donor. The amount of starting material, that is, the umbilical cord, of course, affects the total number of cells obtained in the subsequent phases of the method of the present invention. Unless specifically stated otherwise, all amounts (eg, cell numbers) given herein refer to a full scale run. The term "full-scale" does not conflict with the term "portion" of stromal Wharton's jelly, provided that reasonable efforts are made to extract the maximum possible amount of stromal Wharton's jelly from one umbilical cord.

The outermost layer of the umbilical cord (also referred to as cord lining, cord tissue, or cord lining, labeled 1a in the diagram of Figure 1) is not a desirable source of cells in the method of the invention. Since the umbilical cord itself is an extension of the placenta, the lining of the umbilical cord is an extension of the amniotic membrane that covers the placenta. The umbilical cord lining has two layers: the amniotic (or epithelial) layer (also called the umbilical cord lining) and the subamniotic (or mesenchymal) layer. The human umbilical cord, for example, is covered with one/several layer(s) of squamous cuboidal epithelial cells (Copland et al., Placenta, 2002; vol. 23, p. 311-321; Mizoguchi et al., J. Dermatol. Sci., 2004 , vol. 35, pp. 199-206); it is undesirable to isolate these cells in the method of the invention. In one embodiment of the method according to the invention, it is desirable not to isolate either cells from the amniotic layer or cells from the subamniotic layer, and therefore such cells are not used as starting material in the following optional cell expansion.

The architecture of the internal tissue of the umbilical cord consists of two arteries and one vein, as well as a surrounding matrix of mucous connective tissue (stromal Wharton's jelly). Wharton jelly has been described to contain fibroblast-like cells (Parry, 1970, J. Anatomy, vol. 107, p. 505-518) and sometimes mast cells. In addition, Wharton's jelly contains an amorphous ground substance rich in proteoglycans, mainly hyaluronic acid.

Preferably, the umbilical cord used in the method of the present invention is obtained from term delivery, also referred to as "term delivery". More specifically, the human umbilical cord used in the method of the present invention is obtained at delivery occurring within 38-42 weeks of gestation, more preferably within 39-41 weeks of gestation. In one embodiment, the human umbilical cord is obtained from a normal (vaginal) birth; in an alternative embodiment, the human umbilical cord is obtained from an artificial birth, such as a caesarean section.

In one embodiment, the collection of the umbilical cord at the birth of a human or animal is not part of the present invention. In this embodiment, the method of the invention is started after the umbilical cord has been obtained. In other words, the method according to the invention is put into practice using as starting material the umbilical cord that was obtained before step (a).

Segmentation of the umbilical cord

In addition, the inventors have found that it is preferable to mechanically separate the umbilical cord as described herein. The division preferably includes segmentation and division into sections, as described in this document.

In the first step, it is preferable to segment the umbilical cord as described herein. Segmentation is optional, but preferred. In particular, segmentation facilitates subsequent steps and promotes reliable reproducibility. In addition, segmentation increases the efficiency of isolation of stromal Wharton's jelly in the subsequent sectioning step. Thus, the segmentation is preferably carried out before the step of dividing the umbilical cord into sections (segments) described below.

Segmentation according to the present invention, as a rule, is carried out by physical methods, for example, using scissors or a scalpel. By segmenting the umbilical cord, segments of the umbilical cord are obtained (for an illustration, see, for example, example 1). Used in this document, the term "segments" means such cut pieces of the umbilical cord. Preferably, the segmentation is performed perpendicular to the umbilical cord. As used herein, the term "perpendicular" in relation to the umbilical cord means that the direction of the incision of the umbilical cord is at an angle of about 90° relative to the direction of the umbilical cord. To determine the angle, the umbilical cord is preferably placed in a stretched state, that is, without curls or bends. "About 90°" means 75°-105°, preferably 80°-100°, more preferably 85°-95°. A qualified person will be able to cut the umbilical cord perpendicularly even without an angle measuring device, based on their own experience. However, a device can be used to determine the angle, if desired.

More preferably, each perpendicularly cut segment has a length of from about 1 to about 4 cm, preferably from about 2 to about 3 cm, and more preferably about 2.5 cm. ) umbilical cord up to step (b). In particular, it is preferable to remove the vessels, or more precisely, the segments of the vessels, after - preferably perpendicular - segmentation of the umbilical cord.

Segmentation is optional, but preferred, and thus is preferably part of the method of the invention. The segmentation is preferably carried out under sterile conditions.

Division into sections to obtain stromal Wharton's jelly

Typically, the mammalian umbilical cord, and in particular the human umbilical cord, is characterized by tissue compartmentalization, with distinct cellular characteristics and extracellular matrix elements. Based on data from structural and functional studies, at least six distinct zones or sections are described (see also Figure 1): they are listed here in order from outside to inside, with the numbers corresponding to those in Figure 1: lining of the umbilical cord (including amniotic epithelium and subamniotic cells) (1), (intervascular) stroma (classic name Wharton's stromal jelly), intervascular stroma (4), perivascular stroma (5), vessels (2 and 3), subamniotic stroma (1b), clefts (not shown) .

It has been accepted in the present field, given the hypothesis that perivascular cells tend to migrate from areas close to the vasculature, that the majority of MSCs in Wharton's jelly originate from the perivascular region (Davies et al., Stem Cells Translational Medicine, 2017, vol., 6, pp. 1620-1630).

However, the inventors unexpectedly found that the sectional approach is preferable for the extraction of MSCs from PC, and that the section containing MSCs with superior properties is Wharton's stromal jelly. The inventors were surprised to find that cells derived from Wharton's stromal jelly, or parts thereof, have excellent properties. Thus, a necessary step in the method of the present invention is the isolation of stromal Wharton's jelly, or part thereof.

Before isolating stromal Wharton's jelly, it is important to flush the umbilical cord, or in particular the segments of the umbilical cord. An aqueous solution is suitable for washing, ideally buffered at or near physiological pH and isotonic, optionally with the addition of antibiotics. For example, HEPES-buffered saline (HBSS) or phosphate-buffered saline (PBS) is suitable, preferably HBSS containing 300 μg/ml gentamicin and 0.15 μg/ml amphotericin B. A typical wash time may preferably be 1-30 minutes , preferably about 10 minutes. During this time, a mechanical movement, such as shaking, is used to properly flush the umbilical cord, or preferably cord segments, with the aqueous rinsing solution. In particular, after segmentation of the umbilical cord, rinsing is important because it allows the removal of blood residues at the boundaries of the segments obtained by segmentation.

Sectioning, preferably of the umbilical cord, or more preferably of the umbilical cord segment(s), is then performed to isolate the stromal Wharton's jelly, as described herein. Thus, the desired stromal Wharton jelly is obtained. And other ways of obtaining stromal Wharton's jelly are suitable as an alternative to the division into sections described in this document, and are also part of the present invention. Preferably, however, Wharton's stromal jelly is prepared by sectioning as described herein.

It turned out that to isolate the desired Wharton's jelly, lateral cutting of the umbilical cord or segment(s) of the umbilical cord is preferable. Preferably, the umbilical cord or segment(s) of the umbilical cord is cut longitudinally, for example with a scalpel. "Longitudinal" means parallel to the length of the umbilical cord. One longitudinal incision per segment is preferred over multiple incisions. A lateral incision, or more specifically a longitudinal incision, makes it possible subsequently to effectively separate the different sections of the umbilical cord, in particular, it makes it possible to isolate the Wharton's stromal jelly, as described below.

As detailed herein and illustrated in the examples provided herein, cells from stromal Wharton's jelly have proven to be of great benefit. Thus, in order to obtain Wharton's jelly, which is to be sectioned and selected for further processing according to the present invention, the perivascular zone is excluded and, therefore, any perivascular Wharton's jelly is excluded. In other words, the Whartonian jelly to be sectioned and selected for further processing according to the present invention is a stromal Whartonian jelly. The fact that Wharton's stromal jelly cells isolated in accordance with the present invention have excellent properties is a surprising finding of the present inventors. Previously by detailed structural, immunohistochemical (e.g. Akerman et al., Gynecol. Endocrinol., 2002, vol. 16, p. 299-306 and Karahuseyinoglu et al., Stem Cells, 2007, vol. 25, p. 319-331 ) and in vitro functional studies (Sarugaser et al., 2005, Stem Cells, vol. 23, p. 220-229; Karahuseyinoglu et al., Stem Cells, 2007, vol. 25, p. 319-331) found that that there are differences in the numbers and nature of cells between the subamniotic (1b), intervascular (4) and perivascular (5) regions, and that the density of cells in a particular space differs in the subamniotic, intervascular and perivascular regions: cell density is significantly lower in the subamniotic regions, with At the same time, cell density is maximal in perivascular areas (Takechi et al., 1993, Placenta; vol. 14, p. 235-245; Karahuseyinoglu et al., 2007, Stem Cells, vol. 25, p. 319-331). It should be noted that in this paragraph, the term "cell density" is used in a non-conventional way in relation to three-dimensional space, since in the rest of this document this term is used in relation to two-dimensional area. For example, in earlier studies, areas adjacent to the serosa and adjacent to the vessels were proposed as a source for cord-derived stromal cells (eg, US 2004/0136967 A1). While it has been previously shown that cells can be isolated from Wharton's jelly and that different cell populations exist in Wharton's jelly, the specific choice of stromal Wharton's jelly of the present invention is a more specific choice than previously published approaches (e.g., US 2004/0136967 A1) , and differs from the choice proposed by other researchers (for example, WO 2004/072273 A). Thus, sectioning prior to physical extraction of cells differs sharply from proposals in the literature whereby tissue is not usually carefully selected and sectioned prior to cell isolation, but the desired cells are isolated from the cell population, for example, by selectively killing unwanted cells (negative). selection, see for example US 2004/0136967 A1). To the present inventors' knowledge, it has not been specifically known that cells derived from umbilical cord stromal Wharton's jelly are superior to perivascular cells in uniformity, size (volume and surface area, respectively), differentiation potential, and stemness. Thus, contrary to conventional wisdom in the art, the present inventors have found that cells derived from stromal Wharton's jelly are superior in many respects. Some of these aspects are illustrated in the experimental examples in this document. In general, it can be assumed that cell populations with uniform morphological and functional properties may be suitable for the development of a relatively uniform cell product. Without wishing to be bound by a particular theory, it is possible that the uniformity can be explained by the fact that Whartonian stromal jelly generally contains fewer, if any, pericytes and endothelial cells than the perivascular region.

The inventors were surprised to find that cells derived from umbilical cord stromal Wharton's jelly have particularly many advantages. Subamniotic stroma (typically containing Wharton's jelly) (1b) can be obtained by scraping the inner surface of the amniotic epithelium (1a) by mechanical means; however, precautions should be taken to avoid rupture or fractionation of the amniotic epithelium, which may lead to undesirable entrapment of a portion of the amniotic epithelium.

Advantages include homogeneity of cell populations and properties similar to those of stem cells. In particular, these advantages can be achieved in the absence of cord vessels and perivascular area, and therefore in the absence of cells from cord vessels and perivascular area.

Thus, it is preferred if step (a) isolating the stromal Wharton's jelly, or a portion thereof, from the umbilical cord comprises sectioning the umbilical cord, or segments thereof. Preferably, it includes the division into sections of segments of the umbilical cord. During the sectioning step, the desired stromal Whartonian jelly is separated from the vessels with the perivascular zone, which may contain the perivascular Whartonian jelly. During the sectioning step, the Whartonian stromal jelly is also separated from at least the amniotic epithelium. Care must be taken to avoid rupture or fractionation of the amniotic epithelium, which may lead to undesirable entrapment of a portion of the amniotic epithelium.

Sectioning is preferably carried out under sterile conditions.

Sectioning is a necessary part of step (a) of the method of the present invention, that is, the step of isolating stromal Wharton's jelly, or a portion thereof, from the umbilical cord. Thus, in one embodiment, isolation of stromal Wharton's jelly, or a portion thereof, from the umbilical cord is accomplished by subjecting the umbilical cord to physical separation, wherein the vessels and perivascular region of the umbilical cord, as well as at least the amniotic layer of the lining of the umbilical cord, are discarded, and the stromal Wharton's jelly, or part of it, save.

It turned out that for sectioning, or more precisely, sectioning by mechanical separation, it is very useful if the umbilical cord was previously segmented as described above, and, in addition, cut longitudinally, as described above. Thus, in a preferred embodiment of the present invention, step (a) of the method includes both the segmenting step described above and the sectioning step described.

A suitable method for isolating stromal Wharton's jelly, or a portion thereof, from the umbilical cord involves removing blood vessels and the perivascular region from the umbilical cord. Preferably, the perivascular zone, which according to the literature contains Wharton's perivascular jelly, is also separated from the lining of the umbilical cord, in particular at least the amniotic layer of the lining of the umbilical cord.

Thus, the sectioning of the present invention is characterized by the physical removal of vessels and the perivascular region from the umbilical cord. In accordance with the present invention, the vessels and the perivascular region can be removed before step (a) or as part of step (a), which is preferable. One technically convenient method for removing the vessels and perivascular region, which is preferred by the present invention, is the separation of the vessels and perivascular region from the umbilical cord. For example, the blood vessels of the umbilical cord (two veins and an artery) are separated using sterile forceps.

The inventors have found that the separation of the vessels, for example, by pulling the vessels, also leads to the separation of the perivascular region. In a preferred embodiment, the perivascular region is removed completely; this means that no cells belonging to the perivascular region remain in the umbilical cord (segments) after removal of the vessels. Any other method is equally suitable, provided that the perivascular region is completely removed. The zone proximal to the outer wall of the umbilical cord vasculature is the perivascular zone. The perivascular zone is typically within a zone extending up to about 3 mm or less from the outer wall of the vessels. The perivascular zone is removed along with the vessels when the vessels are separated. Any Wharton's jelly that may be in the perivascular region proximal to the outer wall of the umbilical vasculature may be termed a perivascular Wharton's jelly. Perivascular Whartonian jelly is typically located in a zone extending up to about 3 mm, or less, from the outer wall of the vessels. Any perivascular warton jelly is removed along with the vessels when the vessels are separated.

Thus, in a preferred embodiment, the vessels are separated from the umbilical cord or umbilical cord segment(s). Separation can be carried out by grasping the vessel(s) with mechanical means, such as tweezers or any other suitable means for this purpose, and pulling the vessel(s). Preferably, the vessels are drawn one after the other, but this is not a requirement. When separating the vessels, the umbilical cord, or a segment thereof, is usually fixed, for example, by holding it with mechanical means, such as tweezers or any other means suitable for this purpose. Thus, it is possible to apply mechanical force and remove the vessels. Removal should be done with care in the sense that vascular opening and/or loss of vascular or perivascular cells should be avoided. Removal should be done with care in the sense that pulling of the umbilical cord matrix should be avoided or alternatively minimized.

Preferably, after the vascular removal step, the umbilical cord, or the corresponding segment(s), is re-examined to ensure that the vessels (and thus also the SSVR) have been completely removed.

Vessels, including any tissue adjacent to them, are discarded after separation. This means that the vessels, including any tissue adjoining them, are not further subjected to a subsequent enzymatic treatment step after separation. In the method of the present invention, Wharton jelly is not used as a source for cell isolation. Thus, no perivascular Whartonian jelly is used as a source for cell isolation. For illustrative purposes, Figures 1 and 2 show Wharton's perivascular jelly (5 in Figure 1).

In accordance with the present invention, the vessels and the area around the vessels are discarded. The area around the vessels has a diameter of at least about 2.0 mm, while measuring the distance away from the outer surface of the vessel (see figure 2), for example, a diameter of 2.0 mm, preferably a diameter of 2.5 mm, and more preferably 3, 0 mm. Thus, at least a region at a distance of about 2.0 mm, for example, 2.0 mm, preferably 2.5 mm, and more preferably 3.0 mm, around the vessels of the umbilical cord is not used as a starting material for the isolation of cells of the present invention. The preferred method for conveniently accomplishing this is to physically remove ("separate") the vessels from the umbilical cord, or more specifically, the vessel segments from the umbilical cord segments, as described herein. Thus, in a preferred embodiment, the vessels are physically removed from the cord, or the vessel segments are physically removed from the cord segments. The latter option is preferred; this requires segmentation of the umbilical cord, as described herein, before removing the vessels from the segments.

After removal of the vessels and the perivascular region, Wharton's stromal jelly is prepared from the vascular cord, or vascular sections of the umbilical cord, as follows: preferably, isolation of Wharton's stromal jelly, or a portion thereof, also includes the step of physically separating the subamniotic stroma (1b) from the lining of the umbilical cord ( 1a). This may include separating the Wharton's jelly from the lining of the umbilical cord, in particular at least the epithelial layer of the lining of the umbilical cord. In this way, a stroma (containing Whartonian stromal jelly) is obtained, free of the corresponding umbilical cord lining material. Preferably, the step of physically separating the stroma (possibly including Wharton's jelly) from the cord lining, in particular at least the epithelial layer of the cord lining, is also performed on the segments obtained as described above. For practical reasons, the step is usually performed on the segments in turn (ie, one segment after the other), although the rapid implementation of this step is preferred.

Wharton's stromal jelly tends to have a relatively loose, jelly-like consistency; thus, it can be mechanically separated from the vascularized umbilical cord or, preferably, the vascularized umbilical cord segments.

The mechanical separation is preferably a scraping. For scraping, you can use any tool, preferably a mechanical tool suitable or designed for this purpose. Removal is preferably carried out by mechanical means; suitable mechanical means are described in the art, for example, in US20080181967 A1, WO2008146992 A1 and WO2007080591 A2. In one embodiment, mechanical means are used to cut pieces of matrix tissue from the PC segments. In one embodiment, the inner surface of the umbilical cord (segments) is scraped off by mechanical means. In one embodiment, the amniotic epithelial membrane is removed mechanically, for example by mechanical means. If the scraping is done gently, which is preferable, the epithelial layer is not damaged and thus no cells of the epithelial layer are trapped in the scraping.

It is important not to include fragments of the amniotic epithelial membrane when collecting matrix tissue. To do this, cutting the lining of the umbilical cord should be avoided, or minimized. In particular, the physical separation of the stromal Wharton's jelly from the umbilical cord lining preferably consists of scraping off the Wharton's jelly, preferably without mechanically cutting the umbilical cord lining, or only minimally cutting the umbilical cord lining. "Mechanical cutting" includes cutting, squeezing, or otherwise breaking tissue integrity through the application of mechanical force. In a preferred embodiment, "minimum degree" means one single longitudinal incision, preferably along the length of the umbilical cord. In this way, the umbilical cord, or segment of the umbilical cord, as the case may be, is opened longitudinally, allowing access to the interior of the umbilical cord.

Optionally, cells from the subamniotic layer are also not obtained. To achieve this, scraping is performed with great care, without the use of unnecessary mechanical forces near the lining of the umbilical cord. In this embodiment, the entire lining of the umbilical cord (including the amniotic layer and the subamniotic mesenchymal layer) is undesirable.

In any case, the amniotic (epithelial) layer is undesirable. Any unwanted cord lining material remaining after mechanical removal of the desired stromal Wharton's jelly is discarded.

In this way tissue of stromal Wharton's jelly is obtained.

Based on the beneficial properties of the cells isolated according to the present invention, the inventors conclude, however, without being bound by any particular theory, that less mature cells with a beneficial ability to proliferate are located relatively far from the vessels and thus closer to the amniotic surface, while the more differentiated cells are closer to the umbilical cord vessels. In conclusion, the inventors chose Wharton's stromal jelly as the starting site for cell isolation of the present invention.

In a preferred embodiment, step (a) isolating stromal Wharton's jelly, or a portion thereof, from the umbilical cord comprises a sectioning step described herein and a grinding step described below.

In a more preferred embodiment, step (a) isolating stromal Wharton's jelly, or a portion thereof, from the umbilical cord comprises the segmenting step described above, the sectioning step described herein, and the grinding step described below.

Reducing the size of tissue fragments by mechanical processing

Preferably, the Whartonian stromal jelly obtained by sectioning is subsequently subjected to mechanical processing, the purpose of which is to reduce the size of the tissue fragments. Machining aims to break the physical bonds in the stromal Wharton's jelly so that the size of the stromal Wharton's jelly is reduced. Mechanical processing is useful for subsequent enzymatic processing, described below. The relatively small and relatively uniform size of Wharton's stromal jelly fragments is associated with several advantages: increased surface area and thus accessibility of material for the enzyme(s) used for enzymatic processing, and the ability to find platelet lysate, which is known to , contains growth factors and other elements suitable for MSCs, in close proximity to almost all of the isolated Wharton's jelly.

Preferably, mechanical processing is carried out by finely crushing the fragments with scissors and/or a scalpel, however, any other means suitable for mechanically crushing the fragments can be used, provided that the cells are practically not damaged or destroyed mechanically. For example, suitable instruments are available from Miltenyi Biotec (Bergisch Gladbach, Germany). In any case, the mechanical treatment is preferably carried out under sterile conditions.

The mechanically treated (eg shredded) tissue is optionally added to and dispersed in the aqueous solution to prevent said tissue from drying out. The aqueous solution is an aqueous solution ideally buffered at or near physiological pH and isotonic, optionally with antibiotics added. For example, HEPES buffered saline (HBSS) or phosphate buffered saline (PBS) is suitable, preferably HBSS. Optionally, the aqueous solution contains antibiotics at appropriate concentrations.

Preferably, the mechanically processed (eg, shredded) tissue is subsequently collected (combined) in an appropriate sterile dish, such as a petri dish. After tissue isolation, and prior to enzymatic digestion, which will be described below, the total weight of ground tissue obtained in a full scale cycle is typically 14 grams or more, such as 16 grams or more, or 18 grams or more.

Enzymatic processing

In accordance with the present invention, the cells are released from the umbilical cord, or its segments, or its sections, or the ground tissue obtained from it, by enzymatic treatment. In contrast to earlier teachings (e.g., Conconi et al., 2011, Open Tissue Engineer. Regen. Med. J., vol. 4, p. 6-20), the authors of the present invention found that enzymatic processing, especially carried out in the conditions described as preferred herein, ie, mild conditions, results in suitable MSCs free of signs of cell damage.

The tissue shredding embodiment is preferred. Thus, preferably, the cells are released as a result of enzymatic treatment from ground tissue obtained from the umbilical cord, or its segments, or its sections.

In particular, in step (b) of the process according to the invention, the Wharton stromal jelly, or a portion thereof, is subjected to an enzymatic treatment, thereby releasing at least one cell from the Wharton stromal jelly, or a portion thereof. As a rule, many cells are released. Stromal jelly, or part of it, is subjected to enzymatic cleavage. In particular, cells are released from stromal Wharton's jelly, which is a specific section of the umbilical cord, and which can be obtained during the previous step (a) described above. The step of the method of the present invention which includes enzymatic treatment may be referred to as "enzymatic treatment", "enzymatic digestion", or the like, or simply "step (b)".

The enzymatic treatment may optionally occur in parallel and/or sequentially with the physical isolation of stromal Wharton's jelly from the umbilical cord; however, the serial version is preferred. More precisely, it is preferable if step (b) of the method according to the invention follows step (a) of the method according to the invention. In such an embodiment, step (b) is the only step in the method of the present invention during which the enzymatic activity is used.

The approach used by the present invention to release cells from umbilical cord tissue, in particular stromal Wharton's jelly, differs markedly from prior art approaches in which umbilical cord tissue is cultured as such and the cells grow from the culture, for example by migrating from it. or cell division, or both (US 2004/0136967 A1). However, in accordance with the present invention, at least one cell, or preferably most of the cells contained in the stromal Wharton's jelly, is/are released by enzymatic treatment, and not by migration from the umbilical cord or cell division. This is a significant advantage in terms of isolation time: instead of several days (eg 2004/0136967 A1), cells can be isolated from the umbilical cord within hours, as detailed below.

An enzymatic treatment is a treatment that releases cells from the umbilical cord, or segments or sections thereof, when treated with at least one enzyme. A suitable enzyme is a protease. Thus, the cells are preferably released from the umbilical cord, or segments thereof, upon treatment with at least one protease. A protease (also called a peptidase or proteinase) is any enzyme that carries out proteolysis; protein catabolism by hydrolysis of peptide bonds. Preferably, the protease is capable of cleaving at least one extracellular matrix protein, preferably collagen. Most preferably, the enzymatic treatment in step (b) is a treatment involving exposure to a collagenase enzyme. In other words, at least one protease is preferably a collagenase. Other proteases are also optionally present. In one embodiment, virtually no other protease is present other than collagenase. "Substantially no other protease" means that the added collagenase preparation has been purified to the maximum reasonable extent possible, and/or that the activity of other proteases in the collagenase preparation cannot be detected by conventional laboratory methods.

A particularly suitable and thus preferred cell extraction enzyme of the present invention is collagenase (EC 3.4.24.7). The term "collagenase" refers to an enzymatic activity capable of breaking (more specifically, hydrolyzing) peptide bonds in the extracellular matrix protein collagen. Collagen is present in the extracellular matrix of various tissues, including the umbilical cord. Collagenase contributes to the destruction of extracellular structures. Collagenase of both eukaryotic and prokaryotic origin is available, although a prokaryotic protease, in particular from bacteria such as Clostridium sp., is preferred in the present invention. Collagenase can be isolated from its natural source, or obtained by recombinant methods, for example, in E. coli. In some embodiments, collagenase from its natural source, such as Clostridium sp., is preferred. For example, collagenase can be obtained from Clostridium histolyticum.

Unless the context clearly dictates otherwise, "collagenase" as used herein means enzymatic activity, while "collagenase preparation" means a product, commercial or not, that has collagenase activity. Collagenase activity is provided by the collagenase enzyme - pure or not - present in the collagenase preparation.

Collagenase preparations are commercially available. In some embodiments, the collagenase preparation used is sterile or GMP grade. Preferably, the collagenase preparation is an NB4 collagenase preparation or an NB5 collagenase preparation or an NB6 collagenase preparation, all from SERVA (Heidelberg, Germany), or any other collagenase preparation equivalent to any of the above. Equivalence can be determined using the method described in Example 2A, in parallel with the NB4/NB6 collagenase preparation and with the collagenase preparation to be equivalenced, optionally, in a titration experiment to determine equivalent collagen cleavage activity.

Optionally, the collagenase preparation used does not contain any enzymatic activity other than collagenase activity. It is particularly preferred if there is no hyaluronidase activity present. In other embodiments, hyaluronidase activity is present. It has previously been reported that combining collagenase with hyaluronidase may be beneficial (eg, Bailey et al., Tissue Eng., 2007, vol. 13, p. 2003-2010). However, in the experimental examples of the present invention, satisfactory results were obtained without the addition of hyaluronidase. Thus, according to the present invention, it is preferable if additional hyaluronidase activity is absent during the enzymatic degradation process.

In some embodiments, there is no activity of any protease other than collagenase. In other embodiments, the activity of one or more non-collagenase proteases is present. Other proteases may be specific proteases or non-specific proteases, endo- or exopeptidases. Thus, optionally, other enzymatic activities, such as those of other proteases in particular, can be used in addition to collagenase. Such other enzymatic activities may either be present in the collagenase preparation or may be added separately. For example, clostripaine (EC 3.4.22.8, also called clostridiopeptidase B, Clostridium histolyticum protease B, alpha-clostridipine, clostridiopeptidase, Arg-C endoprotease), a protease that cleaves proteins at the carboxyl peptide bond of arginine, or an equivalent enzyme may be present. In another, non-mutually exclusive example, caseinase c (EC 3.4.24) may be present. In another, non-mutually exclusive, embodiment, trypsin (EC 3.4.21.4), a serine protease, may be present.

Optionally, the collagenase preparation used has no enzymatic activity other than proteolytic activity, ie collagenase activity and proteolytic activity other than collagenase activity. However, in one embodiment, the enzymatic treatment is also carried out in the presence of hyaluronidase, but the presence of hyaluronidase is optional.

Accordingly, an aqueous solution is prepared containing water, a degrading enzyme, ie a protease, more specifically collagenase, and optionally but preferably a buffering agent. The buffering agent ensures that the pH value is maintained within the desired pH range. You can use any buffer that is suitable to keep the pH in the desired range.

An aqueous solution containing an enzyme, a buffering agent and water, and optionally additional ingredients, is referred to as a digestion buffer. In some embodiments, the digestion buffer does not contain a metal chelating agent such as EDTA or EGTA.

Preferably, the digestion buffer has a pH value of 6.0-9.0, more preferably 7.0-8.0. This value is not only close to the physiological pH value, but the corresponding enzymes have also been described to have a pH optimum in the range of pH 7.0 to pH 8.0. Thus, the digestion is preferably carried out in this pH range. The pH value is preferably determined at the temperature at which the splitting will take place.

Optionally, calcium ions are present in the digestion buffer; for example, a water soluble calcium salt such as calcium chloride may be added during the preparation of the digestion buffer. For example, when the enzymatic treatment is a collagenase treatment, the presence of calcium ions is useful and therefore preferred.

Optionally, heparin is present in the digestion buffer. Heparin is known to be a suitable additive for liquid formulations containing platelet lysate to avoid plugging (eg, Lohmann et al., 2012, PLoS ONE, vol. 7e37839). Suitable concentrations of heparin in the digestion buffer of the present invention are 0.1-100 U/ml, eg 1-10 U/ml, eg 2 U/ml heparin. The number of ml refers to the total volume of digestion buffer. One unit of heparin (the "Howell unit") is an amount roughly equivalent to 0.002 mg of pure heparin, which is the amount required to keep 1 ml of feline blood liquid for 24 hours at 0°C ("Online Medical Dictionary", 2000, Center for Cancer Education).

To start digestion, a digestion buffer is added to the tissue to be digested - or vice versa. It is important to mix the enzyme-containing aqueous solution, i.e., digestion buffer, and the tissue. Preferably, the digest buffer/tissue mixture is gently shaken immediately to ensure proper mixing. When the buffer is added, enzymatic processing begins.

Preferably, the previously isolated tissue from Wharton's stromal jelly and the digestion buffer are mixed in a specific ratio. This is due, in particular, to the advantages of process reproducibility and digestion under mild conditions, if necessary.

Ratio=X mg tissue/1 ml digestion buffer

Since the minced tissue is optionally stored in buffered saline (preferably HEPES buffered saline, HBSS) immediately prior to mixing with the digestion buffer to prevent drying of the tissue, the specific ratio in this embodiment is determined as follows:

Ratio=X mg (tissue+buffered saline) / 1 ml digestion buffer

("Buffered saline" should not be confused with "degrading buffer". "Saline buffer" usually does not contain a degrading enzyme, in particular, does not contain collagenase.)

In both formulas above, X is preferably in the range of 10-1000, preferably 50-500, more preferably 80-200, and most preferably substantially 100. Thus, in a preferred embodiment, 100 mg of minced tissue/HBSS is mixed with 1 ml of digestion buffer .

Unless otherwise indicated, collagenase treatment is typically carried out at a temperature of 35-39°C, more preferably 36-38°C, and most preferably at a temperature of about 37°C. Thus, preferably, the enzymatic digestion is carried out at a temperature of 35-39°C, for example 38-38°C, preferably 37°C. The umbilical cord tissue described herein is subjected to collagenase treatment for a certain time interval (duration). Suitable time intervals are described in this document.

Preferably, the enzymatic digestion is carried out with stirring. For example, continuous gentle agitation at about 50-100 rpm, such as on an orbital shaker, has been found to be suitable for digestion.

In the most preferred embodiment, protease digestion is carried out under mild conditions. The inventors of the present invention were surprised to find that in one embodiment, a suitable cleavage time is 1 hour. More generally, in contrast to the results of several earlier studies, relatively mild conditions are suitable for digestion in the process of the present invention. Among earlier studies, for illustrative purposes, mention may be made of WO 2004/072273 A and Sarugaser et al., 2005, Stem Cells, vol. 23, p. 220-229: according to WO 2004/072273 A, it is recommended to use collagenase of unspecified origin and concentration, for example at a concentration of 0.1 mg/ml or 1 mg/ml; recommended collagenase digestion time is 12-36 hours, eg about 24 hours; according to Sarugaser et al. (above), using 1 mg/ml collagenase from Sigma, C-0130); the cleavage time by collagenase is 18-24 hours. Thus, the mild conditions used in the context of the present invention are different from those described in the earlier literature.

As used herein, the term "mild conditions" or "gentle conditions" means that at least any two, but most preferably all three of the following conditions: (1) digestion time, (2) enzymatic activity, and (3) platelet the lysate are as described below. Enzymatic activity is, of course, related to the type of enzyme used and the amount of enzyme used relative to the volume in which it is used (enzyme concentration) or relative to the amount of tissue being digested.

Unless the context clearly dictates otherwise, all embodiments of (1) cleavage time, (2) enzymatic activity, and (3) platelet lysate can be combined with each other. Thus, in general, the factors that determine mild conditions are short enzyme digestion time, low enzyme concentration, and the presence of hTL.

In one embodiment, a short enzyme digestion time, described below, is combined with a low enzyme activity, described below. The preferred embodiments of short enzyme digestion time described below can also be combined with the preferred embodiments of low enzyme activity described below.

In one embodiment, the short enzyme digestion time described below is combined with the presence of a platelet lysate described below. The preferred short enzyme digestion time embodiments described below can also be combined with the preferred platelet lysate embodiments described below.

In one embodiment, low enzymatic activity, described below, is combined with the presence of a platelet lysate, described below. The preferred low enzyme activity embodiments described below can also be combined with the preferred platelet lysate embodiments described below.

In a particularly preferred embodiment, the short enzyme digestion time described below is combined with low enzymatic activity described below and also with the presence of a platelet lysate described below. The preferred embodiments of short enzyme digestion time described below, the preferred embodiments of low enzyme activity described below, and the preferred embodiments of platelet lysate described below can also be combined with each other. This embodiment is implemented in Example 2A.

Variations in the specific values of (1) cleavage time specified herein, (2) enzymatic activity specified herein, and (3) platelet lysate specified herein are also possible without deviating from the scope of the present invention. For example, it is possible to reduce the digestion time specified herein by, for example, 50%, together with an increase in the enzymatic activity specified herein, for example, by 50%. The skilled person can anticipate and practice such variations based on his own experience and based on the general principles of enzymatic kinetics, according to which a lower enzymatic activity may still be sufficient to cleave a given amount of substrate with a corresponding increase in cleavage time, and vice versa. For example, it is possible to increase the cleavage time specified herein, for example, by 50%, together with a decrease in the enzymatic activity specified herein, for example, by 50%. The corresponding amended terms may still be referred to as soft terms. Collagenase is usually not autocatalytic; thus, variations are generally possible without causing a decrease in the amount of collagenase in the additional cleavage time.

By applying mild conditions, serious damage to isolated MSCs can be largely avoided. Mild conditions make it possible to obtain a much higher proportion of viable cells than previously used methods.

Although the method of the present invention in typical embodiments results in a lower total number of cells than the reference method (see Figure 4B and 4C), the cells isolated by the method of the invention in typical embodiments have a low percentage of apoptotic cells (see Figure 4B and 4D). This is an advantage, for example, for optional propagation, which will be described below.

(1) The inventors of the present invention have found that a short enzymatic digestion time is preferable in the method of the invention. In one example, as originally found by the authors of the present invention, the short time of enzymatic cleavage is 1 hour. Based on this, in general terms, in a preferred embodiment, step (b) is carried out for a total of less than 5 hours, more preferably less than 4 hours, and most preferably less than 3 hours. Less than 3 hours includes time intervals from 0.5 to 2.5 hours and from 1 to 2 hours, as well as any time interval with an upper limit of less than 3 hours, for example, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours and 2.95 hours. All such time intervals may be called "fast" or "short", although "fast" and "short" are also relative terms, eg 2 hours is certainly faster/shorter than 4 hours. Rapid enzymatic treatment avoids problems with dehydration and other possible problems associated with exposure of cells to an unnatural environment for long periods of time; the rapid enzymatic processing also overcomes the difficulties described in Seshareddy et al. (2008, Meth. Cell Biol., vol. 86, p. 101-119).

It has been suggested that short enzyme treatment times may be advantageous, particularly in the case of co-cleavage by hyaluronidase and collagenase (Can et al., Stem Cells, 25:2886-2895, 2007). Somewhat surprisingly, in the present invention, a short treatment time is also advantageous in the absence of hyaluronidase. This is remarkable in two respects: first, in the absence of hyaluronidase, one would normally not expect any undesirable effect of hyaluronidase, such as extensive undesirable cleavage of cell surfaces directed towards the extracellular environment; secondly, without being bound by a particular theory, it is clear that collagenase acts only on the extracellular matrix, and not on the desired cells themselves. However, the benefits of mild treatment, including short treatment times, for cell viability are quite remarkable. In one embodiment, the enzymatic digestion is a mild enzymatic digestion without the addition of hyaluronidase. In one embodiment, the enzymatic digestion is a mild enzymatic digestion in the absence of hyaluronidase. In a preferred embodiment, step (b) is carried out for a total of less than 5 hours, more preferably less than 4 hours and most preferably less than 3 hours, and without the addition of hyaluronidase, or preferably in the absence of hyaluronidase.

Ideally, both steps (a) and (b) are carried out quickly. Thus, cells from the umbilical cord can be obtained in a short time. This has an advantage over some of the earlier studies that used either long time frames for enzymatic digestion of umbilical cord tissue, or growth of cells from umbilical cord tissue, or both. In a preferred embodiment, steps (a) and (b) are carried out in total for less than 6 hours, more preferably less than 5 hours, more preferably less than 4 hours, and most preferably less than 3 hours. Less than 3 hours includes time intervals from 0.5 to 2.5 hours and from 1 to 2 hours, as well as any time interval with an upper limit of less than 3 hours, for example, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours and 2.95 hours. Obviously, the short combined duration of steps (a) and (b) also means that step (b) itself is short. The present inventors have found that, in one embodiment, a digestion time of about 1 hour is preferred.

(2) The inventors also found that the degree of enzymatic activity matters. Enzymatic activity is, of course, related to the type of enzyme used and the amount of enzyme used relative to the volume in which it is used (enzyme concentration) or relative to the amount of tissue being digested.

In particular, the inventors have shown that the degree of collagenase activity matters. For purposes of describing the present invention, collagenase activity is indicated in Wunsch units. 1 Wunsch unit (U) (Wunsch unit, or PZ unit) catalyzes the hydrolysis of 1 µmol 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucylglycyl-L-prolyl-D-arginine per minute at 25°C, pH 7.1 ( Wünsch et al., 1963, Hoppe-Seyler's Z. Physiol. Chem. 333, 149-51).

The method according to the present invention, as a rule, is characterized by a relatively low activity of collagenase in the enzymatic processing step (b). In this document, the indicated degree of collagenase activity is indicated for the time point at which the collagenase is added. For example, if collagenase loses activity over time in step (b), no additional collagenase is added. However, collagenase does not normally cleave itself and is thus relatively stable, and once added, collagenase can be reasonably expected to be present throughout the enzymatic processing step (b).

Collagenase activity is conveniently indicated relative to the total volume of digestion buffer. In one embodiment, the digestion buffer contains collagenase with a specific activity of 0.01 Wunsch U/ml to 10 Wunsch U/ml, preferably 0.05 Wunsch U/ml to 5 Wunsch U/ml, preferably 0.1 Wunsch U/ml, preferably 0.1 Wunsch U/ml ml to 1 Wunsch Unit/ml, preferably from 0.15 Wunsch Unit/ml to 0.5 Wunsch Unit/ml, preferably, for example, about 0.1 Wunsch Unit/ml or about 0.2 Wunsch Unit/ml. In one embodiment, the lower limit of collagenase activity is not specifically defined, provided that the added collagenase activity is sufficient to release mesenchymal stromal cells from tissue in 5 hours or less. In this and other embodiments, the upper limit of collagenase activity can be defined as at most 0.5 Wunsch U/ml, more preferably at most 0.25 Wunsch U/ml, and specifically 0.18 Wunsch U/ml or 0.09 U Wunsha / ml.

In a preferred embodiment, collagenase is present during the enzymatic treatment step (b) with a specific activity of at most 0.5 Wunsch/ml, more preferably at most 0.25 Wunsch/ml and specifically 0.18 Wunsch/ml relative to digestion buffer . Even more preferably, the collagenase activity used in step (b) of the method of the present invention is in the range of 0.18-0.09 Wunsch units/ml digestion buffer (see also Example 1). This activity is different from the collagenase activity used according to WO 2004/072273 A and Sarugaser et al., 2005, Stem Cells, vol. 23, p. 220-229. Indeed, in one embodiment, the collagenase activity in step (b) of the method of the present invention can be determined to be lower than the collagenase activity described in WO 2004/072273 A and Sarugaser et al. (higher).

Alternatively, collagenase activity is conveniently indicated relative to the mass of tissue to be digested. (The weight of said fabric can be appropriately determined by weighing the container it is in before and after adding said fabric thereto). Said tissue, optionally contained in the container, optionally includes buffered saline (eg, HBSS). In one embodiment, a suitable ratio is in the range of 0.01-10 Wunsch units collagenase/100 mg tissue, preferably 0.05-5 Wunsch units collagenase/100 mg tissue, preferably 0.1-1 Wunsch units collagenase/100 mg tissue, preferably 0.15-0.5 Wunsch units of collagenase/100 mg of tissue, preferably, for example, about 0.1 Wunsch units of collagenase/100 mg of tissue, or about 0.2 Wunsch units of collagenase/100 mg of tissue. In one embodiment, the lower limit of collagenase activity is not specifically defined, provided that the added collagenase activity is sufficient to release mesenchymal stromal cells from tissue in 5 hours or less. In this and other embodiments, the upper limit of collagenase activity can be defined as at most 0.5 Wunsch U collagenase/100 mg tissue, more preferably at most 0.25 Wunsch U collagenase/100 mg tissue, and specifically about 0.16 Wunsch U collagenase collagenase/100 mg tissue or about 0.08 Wunsch units collagenase/100 mg tissue, or any value in the preferred range. According to the present invention, it is preferable if the ratio of collagenase activity to total tissue mass is lower than for the collagenase activity described in WO 2004/072273 A and Sarugaser et al., 2005, Stem Cells, vol. 23, p. 220-229.

(3) The inventors have further found that the presence of platelet lysate in the enzymatic digestion process has advantages. This was an amazing discovery of the authors of the invention. There is a certain prejudice in the art that collagenase does not function properly in the presence of platelet lysate. For example, it has been previously described that collagenolytic activity is inhibited by normal human plasma due to inhibitors such as α ₁ -antitrypsin (Chesney et al., J. Clin. Invest., 1974, vol. 53, p. 1647-1654). However, the inventors have found that collagenase digestion is effective even in the presence of platelet lysate. In fact, collagenase digestion is effective even at the very low amounts of collagenase preferred in the present invention (see above). Thus, preferably, the platelet lysate is present during the enzymatic treatment. Platelet lysate can be obtained from commercial sources or prepared independently. Many hospitals, blood banks, and research institutes have established protocols for making platelet lysates, and such lysates are generally appropriate in the context of the invention. For example, the lysate can be obtained by the freeze/thaw method. Such lysates can be prepared by freezing a suspension of platelets followed by thawing of the material, although other freeze/thaw regimens are also suitable provided they result in platelet cytolysis. When using freeze/thaw, this method causes swelling and destruction of cells, presumably due to the formation of ice crystals, followed by contraction when thawed. Thus, the cyclic swelling and contraction eventually causes the cells to rupture. It may be preferable to use multiple cycles for more complete cytolysis, however "more complete" cytolysis is not necessarily needed. There may be varying degrees of platelet cytolysis, such as at least 30%, at least 50%, at least 70%, at least 90%, or up to 100% cytolysis, in terms of platelet count. Optionally, unlysed material and other debris are removed from the platelet lysate prior to use.

Preferably, the platelet lysate contains lysed mammalian platelets and optionally mammalian plasma. In the second case, the platelet lysate is a composition containing platelet lysate and plasma. Reasons for containing plasma include embodiments in which the platelets were not completely separated from the plasma prior to their lysis and/or in which the platelets were (re)suspended in the plasma prior to their lysis. Preferably, the concentration of mammalian plasma in the composition is less than about 30%, less than about 20%, or less than about 10% of the total volume. Preferably, the concentration of mammalian plasma in the platelet lysate is from about 0% to about 10%, for example, from about 1% to about 10%. Optionally, the platelet lysate contains concentrated platelets. Preferably, the mammalian platelet lysate is a human platelet lysate. Preferably, the platelet lysate is substantially free of mammalian platelet membrane.

The platelet lysate may be from a single donor or may be a lysate of pooled donor platelets isolated from whole blood or by apheresis. Preferably, the apheresis is based on the separation of substances having different densities, for example, by centrifugation, and/or the apheresis includes adsorption on beads coated with adsorbent material and filtration.

Optionally, the platelet lysate used in the present invention may be obtained by lysis of a platelet-containing suspension, the platelets optionally being suspended in plasma. Said platelet-containing suspension preferably contains from 1x10 ⁸ platelets/ml to 5x10 ⁹ platelets/ml, preferably from 3.8x10 ⁸ platelets/ml to 1.2x10 ⁹ platelets/ml, averaging about 7, 9×10 ⁸ platelets/ml.

It is also contemplated that the presence of platelet lysate contributes to the mildness of the enzymatic digestion conditions. Surprisingly, the inventors found that the presence of platelet lysate did not adversely affect cell breakdown (see Examples 2A and 3 for illustration), although platelet lysate had previously been reported to adversely affect the activity of several enzymes, including proteases. Thus, an important cornerstone of the method of the present invention is that the enzymatic treatment, more specifically collagenase digestion, can take place in the presence of platelet lysate.

Preferably, the platelet lysate is present in the enzymatic digestion process in a percentage volume of from 0.01% to 30%, for example, from 0.05% to 20%, preferably from 0.1% to 10%, more preferably from 0.5% to 5%, more preferably 0.8% to 2%, and most preferably about 1%. "Percent volume" means the total volume of platelet lysate present in the total volume of digestion buffer.

The inventors have found that the presence of platelet lysate, or derivatives thereof, during the enzymatic digestion, in particular in the above ratio, is beneficial.

The term "platelet lysate" as used herein encompasses both platelet lysate directly obtained by platelet lysis and its derivatives such as heat-inactivated platelet lysate, filter-sterilized platelet lysate, and so on. In some cases, the use of derivatives may be necessary or recommended, such as for regulatory reasons. In a more preferred embodiment, the platelet lysate is substantially free of contaminants. A substantially contaminant-free platelet lysate is defined herein as a sterile platelet preparation substantially free of infectious agents such as viruses (encapsulated and unencapsulated), parasites, bacteria and endotoxins, i.e. platelet lysate containing such infectious agents. in an amount that is not detectable by culture, serological or molecular identification systems, or by the LAL test (endotoxins). The use of pathogen-inactivated platelet lysate (PI-PL) is highly recommended, see for example WO 2013/042095 A1 and Iudicone et al., 2014, J. Transl. Med., 12:28.

There are several advantages associated with the presence of platelet lysate during the enzymatic treatment that were unexpected and are described below.

First, the inventors have found that the presence of human platelet lysate does not block the enzymatic activity of the collagenase enzyme, at least not to the extent that would prevent the desired enzymatic release of cells from the stromal matrix.

Secondly, the inventors found that cells obtained by digestion in the presence of platelet lysate, or derivatives thereof, have excellent viability (see, for example, figure 4A).

Thirdly, the inventors have found that cells obtained by digestion in the presence of platelet lysate or derivatives thereof in the method of the present invention have excellent proliferative properties. Thus, preferably, in step (b), the Whartonian stromal jelly, or a portion thereof, is subjected to an enzymatic treatment in the presence of mammalian platelet lysate (TL) or derivatives thereof, more preferably human platelet lysate (hTL) or derivatives thereof.

While platelets contain bioactive growth molecules and factors such as coagulation factors, adhesion molecules and proteoglycans, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), HGF, vascular endothelial growth factor (VEGF), insulin-like factor growth 1 (IGF-1), TGF-β1 and others are also known to contain protease inhibitors (Astori et al., Stem Cell. Res. Ther., 2016, vol. 7, 93). Given this information, the conclusion that platelet lysate can be appropriately present during the enzymatic treatment is surprising.

In general, certain extraction conditions that differ from prior art protocols for collagenase digestion have been shown to be beneficial for cell viability and cell recovery, and thus provide a surprisingly successful isolation method. In particular, the isolation method of the present invention can produce cells with a higher yield than the method described in Seshareddy et al. (2008, Meth. Cell Biol., vol. 86, p. 101-119). More specifically, in accordance with the present invention, it is possible to achieve a cell yield that is 10-25 times higher than the cell yield described in Seshareddy et al. (higher). Due to the excellent viability of the cells isolated according to the present invention, the cells can be efficiently propagated in vitro, if necessary.

Preferably, only one collagenase digestion step is carried out in the method of the present invention. This means that tissue obtained from the umbilical cord, as described herein, is subjected to a collagenase treatment for a certain period of time, and after this period of time, cells physically isolated or detached from the tissue are removed, however, the remaining tissue is not subjected to one or more additional rounds of collagenase digestion. Rather, it is preferably discarded after the period of time has elapsed and the cells have been recovered.

At the end of the digestion period, the digestion mixture can be diluted with a protein-containing solution, such as minimal medium supplemented with platelet lysate. The moment of adding the protein-containing solution determines the end of the digestion time. The type of minimal medium is not particularly limited as long as the medium is suitable for culturing mammalian cells, preferably, however, mesenchymal stem cells and/or mesenchymal stromal cells. The protein-containing solution preferably contains heparin.

The preferred minimal medium is MSCBM CD (Minimum Chemistry Medium for Mesenchymal Cells, Lonza) and the preferred supplement is 5% (v/v) human platelet lysate (hTL).

It is believed that the addition of a protein-containing solution, such as human platelet lysate, leads to inhibition of enzymatic activity. Without being bound by this particular theory, the addition also dilutes the enzyme. Preferably, at least 1 volume of the protein-containing solution is added relative to the volume of the digestion mixture. The protein-containing solution, of course, must be a solution that is suitable for cell culture purposes, that is, illustratively, it must be sterile, optionally, GMP categories, and so on.

Preferably, the liquid containing the cleaved tissue (cleaved liquid) is subsequently subjected to separation. As used herein, the term "separation" in relation to said fluid after digestion is not particularly limited, and generally means that cells can be separated from non-cleaved tissue, incompletely digested tissue, and other debris by appropriate methods, including filtration and centrifugation. In one embodiment, said cleavage fluid is filtered to separate released cells from non-cleaved tissue, incompletely cleaved tissue, and other debris, e.g., by filtration, e.g. Bue at al., 2007, Veterin Res. Comm, vol. 31, pp. 289-292). For example, a filter pore size (network mesh) of 50-150 µm, eg 70-100 µm, is suitable. Optionally, the filter material is gauze; you can use several layers of gauze. Thus, preferably, the liquid containing the cleaved tissue is filtered through cheesecloth. The filtrate is called the "split mixture". Another suitable separation method is low speed centrifugation; in this case, the cells remain in the supernatant while undigested tissue, incompletely digested tissue, and other debris settle to the bottom; in this case, the supernatant is referred to as a "split mixture". Optionally, filtration and low speed centrifugation can be combined in any order. Alternatively, separation is carried out either by filtration alone or by low speed centrifugation alone, with filtration alone being preferred.

Preferably, the digested mixture is subsequently centrifuged, preferably after filtration. The skilled person can adjust the centrifugation conditions to achieve conditions suitable for the precipitation of mesenchymal stromal cells without adversely affecting their proliferative properties and viability. For example, the digested mixture can be centrifuged at 680 x g for 15 minutes using 250 ml centrifuge tubes. At the end of centrifugation, the supernatant is removed and the pellet containing the desired cells is resuspended. Ideally, in embodiments where the method of the present invention includes one or more expansion steps, the cell pellet is resuspended in the same growth medium as used in the (first) expansion step. Propagation according to the present invention will be described in more detail below.

Typically, the digested mixture contains a plurality of cells, that is, a cell population. Preferably, the cell population obtained by the enzymatic treatment described herein is used directly as received, that is, without any negative selection (selective destruction of unwanted cells). A proper, but not required, use is for cell expansion, which is described below.

An illustrative example of a selection method is given in example 2A. As shown in example 3, the use of platelet lysate does not adversely affect the activity of collagenase, providing the release of cells, and their viability. This is surprising since platelet lysate has previously been reported to adversely affect the activity of several enzymes, including proteases.

The enzymatic treatment is preferably carried out under sterile conditions.

The selection of a single cell, if necessary, can be carried out, for example, by serial dilution. In this way, an isolated single cell can be obtained. Serial breeding is an optional step after step (b); however, obtaining a single isolated cell is not the main purpose of the invention; rather, it is preferred if step (b) is followed by step (c) of expanding the cell(s) as described below.

reproduction

Cells obtained by isolation, in particular in steps (a) and (b) of the method according to the invention, can be propagated (i.e., propagated). Cells are propagated ex vivo. Ex vivo propagation allows the skilled artisan to select specific culture conditions. They are described below.

The purpose of propagation is to increase the number of previously isolated cells that have beneficial properties. As is customary in the art, propagation takes place in a culture medium. Unless specifically stated otherwise, conditions suitable for propagation include those generally known for culturing mammalian cells, more particularly for the propagation of mammalian cells.

Reproduction is a period of the cell culture/production process, which is mainly characterized by a high degree of growth and high mitotic activity. In accordance with the present invention, propagation serves in particular the purpose of increasing the number of cells, which means obtaining an increased number of cells that are preferably mitotically active, and more preferably in an exponential growth phase. By multiplication, larger cell populations can be obtained. The cell propagation step of the present invention may be a single step in which the cells are allowed to proliferate under appropriate conditions: however, more preferably, the propagation of the present invention comprises multiple passages, as further described below.

Propagation is generally carried out under sterile conditions, and generally also under conditions necessary or useful or recommended for culturing mammalian cells.

This embodiment of the invention is based on the fact that a particular section of Wharton's jelly, ie Wharton's stromal jelly, contains cells that can be isolated efficiently and reliably and subsequently propagated efficiently and reliably. Thus, after cell isolation, their population is propagated by mitosis.

Thus, optionally and in the most general sense, the method of the present invention includes one or more additional steps. In particular, in preferred embodiments, implementation of the present invention includes the step of increasing the number of cells, using the ability to divide in the cells obtained in step (b). In such preferred embodiments, step (b) is followed by step (c); step (c) includes propagation of at least one cell obtained in step (b). Step (c) is also referred to herein as the "propagation step" or simply "propagation".

Propagation conditions are chosen to be suitable for ex vivo growth and cell division of mesenchymal stromal cells. Thus, a culture enriched in mesenchymal stromal cells is obtained. Without being bound by a particular theory, it can be foreseen that either the culture conditions favor the selection of a specific subset of the isolated cells, thereby enriching them, or the cells change their phenotype.

Propagation according to the present invention with isolation according to the invention leads to synergistic results as described below: although the total number of cells isolated under the preferred mild conditions of the present invention can be (and usually is) lower than the total number of cells extracted by the method proposed in US 2004 0136967 A1 and Seshareddy et al., Meth. Cell Biol., 86:101-119, 2008, the inventors have found that cells isolated under the preferred mild conditions of the present invention are particularly suitable for expansion, for example, due to their high viability (see figure 4B for illustration) and also due to their high proliferative potential (see figure 9 for illustration), and thus large numbers of cells can be obtained by expanding the SHS-derived cells isolated in accordance with the present invention. In other words, the preferred isolation method of the present invention, in conjunction with propagation of the present invention, has the synergistic advantage that less starting material and/or fewer passages are required to achieve a particular desired number of cells, since most of the cells to be propagated are viable, and they proliferate especially efficiently.

Preferably, the umbilical cord cells obtained in accordance with the present invention are the only cells that are transferred to the expansion step. In such embodiments, it is particularly preferred if no nurse cells are present during the expansion step.

Propagation usually takes place in a culture vessel. More specifically, the cells to be propagated are seeded in a culture vessel. Typical culture vessels are vials, plates and pouches (where pouches typically consist of multiple plates stacked one on top of the other). Particularly suitable culture vessels in the context of the present invention are culture vessels that allow cells to attach to the bottom of the culture vessel; such vessels are vials, tablets and bags with a flat bottom surface. Preferably, the culture vessel has at least a plastic bottom surface. More preferably, the culture vessel is a plastic culture vessel. The culture vessel preferably has a flat bottom surface ("flat bottom").

The type of plastic is not particularly limited as long as it is suitable for cell culture; however, hydrophobic polymers such as, in particular, polystyrene, optionally surface-modified, are preferred. The surface modification can be carried out by a plasma method, which creates the availability of various functional chemical groups on the polystyrene surface, and which is suitable for ensuring cell attachment, as a rule. Such techniques are known in the art, and surface-modified plastic vessels for culturing adherent cells are commercially available.

Preferably, however, the surface of the cell culture vessel is not coated with protein prior to use. In particular, it is not coated with proteins, for example cell matrix proteins such as collagen and polylysine.

Preferably, propagation is carried out using multiple passages. Details of the passages are given below.

Growth medium

The growth medium for propagation of the present invention will typically contain a minimal medium and one or more additives. In a broad sense, the type of minimal environment used has no specific restrictions. Typical minimal media are water-based and have a defined chemical composition. "Defined chemical composition" means that the minimum medium consists of specific ingredients in specific quantities; The advantage of a minimal medium with defined chemistry is that it can be prepared reproducibly, without variation due to experimental conditions, different laboratories, and so on. The minimal medium is usually sterile. Minimal mammalian cell growth media are commercially available from various commercial vendors.

Typically, the minimal medium contains ingredients including amino acids, salts (such as calcium chloride, potassium chloride, magnesium sulfate, sodium chloride, and sodium monophosphate), glucose, and vitamins (such as folic acid, nicotinamide, riboflavin, B ₁₂ ), at least at least one buffering agent (such as, for example, HEPES) and, as a rule, also a dye (for example, phenol red); however, this is optional. Most commercially available minimal media include phenol red as a pH indicator (dye) to allow constant monitoring of pH.

Preferably, the minimal growth medium is one that does not require the addition of an attachment matrix for cell seeding.

Preferably, the minimal medium is serum-free. Serum-free means that it contains neither human nor animal serum. More preferably, the minimal medium is a chemically defined minimal medium that is serum-free.

Preferably, the minimal environment has a certain chemical composition. A minimum chemistry defined medium is a minimum medium suitable for in vitro cultivation of human or animal cells in which all chemical components are known. In particular, in an environment with a certain chemical composition, all components must be identified and have known concentrations. Therefore, the chemically defined medium must be completely free of animal components and cannot contain serum or serum proteins, such as fetal bovine serum, bovine serum albumin or human serum albumin. The minimum environment with a certain chemical composition includes only recombinant proteins and/or hormones. To achieve this, the chemically defined medium does not contain components of human or animal origin, such as proteins and/or growth factors; in particular, it does not contain albumin of human or animal origin, or growth factors of human or animal origin. Preferably, it contains recombinant albumin and/or recombinant growth factors, usually derived from sources other than human and animal, such as plant cells and bacteria, and/or synthetic chemicals such as, for example, polyvinyl alcohol.

In some embodiments, the implementation of the minimum environment with a certain chemical composition contains

(a) the usual minimal medium (such as DMEM, F12 or RPMI 1640 containing amino acids, vitamins, inorganic salts, buffers, antioxidants and energy sources),

And

(b) one or more serum replacement ingredients, optionally selected from, but not limited to, the following open list: recombinant albumin, chemically defined lipid(s), recombinant insulin, and/or metal ion(s) such as zinc, recombinant transferrin or iron, selenium, and an antioxidant thiol such as 2-mercaptoethanol or 1-thioglycerol.

The minimum medium may, for example, be selected from a list including, but not limited to, Eagle's Minimum Supporting Medium (EMEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha-MEM, RPMI-1640, IMDM, and others. If necessary, one or more serum replacement ingredients are added to these media. In this way a serum-free minimal medium is obtained.

More preferably, the minimal medium is a minimal medium suitable for culturing mesenchymal stromal cells and/or mesenchymal stem cells. Preferably, the minimal growth medium is suitable for multiple passages in the propagation of MSCs, in particular human MSCs, more specifically derived from stromal Wharton's jelly (preferably human) MSCs.

Media suitable for culturing mesenchymal stromal cells and/or mesenchymal stem cells are commercially available and some are specifically sold for this purpose. For example, TheraPEAK™ MSCBM CD (hereinafter referred to as MSCBM CD for simplicity) is a chemically defined minimal medium that is suitable for mesenchymal stem cells as described by its manufacturer Lonza (Wakersville, MD, USA; cat. no. 95062- 688). Indeed, the present inventors have found that this minimal environment is particularly suitable. Thus MSCBM CD is the preferred minimum medium for use in the present invention.

All minimal environments that are practically equivalent to MSCBM CD are equally preferred. The term "substantially equivalent" is explained below. The similarity of the composition of the minimum environment in terms of ingredients and their relative amounts can be checked using a combination of chromatographic and/or spectrometric methods, or the like.

As a rule, the choice of a suitable culture medium is very important for the overall performance of cell culture. This issue was resolved by selecting MSCBM CD and minimal media practically equivalent to it as particularly suitable minimal media for the present invention. MSCBM CD is a minimal chemistry defined medium for mesenchymal cells and is commercially available from Lonza (Lonza, Wakersville, MD, USA, cat. no. 95062-688). Variations of MSCBM CD, such as, in particular, minimum media consisting of, or containing the same components as MSCBM CD, albeit, for example, in different quantities or proportions, or minimum media consisting of, or containing components, in part or completely different from the components of MSCBM CD, but having a substantially equivalent effect on MSC growth, when used as described herein, can equally be used in the present invention.

Other minimal media are also suitable for propagating MSCs in the method of the present invention. For example, the inventors have found that DMEM is a minimal medium that can also be used when it is part of a growth medium also containing at least one additive(s) described below (data not shown).

In important embodiments of the present invention, the growth medium includes a minimal medium and at least one additive(s), in particular platelet lysate. Thus, preferably, at least one cell is propagated in a growth medium based on minimal medium and containing at least one additive. Platelet lysate is the preferred supplement.

The term "based" means that the minimum medium, eg MSCBM CD, is supplemented with at least one additive. If additives are added in liquid form, then the minimum medium is made slightly diluted by the addition of at least one additive; however, slight dilution does not pose any problems. In a preferred embodiment, supplements may be selected from a list including human or animal serum, human or animal platelet lysate, human or animal or plant cell lysate, one or more antibiotics, heparin, adjunct growth factors such as insulin or hydrocortisone, amino acids, such as glutamine or its oligomers.

Preferably, the growth medium contains heparin as an additive. Suitable concentrations of heparin in the growth medium are 0.1-100 U/ml, eg 1-10 U/ml, eg 2 U/ml. U means a unit (Howell unit) of heparin as described above. Heparin can be added to minimal medium.

In a particularly preferred embodiment, at least one cell is propagated in a growth medium based on MSCBM CD Minimal Medium (Lonza, Wakersville, MD, USA; cat. no. 95062-688) or minimal medium substantially equivalent thereto. The MSCBM CD used in the present invention, for example, has proven to be particularly useful in achieving high cumulative population doubling/high cell counts.

Whether or not two minimal media are substantially equivalent to each other is tested for the purposes of the present invention by growing cells, ideally isolated from the same umbilical cord, in a growth medium based on said minimal medium under substantially identical conditions, i.e. , with the same additives (eg, platelet lysate), in the same amounts, in the same culture dishes at the same temperature, and so on, ideally in parallel. In other words, to test whether these two minimal media are practically equivalent to each other, the only variable in the cell growth test is the minimal medium itself. The cells are preferably MSCs derived from stromal Wharton's jelly, isolated as in Examples 1 and 2A and subcultured ("P0") as in Example 2B. The trypsinized P0 cells are grown in parallel in a growth medium based on a first minimal medium (eg, MSCBM CD) and in a growth medium based on a second minimal medium (tested for significant equivalence). Two minimal media are considered to be substantially equivalent when both of the following conditions occur: Mean cumulative population doubling per seed (P1-P8) rates are substantially the same (maximum difference 10% ± deviation) and the rate of senescence of cells obtained after passage 8 (for information see example 11) is practically the same (maximum difference 10% ± deviation).

In an alternative embodiment, the growth medium is based on a minimal medium which, although not substantially equivalent to MSCBM CD, is characterized by the following: Average cumulative population doubling per seed (P1-P8) is at least 70% compared to growth in growth medium , otherwise substantially identical, but based on MSCMB CD minimal medium, and the rate of senescence of cells obtained after passage 8 (for information, see Example 11) is 150% or less compared to the rate of senescence of cells growing in parallel in the growth medium, otherwise almost identical, but based on the minimal MSKMB CD environment. DMEM (commercially available from many vendors) may be an embodiment of such a minimal environment.

Preferably, the growth medium supports multilineage differentiation of MSCs, in particular the MSCs of the invention. If necessary, the minimal medium is supplemented with additives to direct cell differentiation towards a specific lineage. Tests for multilinear differentiation are described below.

Preferably, the platelet lysate, or derivative thereof, is present in the growth medium. Thus, the growth medium useful in the present invention preferably contains a platelet lysate. More specifically, the present inventors have shown that the combination of MSCBM CD minimal medium, or minimal medium substantially equivalent thereto, with additional platelet lysate is particularly suitable and such a growth medium is most preferred. Preferably, if platelet lysate is present, no human or animal serum is present in the growth medium, and more preferably, no human, animal or plant cell lysate other than platelet lysate is present. The use of platelet lysate in the growth medium of the present invention instead of human or animal serum overcomes the problems associated with the traditional use of animal sera in terms of transmission of prions, viruses or induction of immunological reactions in recipients after infusion, as well as regulatory restrictions in accordance with the requirements of good manufacturing practices (GMP).

If the platelet lysate is present in the growth medium, or more specifically has been added to a minimal medium (such as Lonza's MSCBM CD, for example, or a minimal medium substantially equivalent thereto), then the platelet lysate may also be referred to as a minimal medium supplement.

The platelet lysate suitable as an additive in this embodiment generally corresponds to the platelet lysate described for the embodiment with the addition of platelet lysate in the enzymatic digestion process described above, however, ideally, the following four additional more preferred embodiments are also performed , alone or in combination:

In a first particularly preferred embodiment, the platelet lysate is from the same species (but usually not from the same individual) as the umbilical cord. The advantage associated with culturing in the presence of platelet lysate from the same species is surprising, in particular in view of the prejudice in the art against the use of platelet lysate from the same species for culturing stromal cells: for example, it has been reported that equine platelet lysate is generally not useful for culture of equine mesenchymal stromal cells (Russell et al., 2016, Equine Vet. J., vol. 48, p. 261-264), and that canine platelet lysate is inferior in results to fetal bovine serum in the isolation and expansion of canine mesenchymal stromal cells (Russell et al., PLOS One, 2015, vol. Vol. 10, e0136621). It is particularly preferred if the platelet lysate used contains platelets from multiple donors included in such preparation to compensate for individual variability and to obtain a more standardized and reproducible platelet lysate preparation; pooling of platelets derived from buffy coats of whole blood is a standardized procedure, see eg Iudicone et al., 2014, J. Transl. Med., 12:28. Preferably, the platelet lysate is obtained from the blood of a mammal. Depending on the purpose, an appropriate mammal can be selected as the blood source. Mammals that are intended for use in the present invention can, in principle, be any animal, young or adult, from which you can get the necessary and reasonable number of platelets. In some embodiments, adult mammals are primarily used. Examples of non-human animals are animals raised for slaughter, other farm animals such as cattle, pigs, sheep or poultry. Mammalian platelet lysate is preferred, human platelet lysate (abbreviated "hTL") is particularly preferred. In some embodiments, the mammal is a human. Generally, the platelet lysate added is preferably from the same species as the umbilical cord/cells. In particular, for culturing cells derived from human umbilical cord, it is preferable to add human platelet lysate. Platelet lysate is generally allogeneic to the cells. In some embodiments, the blood donors are healthy and meet the requirements set forth by the relevant government regulatory authorities. For example, the blood may meet the requirements that government food and drug control agencies place on products intended for human consumption or medical use, or meet the relevant regulations in the geographic area in which the present invention is applied. The human platelet lysate can completely or partially (preferably completely) replace fetal bovine serum. In most preferred embodiments, the platelet lysate is from human donors. In these embodiments, donors are preferably about 50 years old, about 45 years old, about 40 years old, or less.

In a second particularly preferred embodiment, the platelet lysate has an increased growth stimulating effect on cells, in particular Wharton's jelly-derived MSCs of the present invention, when used as an additive in a cell culture medium, compared to bovine serum, in particular fetal serum. bovine serum (EBS) at the same concentration. In some embodiments, the composition has an effect that stimulates an increase of at least 20% in cell growth compared to fetal bovine serum (FBS) at the same concentration.

In a third particularly preferred embodiment, the concentration of platelet lysate in the growth medium is from about 0.1% (v/v) to about 20% (v/v), preferably from about 0.5% (v/v) to about 10% v/v), more preferably from about 1% (v/v) to about 5% v/v), e.g. 1% (v/v), 2% (v/v), 3% (v/v) , 4% (V/V) or 5% (V/V). A concentration of about 5% (v/v) has proven to be particularly useful (see examples herein) and therefore preferred, particularly in combination with a minimal chemistry-specific medium suitable for mesenchymal stem cells, such as MSCBM CD (Lonza), or a minimal environment almost equivalent to it.

In a fourth particularly preferred embodiment, the platelet lysate that is added to the growth medium contains platelet derivatives. Alternatively, the platelet lysate is substantially free of mammalian platelet membranes and/or other cellular debris. As used herein, "substantially free" means that only an undetectable amount of platelet membranes is present, or the amount of platelet membranes cannot be further reduced by any reasonable, commercially viable method, or the amount of platelet membranes does not substantially interfere with the use of platelet lysate. . The platelet membranes can be separated from the platelet lysate by any suitable methods known in the art, such as density gradient centrifugation.

Some useful embodiments are described in Examples 0 and 2.

Preferably, the platelet lysate, or derivatives thereof, is/are present during all or part of the propagation step (c). Thus, preferably in step (c), at least one cell is propagated in a growth medium containing platelet lysate (PL) or derivatives thereof, preferably human platelet lysate (hTL) or derivatives thereof.

Without wishing to be bound by a particular theory, it is believed that the presence of platelet lysate provides nutrients and growth factors and also contributes to an environment that allows cells to proliferate efficiently. Without wishing to be bound by a particular theory, it can also be assumed that the presence of a platelet lysate contributes to the neutralization or inhibition of any enzymatic activity remaining after the enzymatic extraction, and thus the mildness of conditions during the subsequent expansion phase. Human platelet lysate (hTL) is preferred for use with human MSCs.

Thus, in the present invention, platelet lysate (TL) can serve as an alternative to animal serum in the cultivation of mesenchymal stromal cells (for illustration and more details, see also WO 2013/042095 A1). Platelets are known to be a rich source of numerous growth factors such as PDGF (platelet growth factor), b-FGF (basic fibroblast growth factor), VEGF (vascular endothelial growth factor) and TGF-β (transforming growth factor β).

Optionally, one or more antibiotics are present in the growth medium. In this case, one or more antibiotics may supplement the minimal medium as supplements. Suitable antibiotics in the context of the present invention include, for example, amphotericin and gentamicin, among others. The concentrations of one or more antibiotics in the growth medium are preferably within the concentration ranges commonly used in the art. Alternatively, there are no added antibiotics in the growth medium.

Preferably, no additional ingredients other than those listed above are added to the minimal medium. In particular, it is particularly preferred if at least one cell is propagated in a growth medium free of any added serum, in particular free of bovine serum such as fetal bovine serum (FBS). For the purposes of this embodiment, platelet lysate does not fall under the definition of serum; in other words, even if a small amount of serum, or trace amounts of serum, may be present in the platelet lysate added to the growth medium, it can be said that the growth medium does not contain "added serum", this is a clarification that the serum is not added as an ingredient for cell culture in the classical sense. In the context of the present invention, EBS is an undesirable additive, for example, due to the fact that it carries the risk of transmission of viral and prion diseases. On the contrary, the method of the present invention is effective in the presence of platelet lysate, for example, as a replacement for PBS. For the growth of human MSCs, human platelet lysate is a preferred supplement over FBS.

Preferably, no attachment matrix is added, or present, when cells are passaged to grow on the plate. Thus, no attachment matrix is added or present when seeding the cells and/or no attachment matrix is added as an additive to the minimal medium.

Passages

In a preferred embodiment, cells are propagated over one or more passages.

The term "passage" refers to a culture of cells in a culture vessel (of a certain type) in which the cells are seeded to start the corresponding passage. Usually, the cells are attached to the specified culture vessel during their cultivation, and the cells usually proliferate in the specified culture vessel during their cultivation. The end of the passage is the detachment of normally attached cells (eg by trypsin treatment). The cells are then optionally seeded in a new culture vessel of the same or different type; sowing into a new culture vessel is the beginning of the next passage. Thus, when cells after such expansion are transferred from a culture vessel to another culture vessel, the cells present in that other culture vessel represent a new passage.

The term "cell harvest" refers to the process of obtaining substantially all of the cells grown in the culture vessel to the end of the passage.

One culture vessel can be used for each passage; more preferably, however, multiple culture vessels are used in parallel for each passage. The cells are then cultured in parallel in several culture vessels under substantially identical conditions and for substantially the same time. Preferably, the seed density in several culture vessels is substantially the same.

At the beginning of each passage, cells (not attached/isolated, preferably at the desired number/density) are added to the culture vessel containing the growth medium.

Although antibiotics are not essential for cell growth, they are often used to control the growth of bacterial and fungal contaminants. This is particularly preferred in early passages, but is not preferred in later passages, for example from P2 onwards, or from P1 onwards. Thus, even if antibiotics are present at the first passage after cell isolation ("P0"), antibiotics are preferably discontinued at all passages after P0; in other words, the growth medium without the addition of antibiotics can be used for P1 and all subsequent passages. Thus, in the present invention, the antibiotics are preferably present as an additive in the first culture medium (eg, P0) after cell isolation from the umbilical cord, but not in later cell cultures, ie, P0 subcultures.

At the end of each passage, when the cells have reached a percentage confluency that meets criterion 3 in the table below, the cells are harvested. For this purpose, the growth medium (and any non-adherent cells contained therein, if any) are removed by aspiration, the adherent cells are optionally washed with an appropriate sterile buffer, and optionally, but preferably, separated by an enzyme, for example, by treatment with trypsin, optionally counted, optionally but preferably concentrated by centrifugation, and optionally but preferably resuspended, preferably in a growth medium suitable for MSCs described herein. Resuspended non-adherent cells are then optionally used to initiate culture in a subsequent passage.

Passages are consecutively numbered as detailed herein below, starting at passage 0 (P0). P0 is the first passage after cell generation, preferably by enzymatic digestion as described above. Thus, P0 can be called "primary culture". Cells for primary culture inoculation are derived from umbilical cord Wharton's jelly, mechanically and/or enzymatically dissociated tissue as described above, or alternatively, but much less preferably, from growth of cells migrating from stromal Wharton's jelly tissue.

By growing cells in different passages, cells can proliferate to higher cell numbers and/or higher uniformity and/or less contamination with unwanted cells than would be possible using only one passage. Cells can be propagated under certain conditions or other desirable conditions.

It is generally accepted that umbilical cord stromal cells (PCs) are heterogeneous in terms of structure, biological properties, and localization in PCs. For example, Figure 7A shows that cells freshly isolated from PC, both in accordance with the present invention and in accordance with the state of the art, are heterogeneous in terms of displaying certain surface markers.

As a rule, not all cells isolated from stromal Wharton's jelly (or from any other area of the umbilical cord) are clonogenic. In other words, not all isolated cells are colony forming units (CFU). However, by growing the cells isolated in accordance with the present invention, enrichment for desired mesenchymal stromal cells that are clonogenic can be achieved over several passages. Thus, in one embodiment, the propagation of the present invention is characterized in that the percentage of MSCs increases over time. For example, the percentage of MSCs at the end of P0 may generally be higher than the percentage of MSCs at the beginning of P0.

In the present invention, multiple culture passages have the advantage that enrichment for MSCs relative to other (undesired or contaminant) cells such as erythrocytes can be achieved because (a) non-attached (and therefore non-MSCs) cells are removed along with the growth medium at the end of the culture passage, (b) cells with proliferative properties inferior to those of MSCs will hardly proliferate as well as MSCs, and thus, over several cumulative passages, the cell population becomes enriched in MSCs, and eventually (usually from P1 onwards) is practically pure in relation to MSCs.

As a general rule, it is possible to arithmetically convert the number of passages to cumulative population doubling rates, using certain or other well-known conditions.

Cells are preferably propagated for several passages, preferably from more than one to less than fourteen passages. For example, cells are cultured for three to six passages.

In one specific embodiment, freshly isolated cells after enzymatic digestion first undergo a primary culture passage (for exemplary illustration, see Example 2B) - the primary culture passage may be referred to as P0; optionally, propagation is repeated in a new passage under the conditions for P0 (repeated culture passage is then referred to as P0+). Subsequent passages, that is, immediately after P0, or after P0+, are numbered as P1, P2, and so on. In a preferred embodiment, the cells are propagated to at least P2 and no longer than P6. In the most preferred embodiment, the cells are expanded to P4.

With regard to cumulative population doublings, it is generally difficult to determine the rates of cumulative population doubling in P0. The main reason is that, in the experience of the present inventors, not all cells seeded at the beginning of P0 proliferate. To determine the rates of cumulative population doubling at P0, it would be necessary to determine the number, or fraction, of clonogenic cells at the beginning of P0, that is, those cells that are able to multiply, giving rise to daughter cells. While such a determination is not entirely impracticable in itself, it is not required for the method of the present invention; instead, in such embodiments, cumulative population doubling rates are determined for all passages except for P0. Although cells initially isolated from the umbilical cord typically include MSCs that are clonogenic as well as other cells (eg, erythrocytes) that are not clonogenic, other cells will be substantially absent after P0, as described below, in part because that such non-clonogenic cells do not produce offspring in P0.

In one embodiment, the cells are propagated for 5-30 cumulative population doublings (not counting the cumulative population doublings in the P0 process), for example, 8-25 cumulative population doublings (not counting the cumulative population doublings in the P0 process). In one preferred embodiment, the cells are propagated for only 5-14 cumulative population doublings (not counting the cumulative population doublings in the P0 process). Such an embodiment is suitable for establishing a primary cell bank.

In an alternative preferred embodiment, the cells are propagated for 14-25 cumulative population doublings (not counting the cumulative population doublings in the P0 process). Such an embodiment is suitable for establishing a secondary cell bank.

In our experience, cumulative population doubling rates during P1 and P2 (collectively P1 plus P2, but not including P0) are generally in the range of 7-15, preferably 9-13, in the most general case. In our experience, cumulative population doubling rates during P3 and P4 (collectively P3 plus P4, but not including P0, P1 and P2) are generally in the range of 4-12, preferably 6-10, in the most general case. Therefore, the cumulative population doubling rates during P1, P2, P3, and P4 (collectively P1 plus P2 plus P3 plus P4, but not including P0), in the most general case, are usually in the range of 9-27, preferably in the range of 15- 23. These numbers do not include rates of cumulative population doubling during P0.

At the beginning of each passage, the cells are transferred to a cell culture vessel. Preferably, the cells are transferred in a certain number of cells to a culture vessel. The amount is preferably determined depending on the two-dimensional surface of the cell culture vessel. Thus, it is possible to start the passage from a certain cell density. Cell densities for the start of culture passages in the context of the present invention are as follows:

To start P0 and P0+ For the beginning of P1 and all passages following P1 Suitable Range 1000-50000 cells/ ^cm2 200-50000 cells/ ^cm2 Preferred Range 8000 cells/ ^cm2 or more 2000-3000 cells/ ^cm2 Definite value Not applicable 2500 cells/ ^cm2

At the beginning of each of P0 and P0+ (if any), all available cells are seeded. Thus, the exact cell density for onset P0 and P0+ (if present) has not been determined. In any case, the cell density to start P0 and P0+ (if available) should be 8000 cells/cm ² or higher.

As is typical for mesenchymal stromal cells, the cell of the invention is typically an adherent cell when growing in a culture vessel, in particular a culture vessel with a plastic bottom surface. Preferably, the cell is adherent, regardless of the choice of growth medium. In any case, it is most preferred if the cell is adherent when cultured in the preferred growth medium of the present invention.

At the end of each passage, the cells are harvested. To do this, they are optionally, but preferably, separated by an enzyme, for example by treatment with trypsin. As is known, trypsin is a commercially available enzyme suitable for proteolytic cleavage of cell adhesion molecules and thereby separating adhering cells from the culture vessel and from each other. Thus, "treatment with trypsin" means exposure to trypsin - or any other enzyme with a cleavage pattern similar to that of trypsin - under conditions suitable for such cleavage. Standard methods for trypsinizing mammalian cells, in particular mesenchymal cells, are also suitable for trypsinizing cells obtained according to the present invention. Trypsin preparations for this purpose are commercially available and can be used according to the manufacturer's instructions. Alternatively, separation with an enzyme is performed using a suitable enzyme other than trypsin.

After separation with an enzyme, for example after trypsin treatment, optionally but preferably, the total number of cells is determined (per flask or per plate or per bag, or per total number of culture vessels used in the corresponding passage).

When it is said herein that cells are propagated to a certain passage, for example, to P4, this means that cell propagation is not continued further or interrupted after the corresponding passage.

After each passage, the following acceptance criteria are preferably evaluated: 1 - yield, 2 - viability, 3 - confluence, 4 - morphology. Acceptance criteria 3 and 4 are determined prior to enzymatic cell separation. Determination of acceptance criteria 1 and 2 is carried out after enzymatic separation of the cells. See below for details.

Acceptance criterion After P0 and P0+ After P1 and all passages following P1 Test 1 Exit
(full scale cycle) See example 2B See example 2C Cell count after enzymatic separation 2 Viability More than 80% viable cells More than 80% viable cells Staining with 7-amino-actinomycin D (7-AAD) after enzymatic separation 3 Confluence At least 80%, preferably 80-90% confluence At least 80%, preferably 80-90% confluence Determination under a microscope 4 Morphology Most cells are small, fusiform and in distinct colonies Most cells are small, fusiform and in distinct colonies Determination under a microscope

Preferably, all four acceptance criteria must be met.

Microscopic determination of confluency is directed to determining the percentage of the surface area of a cell culture vessel covered by cells attached thereto.

If the confluence criterion is not met, the cells are expanded further and monitored regularly to determine the point in time when the confluence criterion is met.

To determine the yield, the cells are isolated with an enzyme, eg trypsinized, and an automated cell count is performed on a certain portion of the cell-containing suspension, eg with a ^{NucleoCounter®} . Then, based on the results for the part, the total number of cells is arithmetically determined.

Microscopic determination of morphology (ie, cell morphology) aims to determine the shape of the cells attached to the bottom surface of the cell culture vessel. It is typical for MSCs if most of the cells are spindle-shaped. For illustrative purposes, a photograph of the bottom surface of a cell culture dish containing such cells is shown in Figure 5C, panel referred to as "CBC".

Viability determination ensures that cells used in cell bank establishment and/or subsequent passage are suitable for further expansion (after freezing and thawing, as appropriate). The cells of the present invention have superior viability compared to MSCs isolated using state of the art methods. Due to this, and due to the expansion of the present invention, large cell populations can be obtained even when the total number of cells initially isolated from umbilical cord stromal Wharton's jelly is relatively low. Thus, the cells obtained according to the present invention are excellent in their proliferative potential.

Freezing

Cells can be frozen, eg flash frozen in liquid nitrogen, by standard methods. It is entirely possible to thaw such frozen cells and, for example, subject them to further culture passages after thawing. You can freeze either the entire batch or aliquots.

When it is said herein that cells are propagated to a certain passage, for example, to P4, this means that cell propagation is not continued further or interrupted after the corresponding passage. In an illustrative, but typical, embodiment, the cells are frozen after the appropriate passage. Re-freezing after repeated passages is possible. For example, cells can be frozen after P2 and/or after P4. Preferably, the cells are frozen after P2 and after P4.

Frozen cells and frozen cell populations are also an object of the present invention. Populations of frozen cells can be called cell banks. Suitable cell banks that can be obtained in the context of the present invention will be described below.

In one embodiment, the cells are frozen after 5-30 cumulative population doublings (not counting the cumulative population doublings in the P0 process), eg, 8-25 cumulative population doublings (not counting the cumulative population doublings in the P0 process). In one preferred embodiment, the cells are frozen after only 5-14 cumulative population doublings (not counting the cumulative population doublings in the P0 process). Such an embodiment is suitable for establishing a primary cell bank. In an alternative preferred embodiment, the cells are frozen after 14-25 cumulative population doublings (not counting the cumulative population doublings in the P0 process). Such an embodiment is suitable for establishing a secondary cell bank.

Standard methods for freezing mammalian cells, in particular mesenchymal cells, are suitable for freezing cells obtained according to the present invention. Preferably, the cells are frozen at temperatures of -70°C or less (eg, -80°C), optionally with prior flash freezing at temperatures of -190°C or less. In one embodiment, the cells are frozen in liquid nitrogen.

Frozen cells, optionally, but preferably, can be stored frozen. Standard methods for storing mammalian cells, in particular mesenchymal cells, are suitable for storing cells obtained according to the present invention. It is preferable to freeze the cells in vials. In one embodiment, the frozen cells are stored in liquid nitrogen.

In a preferred embodiment, cells are aliquoted prior to freezing. Suitable containers for containing aliquots of cells obtained in accordance with the present invention are all containers suitable for containing appropriate quantities (aliquots) of cells and for storage at low temperatures, in particular vials. A vial (also known as a mattress) is a small glass or plastic jar or bottle suitable for cell culture. The nature of the vial suitable for the present invention is not particularly limited, provided that it allows cells to be maintained in the medium as directed, and provided that it is suitable for freezing under conditions in which the cells are kept in a state that allows them to be thawed later. and subculturing. Thus, vials must be freeze-compatible (cryovials) for this purpose. The vials may be made from any suitable material, although glass vials and vials made from plastics such as polypropylene are most typical. The vial can be closed, for this it usually has a cap, and a cryo vial suitable for use in the present invention is preferably suitable for freezing at temperatures of -196°C or less. Capped vial options include screw-top vials (closed with a screw cap or dropper/dropper), stopperable vials (closed with a stopper or plastic stopper) and roll-on vials (closed with a rubber stopper and a metal cap) or, in the case of plastic vials, also vials with "hinged caps". » (snap covers) that snap into place when pressed. The bottom of the vial is preferably flat, although this is not a strict requirement. Preferably, all vials used in one particular isolation/expansion process are identical to each other and filled with identical volumes of cell-containing fluid (cells in the medium, usually in suspension, but not necessarily).

Preferred vials are sized to hold 2-10 ml cells per medium (typically in suspension, but not necessarily), eg 3-8 ml cells per medium, and most preferably about 4 ml cells per medium. Preferred vials are sized to hold 10 ⁶ -10 ⁹ cells per medium, eg 10×10 ⁶ to 100×10 ⁶ cells per medium, and most preferably about 20×10 ⁶ cells per medium.

Frozen and/or stored cells may optionally, but preferably, be thawed. After thawing, the cells can optionally be further cultured (subcultivated), including propagated. Standard methods of thawing and optionally subculturing, in particular mesenchymal cells, are suitable for thawing the cells obtained according to the present invention. For further culturing or sub-culturing, the methods described herein are suitable, including growth medium and so on.

In a preferred embodiment, the cells are frozen after passage 2 (P2). Cells obtained and frozen after P2 are referred to herein as "primary cell bank" or "PCB" or alternatively "cell master bank". In one preferred embodiment, the primary cell bank contains or consists of cells that have been frozen after 5-14 cumulative population doublings (not counting the cumulative population doublings in the P0 process).

In another preferred embodiment, the cells are frozen after passage 4 (P4). Cells obtained and frozen after P4 are referred to herein as "secondary cell bank" or "VKB", or alternatively "working cell bank". In a preferred embodiment, the secondary cell bank contains, or consists of, cells that have been frozen after 14-25 cumulative population doublings (excluding cumulative population doublings in the P0 process).

Preferably, the PCB embodiment and the PCB embodiment are linked such that the cells are first frozen as PCP, then all or part of the PCB is thawed and subcultured, and then PCB is obtained after two further passages (total P4). Preferably, not all of the cells obtained and frozen after P2, eg not all of the PCB, are thawed and subcultured together. Rather, most preferably, one container (typically one vial) obtained and frozen as PCB is propagated and subcultured while the remaining containers (vials) are left frozen as PCB.

It is important to note that in this document, references to individual passages are provided for reference purposes. For example, it is also possible to obtain PCB after a passage other than P2. The preference for obtaining PKB after P2 and VKB after P4 is based on the experience of growing cells in a medium based on the minimum medium MVKBM CD, or a minimum medium practically equivalent to it, under the cultivation conditions specifically described in the experimental examples (in particular, example 2) . When using other culture conditions that are equally acceptable in the context of the present invention, PCB and WKB are ideally obtained after cumulative population doublings corresponding to the cumulative population doublings observed after P2 and P4, respectively, in the experimental examples described herein. , in particular, example 2C.

The selection of a single cell, if necessary, can be carried out, for example, by serial dilution, before or after propagation. Unless such isolation is specifically performed, the method of the invention results in a population of mesenchymal stromal cells. As described below, the yield can be relatively high compared to known methods, in particular due to the excellent viability and proliferability of the cells of the present invention.

Exit

The method of the present invention, including step (c), results in a population of mesenchymal stromal cells. Such a population of mesenchymal stromal cells is described herein. Preferably, the method of the present invention provides a cell population devoid of blood cells. Additional aspects of the cell population obtained in the method according to the invention are described in the third aspect of the invention.

In a preferred embodiment, cells obtained from the cell expansion process of the present invention are aliquoted and optionally frozen. The appropriate time to aliquot and freeze the cells is after completion of any passage and subsequent enzymatic separation of the cells, eg by trypsinization, centrifugation and resuspension in growth medium. The cells (cell population) thus obtained and stored frozen, preferably in aliquots, may also be referred to as a cell bank.

In one preferred embodiment, the cells are frozen after P2. The cells (cell population) thus obtained and stored frozen, preferably in aliquots, may also be referred to as a primary cell bank. In one preferred embodiment, the primary cell bank contains or consists of cells that have been frozen after 5-14 cumulative population doublings (not counting the cumulative population doublings in the P0 process).

In one additional, but not mutually exclusive, preferred embodiment, the cells are frozen after P4. The cells (cell population) thus obtained and stored frozen, preferably in aliquots, may also be referred to as a secondary cell bank. In a preferred embodiment, the secondary cell bank contains, or consists of, cells that have been frozen after 14-25 cumulative population doublings (excluding cumulative population doublings in the P0 process).

The frozen aliquots thus obtained from the respective cell bank can be thawed individually at the desired time point. In other words, it is not necessary - and generally undesirable - to thaw all cell bank aliquots at the same time. In contrast, aliquoting and individually freezing the aliquots provides the advantage that individual aliquots can be taken out of the freezer, thawed and further cultured (subcultured, further expanded) at different individual desired time points.

A full-scale cycle starting from the human umbilical cord, including step (c) and continuing until passage P2, typically results in a primary cell bank (PCB) of at least 1x10 ⁹ cells, more preferably at least 2x10 ⁹ cells, more preferably at least 3×10 ⁹ cells, more preferably at least 4×10 ⁹ cells, more preferably at least 5×10 ⁹ cells, more preferably at least 6×10 ⁹ cells, more preferably at least at least 7x10 ⁹ cells, more preferably at least 8x10 ⁹ cells, more preferably at least 9x10 ⁹ cells, and in some embodiments at least 10x10 ⁹ cells. In one preferred embodiment, the primary cell bank contains or consists of cells that have been frozen after 5-14 cumulative population doublings (not counting the cumulative population doublings in the P0 process).

Due to the excellent proliferative properties of the cells according to the invention, however, the cells can be cultured much longer than P2. This can be done either by transferring some or all of the cells obtained during P2 after enzymatic separation of cells (for example, by trypsinization) to the next passage (P3), or by creating a primary cell bank after P2, followed by thawing one or more flasks of cells from the primary cell bank at the right time to initiate the next passage (P3). In any case, one or more additional passages may follow P3 and optionally a secondary cell bank may be established, for example after P4. Due to the excellent proliferative properties, the total number of cells after a later passage, for example, P4, will generally be significantly higher than the total number of cells after P2, for which the typical yield is indicated above. For example, after P4, the overall cell yield may be at least 10 times higher than after P2, but more specifically, at least 100 times or at least 1000 times higher than after P2. In some embodiments, after P4, the total yield may be at least 10×10 ¹² cells in one full cycle. Optionally, a secondary cell bank is obtained after P4. In a preferred embodiment, such a secondary cell bank contains, or consists of, cells that have been frozen after 14-25 cumulative population doublings (excluding cumulative population doublings in the P0 process).

Optionally, the yield can be further increased by further subculturing, for example, to passage P5 and beyond.

Differentiation potential

The potential of cells according to the invention can be evaluated experimentally. Typically, this is done by placing the cells under conditions that are favorable for differentiation into one or more specific cell types.

Preferably, the cell(s) obtained after step (c) is at least multipotent and optionally pluripotent. In a typical embodiment, the cells obtained after step (c) are multipotent. Multipotency can be tested in differentiation assays, as is known in the art. Under certain conditions, a multipotent cell will differentiate into tissues (adipogenic, chondrogenic, and osteogenic). Examples 12, 13 and 14 show that the cells of the present invention have such abilities. Under certain conditions, the cell will differentiate into different tissues (adipogenic, chondrogenic and osteogenic). Preferably, the multipotent cell obtained by the method of the invention has at least all of the following differentiation potentials: (a) adipogenic; (b) chondrogenic; (c) osteogenic. Examples 12, 13 and 14 show that the cell of the present invention has such abilities. In these examples, in particular, in example 12, specific conditions are described that allow differentiation, despite the rapid multiplication of cells obtained from stromal Wharton's jelly according to the invention.

Optionally, the cell has additional differentiation potentials selected from differentiation potentials typical of mesenchymal stromal cells. However, as is typical for mesenchymal stromal cells, the cell of the invention is generally not totipotent.

However, the cell of the invention is generally not totipotent. However, the cell may optionally be subjected to genetic manipulation and/or nuclear reprogramming, which may affect differentiation potential.

Relationship with other aspects of the invention

The cell described in detail in the second aspect of the invention can be obtained by the method of the invention. The cell population described in detail in the third aspect of the invention can be obtained by the method of the invention. For example, a cell bank can be obtained by the method of the invention. In one embodiment, the cell bank can be obtained by the method according to the first aspect of the invention. In this embodiment, step (c) of the method of the invention is optionally followed by step (d) aliquoting the cells and step (e) storing the cells. Cells can be stored under any suitable conditions, although freezing at temperatures of 180° C. or less is most typical. Cells can be frozen, preferably by flash freezing them in liquid nitrogen. A cryoprotectant may be added before freezing.

Since the aspects of the present invention are related to each other, the embodiments described in relation to the second and third aspects can also be applied to the first aspect, and vice versa. For example, by demonstrating a significant absence of endothelial marker proteins by profiling expression or determining surface markers as described in the second aspect of the invention, it can be experimentally confirmed that the aspect of the method of the first aspect of the invention, namely the removal of the epithelium, has been properly performed.

Particularly preferably, if the cell population obtained by the method according to the first aspect of the invention is the cell population described below in the third aspect of the invention. It is also particularly preferred if the method for producing a cell according to the first aspect of the invention leads to the production of a cell described in the second aspect of the invention; thus, all embodiments and features described herein as relating to the cell according to the second aspect of the present invention are also suitable for describing the characteristics of the method for producing the cell according to the first aspect of the present invention. It is also particularly preferred if the cell obtained by the method according to the first aspect of the invention is the cell described in the second aspect of the invention below.

mesenchymal stromal cell

In a second aspect, the present invention relates to a mesenchymal cell. More specifically, a mesenchymal cell is a mesenchymal stromal cell (MSC). Features and embodiments of the cell are described below.

Some features and embodiments of the cells of the present invention, described below, are associated with the physical characteristics of the cells of the invention. By way of illustration only, and notwithstanding the details disclosed elsewhere herein, physical characteristics include those characteristics that can be directly determined by physical methods, such as gene expression, cell size, shape, and so on. Some of the features and embodiments of the cell of the present invention, described below, are related to the way in which the cell of the invention can be obtained, or obtained. Solely by way of illustration, and notwithstanding the details disclosed elsewhere in this document, cell characteristics associated with the manner in which the cell of the invention may be obtained, or obtained, may include (a) the anatomical origin of the cell, in particular the umbilical cord and stromal wartons jelly as its preferred section, (b) the conditions used to isolate the cell, and optionally but preferably also (c) the conditions used to propagate the cell.

Dedicated cell

Preferably, the cell of the present invention is an isolated cell, in particular an isolated mesenchymal stromal cell. In particular, the mesenchymal stromal cell of the invention is preferably an isolated mesenchymal stromal cell derived from or derived from the intervascular region (see Figure 1 for illustration) of the umbilical cord. An isolated stromal cell can be obtained from the stroma by a method including the isolation described herein. A cell can be obtained from such a cell as a result of reproduction. Thus, in general, when a particular cell is said to "descend from" another cell, it means that a particular cell is derived from said other cell by reproduction (cell division) and/or differentiation.

In one embodiment, the cell is an isolated cell located outside the human or animal body. An isolated cell is typically a cell outside of its physiological in vivo environment. Preferably, the cell is an ex vivo cell. In particular, ex vivo the cell is preferably located outside the umbilical cord. In particular, ex vivo the cell is preferably not in contact with blood vessels, either in direct contact or indirect contact. In one ex vivo embodiment, the cell is in a culture vessel. In one ex vivo embodiment, the cell is in a suitable storage container such as a vial. In one embodiment, ex vivo the cell is at a temperature of from about 35°C to 39°C, preferably in a culture vessel. In one ex vivo embodiment, the cell is in a suitable cryopreservation container, such as a cryo vial, preferably at -180° C. or less. In one ex vivo embodiment, the cell is in a culture vessel. In one ex vivo embodiment, the cell is a metabolically active cell. In one ex vivo embodiment, the cell is a frozen cell.

In one embodiment, the cell is characterized by at least one feature - structural or functional - that is usually absent in a cell from the human umbilical cord. For example, such a feature may be a feature specific to the way in which the cell according to the invention can be obtained. Such a structural or functional feature may be selected from features specifically described herein and/or from features not specifically described herein but inherent in the cell of the invention, for example, inherent in the cell produced by the method of the invention. In one embodiment, the cell is an adherent cell, eg, attached to a culture vessel, such as a culture vessel with a plastic bottom surface.

The isolated stromal cell of the present invention can be propagated in vitro by culturing. Thus, a derived cell can be obtained. A derived cell is also a cell of the invention, provided that it conforms to the claimed characteristics defining the cell of the invention. Thus, due to the properties of the cell according to the invention, similar to those of stem cells, the present invention can cover many generations of cells. Many generations can be obtained by breeding, described in this document. Indeed, a particular embodiment of the cell of the present invention involves ex vivo propagation of the cell. Thus, although a cell is isolated, it is capable of giving rise to the same or similar cells and thus creating a cell population. Such a cell population is described in the third aspect of the invention. Suitable culture conditions are not particularly limited, but include those described in the first aspect of the invention.

A suitable non-limiting cell isolation method, including enzymatic treatment, is described herein in the context of the first aspect of the present invention. Isolation of individual cells, if necessary, can be carried out, for example, by the method of serial dilutions. Thus, preferably, the cell of the invention is a cell in an ex vivo environment, eg in vitro. Various in vitro cell culture techniques may be practiced by the skilled artisan, including those described in connection with the first aspect of the present invention.

Typically, a mesenchymal stromal cell can originate from various tissues such as bone marrow, adipose tissue, and the umbilical cord. Preferably, the cell of the present invention is derived from, or derived from, the umbilical cord, more preferably from the umbilical cord stromal Wharton's jelly. However, the cell according to the invention is not derived from cord blood. The cell is also neither an epithelial nor an endothelial cell.

In one embodiment, the cell of the invention is a freshly isolated cell. A freshly isolated cell is a cell that has been directly obtained by isolation from the tissue from which it originates, for example, in the manner described above. More preferably, however, if the cell according to the invention is a cell that is obtained from freshly isolated cells, mainly, as a rule, by reproduction in vitro. As such, there are no restrictions on the number of passages or cumulative population doublings in vitro immediately after cell isolation; however, in general, a cell of the invention is derived from a freshly isolated cell by in vitro propagation for 12 passages or less, or an equivalent number of cumulative population doublings. In general, any cell that meets the stated limitations is a cell of the invention, regardless of the number of passages or cumulative population doublings that resulted in it after isolation from the original tissue.

As is typical for mesenchymal stromal cells, the cell of the invention is typically an adherent cell when grown in a culture vessel, in particular a culture vessel with a plastic bottom surface. Thus, in one embodiment, the mesenchymal stromal cell of the invention is a surface-attached cell, in particular an in vitro plastic-attached cell. If necessary, the plastic-attached cell can be separated from the culture vessel, for example by trypsin treatment. In this way, non-attached cells can be obtained. The invention also includes such a non-attached cage.

In one embodiment, the mesenchymal stromal cell of the invention is spindle shaped. This usually occurs when the mesenchymal stromal cell is attached to a culture vessel, such as a culture vessel with a plastic bottom surface. In particular, in a population of mesenchymal stromal cells, the majority of cells, eg 50% or more by cell number, are typically spindle-shaped, particularly when attached to a plastic surface in vitro.

When the cell of the invention is not attached, for example when it has been separated by an enzyme, for example by treatment with trypsin, for example when it is in a primary or secondary cell bank, optionally in a frozen state, the cell is usually not spindle-shaped. Thus, the fusiform shape is specifically related to surface attachment under culture conditions.

In one embodiment, the cell according to the invention can be obtained by the method described in the first aspect of the invention. In this embodiment, the cell of the invention is preferably selected from a freshly isolated cell, ie a cell that has not been expanded ex vivo, or a expanded cell, ie a cell obtained by ex vivo propagation of a previously freshly isolated cell.

Gene expression and exposure of surface antigens

The cell of the present invention can be characterized by a gene expression pattern. As used herein, the term "gene expression pattern" means that at least one gene is expressed, or not expressed, or expressed at a certain level, as the case may be. Thus, the fact that a particular gene is expressed, or not expressed, or expressed at a certain level, characterizes the cell according to the invention, for example, in order to distinguish it from a reference cell. The same applies to the expression of more than one gene, such as two genes, three genes, four genes, five genes, six genes, seven genes, eight genes, nine genes, ten genes, and so on. When a cell of the invention is characterized by the expression of one or more genes, it is undesirable to make a specific statement about the expression of any other gene not mentioned in this context. As an illustrative example, when it is stated herein that a cell of the invention expresses APCDD1, this should not be taken as a statement that any particular other gene is also expressed, or not expressed, or expressed at a certain level, if from the context clearly does not follow otherwise.

Typically, gene expression can be tested by various methods, both at the nucleic acid level and at the protein level. Gene expression is relatively frequently tested in the fields of molecular biology, biochemistry, and cell biology; any method known or suitable for this may be used in the context of the present invention. Some methods are described below. Methods related to gene expression can also be called transcriptomic methods. Thus, the gene expression profile of a cell can also be called a transcriptome. In one embodiment, a cell of the invention is characterized based on its gene expression profile.

You can use different terms to describe the analysis of gene expression: for example, you can say that the expression is tested, analyzed, evaluated, investigated, measured, determined, and so on. These terms include both a qualitative definition, that is, the answer to the question whether the corresponding gene is expressed, and a quantitative definition, that is, the answer to the question at what level the corresponding gene is expressed.

Generally, to determine the expression of a gene, a sample comprising a single cell or a plurality of cells (which is preferred) is taken from a cell population, and then the expression of the gene is analyzed in this sample, while the cells in this sample may or may not be killed, depending on circumstances. Since the cell population of the present invention is usually homogeneous, i.e., substantially all of the individual cells that make it up are very similar or similar, facts relating to gene expression in cells found in a sample taken from the cell population (representative sample) as will generally also apply to the rest of the cells in the population, unless the context clearly dictates otherwise and/or the cell population does not appear to be substantially homogeneous.

Determination of the expression of any relevant gene can be either non-targeted determination, i.e., when gene expression is analyzed in a general way (for example, by a broad approach, for example, using microarrays), without focusing on a single gene, or directed determination, when specifically testing whether a particular gene is expressed. In the second case, in particular, the corresponding gene may also be referred to as the "gene of interest".

Several transcriptome technologies can be used to obtain data to analyze gene expression. For example, DNA microarrays measure the relative activity of previously identified target genes. Sequence-based methods such as RNA sec provide information about gene sequences in addition to their level of expression.

The cell of the invention is a cell of a placental animal, preferably a mammal, preferably a primate, more preferably a human. Thus, in one embodiment, the mesenchymal stromal cells (MSCs) of the invention are mammalian cells. Thus, unless specifically stated otherwise, all references to a particular gene refer to the corresponding mammalian gene.

More preferably, the mesenchymal stromal cell of the invention is a primate cell, more specifically a human cell. Thus, more preferably, the mesenchymal stromal cells (MSCs) of the invention are human cells. Thus, in this embodiment, all references to a particular gene refer to the corresponding human gene. In some cases it is obvious from the context, for example in the case of human leukocyte antigens (HLA), in other cases it is not specifically stated in the context, but it is obvious: for example, in the case of a human cell, the expression of APCDD1 means the expression of human APCDD1, and so on. Since the cell of the invention in one preferred embodiment is a human cell, it is preferable if the cell of the present invention in this embodiment expresses human APCDD1.

Preferably, the cell of the invention is not a transgenic cell. Thus, in this embodiment, all indications relating to the expression of a particular gene mean that the corresponding gene is expressed from the genome in which it is encoded, more specifically, in which it is encoded according to the Mendelian principle of inheritance. However, in some alternative embodiments, the implementation of the cell according to the invention is a transgenic cell.

Preferably, gene expression is tested on at least duplicated samples, preferably at least three-fold duplicated samples. In some cases, the cells being tested die, are exposed to non-sterile conditions, or are otherwise damaged during the assay and cannot be used for culture; however, the information gained from these dead, sterile, or otherwise damaged cells is still valuable, especially when the cells come from a homogeneous cell population; it is reasonable to assume that the information thus obtained, in particular information relating to gene expression, may be applicable not only to dead, sterile or otherwise damaged test cells, but also to other cells in the population from which the corresponding test cells originate. It can be assumed that such information is applicable both to the cell population itself and to any individual cell contained in such a cell population. In this context, "homogeneous" means that virtually all cells are very similar or similar.

The sample may consist of a single cell or, alternatively, it may contain multiple cells. There is no clear upper limit to the total number of cells contained in a sample; however, this number is usually much less than the total number of cells contained in the cell population; for example, it is 1/100 or less, 1/100 or less, 1/1000 or less, 1/10000 or less, 1/100000 or less, 1/1000000 or less of the number of cells contained in the cell population.

In the most general terms, gene expression can be tested directly, for example, as a rule, by molecular biology methods, including determining the presence and, if necessary or desired, levels of a gene product, such as a particular messenger RNA (mRNA), or its precursor or degradation product; or, alternatively, gene expression can be tested indirectly, for example, usually by biochemical methods, including determining the presence and, if necessary or desired, levels of a protein encoded by a particular gene, for example, a particular cell surface protein, or its precursor or degradation product. Suitable surface proteins for characterizing the cell of the invention include, in particular, clusters of differentiation (CD).

Any known method for testing gene expression can also be used in the present invention. Suitable methods include methods selected from a list including, but not limited to, gene expression profiling, polymerase chain reaction such as PCR, e.g., preferably, quantitative PCR (qPCR), including RT-qPCR, transcription product sequencing, any type of transcriptome analysis. and others. At the nucleic acid level, preferred methods include the use of microarrays and qPCR.

In one embodiment, gene expression is determined by gene expression profiling. In the field of molecular biology, gene expression profiling means measuring the expression of several, often thousands, of genes, usually all at the same time, to obtain a global picture of cellular gene expression. For example, gene expression profiling is the measurement of the expression of several, typically, but not necessarily, thousands, of genes at the same time to obtain a global picture of the cell. Gene expression profiling makes it possible, for example, to distinguish between the cells of the invention and other cells. It is even possible to simultaneously determine the expression of the entire genome, that is, of each gene present in a particular cell. In many experiments of this type, measurements are made simultaneously for the entire genome, that is, for each gene present in a particular cell. Some transcriptomic technologies can be used to obtain the data needed for analysis. In some embodiments of the present invention, the use of microchips is preferred. Microarrays make it possible to determine the relative expression of genes, including one or more genes of interest, such as, for example, APCDD1. Without wishing to be bound by any particular theory, it is understood that gene expression profiling currently provides the greatest possible global picture of gene expression in a single experiment.

Typically, microarray analysis is a suitable method for profiling gene expression. Indeed, a microchip is preferably used for this purpose in the present invention. As is known, microarray analysis indicates whether certain particular genes are differentially expressed in a cell compared to, for example, a cell obtained by a prior art method. Microchips are commercially available and can be used in the context of the present invention.

In one embodiment, gene expression is determined in a high throughput experiment. In an alternative embodiment, gene expression is determined in an experiment that is not a high throughput experiment, for example, in a specific experiment aimed at determining a particular gene. In one embodiment, gene expression is determined in both a high throughput experiment and in an experiment that is not a high throughput experiment; in such a case, the high throughput experiment is usually performed first; and an experiment that is not a high throughput experiment is subsequently carried out at a later stage, for example, to confirm the results obtained in the high throughput experiment. For example, microarray analysis (high throughput) may be followed, for example, to validate or confirm the expression of specific genes, by qPCR (not high throughput).

Sequence-based methods, including high throughput sequencing such as next generation sequencing (NGS), can be used to determine the expression of the genes of the present invention. For example, RNA sec (RNA sequencing), also referred to as whole-transcriptome shotgun sequencing, can provide information about gene sequences in addition to their level of expression. The RNA sec method uses next generation sequencing (NGS) to determine the presence and amount of RNA in a biological sample at a particular point in time. In one embodiment, gene expression is determined by a sequence-based method such as RNAsec or PCR and any variations thereof.

Expression of some or all of the genes, eg as determined by gene expression profiling, eg using microarrays, can then be confirmed by quantitative RT-PCR (qRT-PCR).

As is known, quantitative polymerase chain reaction (qPCR, also known as real-time polymerase chain reaction (real-time PCR)) is a polymerase chain reaction (PCR) based laboratory method that is suitable for nucleic acid quantification. In particular, quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a preferred method for determining the amount of a transcript (eg, mRNA). Compared to other methods for quantifying RNA, such as Northern blotting, qRT-PCR is generally considered to be a more efficient, sensitive, and quantitative assay for determining RNA levels, and is therefore the preferred method of the present invention. The qRT-PCR method is also a much more sensitive method for determining gene expression than indirect determination of the product of gene expression at the protein level, such as immunological determination, for example, by binding of fluorescent primary or secondary antibodies and flow cytometry. qRT-PCR typically requires specific primers for the corresponding nucleic acid, the sequence of which, however, is usually available from a sequence database, for example, for human genes, or which can be designed by a person skilled in the art based on such information. Commercial qRT-PCR kits and instruments are available and can be used in the present invention. The nucleic acid that serves as a template (also referred to as "target nucleic acid") for determining gene expression in qRT-PCR is a transcription product, typically mRNA.

qPCR is generally carried out for a total of 35-45 consecutive cycles, more preferably 38-42 cycles and most preferably 40 cycles. If there is no fluorescence even after the completion of the entire number of cycles, it is concluded that the target nucleic acid is not present in the analyzed sample.

Preferably, gene expression is determined by qPCR (preferably qRT-PCR), which determines the expression levels of the gene of interest and preferably compared with the expression levels of the "household" gene. For this purpose, the preferred housekeeping gene is the beta actin gene (see, for example, Figure 8).

As is standard in the art, gene expression patterns as determined by qRT-PCR have Ct and dCt values, respectively: typically, in qPCR, Ct (cycle threshold) is defined as the number of cycles required for a fluorescent signal to cross the threshold ( i.e. exceeded the background level). Typically, the Ct value is inversely proportional to the amount of the corresponding (target) nucleic acid in the sample (ie, the lower the Ct value, the greater the amount of the corresponding (target) nucleic acid in the sample). The Ct value is specific for the expression of one gene. Delta Ct (dCt) shows the difference in expression of 2 genes, typically the gene of interest and a housekeeping gene such as the beta actin gene (see, for example, Figure 8).

Preferably, the cell of the invention expresses at least one gene specific for mesenchymal stromal cells.

The cell according to the invention is characterized in particular by the fact that it expresses APCDD1. In particular, the cell expresses APCDD1. The inventors have found that the genes specific for the mesenchymal stromal cells of the present invention are included in a list including, but not limited to, APCDD1 and PPARG, and optionally FLVCR2. More preferably, said at least one stromal cell-specific gene is expressed at high levels relative to the beta actin housekeeping gene compared to the expression ratio or ratio of said at least one gene to beta actin gene in reference cells. . An example of such a phenotype is shown in example 8.

Although doubts have been raised in the literature as to whether non-perivascular MSCs differ from perivascular MSCs (e.g. Davies et al., Stem Cells Translational Medicine, 2017, vol. 6, p. 1620-1630), the present invention provides evidence (e.g. , example 8) that cells from stromal Wharton's jelly obtained according to the present invention differ from perivascular MSCs.

In one embodiment, gene expression is determined indirectly, that is, not by determining the primary transcription product, mRNA, or its precursor or degradation product, but by determining the subsequent specific product formed. Typically, the subsequent specific product formed is the protein encoded by the mRNA; however, it can also be any other specific product, such as, for example, a metabolite specific for the presence of a particular, eg, enzymatically active, protein.

Preferably, the presence of a protein is determined. More preferably, the presence of the protein on the cell surface is determined. In other words, it is determined whether the protein is exposed on the cell surface.

Cells displaying a particular protein on the cell surface can be analyzed, for example, with immunologically active molecules such as specific antibodies and other immunoreactive molecules. The term "cell surface" is used herein in accordance with its usual meaning in this field and, thus, specifically includes the outside of the cell, available for binding by proteins and other molecules. A protein is exposed on the surface of a cell if it is at least partially located on the surface of said cell and is available for binding by antigen-binding molecules, such as antigen-specific antibodies added to the cell. In one embodiment, the cell surface-exposed protein is an integral membrane protein having an extracellular portion that an antibody can recognize. The term "extracellular part", or "exodomain", in the context of the present invention means a part of a molecule, in particular a protein, which is directed to the extracellular space of a cell and is preferably accessible on the outside of said cell, for example, for binding molecules, such as antibodies located outside of the cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof. The term "part" is used herein to refer to a continuous or discontinuous structural element, such as an amino acid sequence. A portion or fragment of a protein sequence preferably contains at least 5, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50 or at least 100 consecutive and/or non-consecutive amino acids of the amino acid sequence that makes up a protein.

A protein that can be detected by an antibody, or other immunoreactive molecule, can also be called an antigen. In some embodiments, the implementation of the cell according to the invention may be characterized by exhibiting - or not exhibiting - one or more specific antigens. In the context of the present invention, such an antigen is preferably displayed on the cell surface. Such an antigen may also be referred to as a "surface antigen". Examples of them are given in example 7.

According to the general principles of cell biology, if an antigen is exposed on the surface of a cell, then the gene encoding the antigen is expressed in the cell. Thus, detection of an antigen that is exposed on the cell surface is an indirect way of demonstrating that the gene encoding the antigen is being expressed. For example, when it is indicated herein that a cell expresses a particular gene encoding an antigen expressed on the cell surface, then the expression of said gene can either be tested directly at the level of expression of the gene product (such as mRNA) or indirectly at the level of the protein exposed on the surface. cells. Although the word "expresses" literally means that a gene is expressed, the word "expresses" can also mean that a protein, in particular a cell surface protein, which is encoded by said gene, is displayed on the cell.

In accordance with the invention, an antigen is displayed on a cell if the expression level is above the detection limit and/or if the expression level is high enough to allow binding by antigen-specific antibodies added to the cell. According to the invention, the antigen is said not to be expressed on the cell if the expression level is below the detection limit and/or if the expression level is too low to allow binding by antigen-specific antibodies added to the cell. Preferably, the antigen expressed in the cell is expressed or displayed, i.e., present, on the surface of said cell and thus available for binding by antigen-specific molecules such as antibodies or other immunoreactive molecules added to the cell. In some cases, a secondary molecule is also added that aids in detection, such as, for example, optionally, a labeled secondary antibody.

An antibody, or other immunoreactive molecule, may recognize an epitope on a cell. The term "epitope" means an antigenic determinant in a molecule such as an antigen, i.e., a portion, or fragment, of a molecule that is recognized by, i.e., binds to, a component of the immune system, for example, that is recognized by an antibody, or other immunoreactive molecule. The detection of an epitope specific for a particular antigen will generally lead to the conclusion that that particular antigen is expressed on the cell being analyzed.

In one embodiment, a cell of the invention can be characterized by immunophenotyping. "Immunophenotyping" generally means that a cell can be characterized by antigen-specific molecules such as antibodies or other immunoreactive molecules that are added to the cell to determine whether the antigen is expressed on the cell. Immunophenotyping involves cell sorting by various methods, including flow cytometry.

The preferred immunophenotyping method is flow cytometry, in particular FACS. An analyte, in particular a cell surface protein, is usually recognized by an antibody or other immunoreactive molecule. The antibody or other immunoreactive molecule is either itself labeled with a fluorophore or is recognized by a fluorophore labeled secondary antibody or other immunoreactive molecule that is added for this purpose.

Preferred analytes suitable for immunophenotyping in the context of the present invention are molecules belonging to a cluster of differentiation (also known as a cluster of differentiation or classification determinant, and herein abbreviated as CD). CD is a term used to identify and study cell surface molecules by cell immunophenotyping. Some examples of clusters of differentiation suitable for characterizing embodiments of the cell of the invention are described herein. Unless the context clearly implies otherwise, when referring to a cassette of differentiation (CD) molecule, it is understood that the expression of the corresponding CD molecule is preferably determined by immunophenotyping; however, determination at the nucleic acid level is also possible. In this document, reference to specific CD molecules in the context of human cells shall mean the corresponding specific CD molecule, following the definitions adopted at Workshops I-IX on human leukocyte differentiating antigens. Example 7 provides an illustrative and useful description of the immunophenotyping of the present invention.

On the other hand, unless the context clearly indicates otherwise, when the expression of any gene other than a cassette of differentiation (CD) molecule or the nucleic acid encoding it is referred to herein, it is understood that the expression of the corresponding gene is preferably determined at the nucleic acid level. preferably in the manner mentioned or described above.

First, the MSCs of the invention are characterized in that they (a) express APCDD1. The expression of APCDD1 is preferably determined by the level of gene expression, most preferably by qRT-PCR. An example is shown in figure 8.

Without wishing to be bound by any particular theory, APCDD1 is believed to be important for the ability of cells to self-renew: the gene product has previously been shown to help maintain high levels of beta-catenin, and high levels of beta-catenin help maintain cell stemness, in particular MSCs ( Viale-Bouroncle et al., Biochem. Biophys. Res. Commun., 2015 vol. 457, pp. 314-317; Yu, Cell Transplant., 2017, vol. 26, pp. 365-377). As shown in example 8, the cells of the present invention differ from those derived from OCBC, in particular, the expression of APCDD1. Thus, the APCDD1-expressing cell of the present invention clearly differs from the MSC described in the prior art, see in particular WO 2004/072273 A and Sarugaser et al., 2005, Stem Cells, vol. 23, p. 220-229.

However, the MSCs of the present invention do not express 3G5. 3G5 is a perivascular cell marker. Thus, the cell of the invention is not a perivascular cell. Thus, preferably, the cell according to the invention does not express any perivascular cell marker, which at the same time is not also a mesenchymal cell and/or stem cell marker.

Preferably, the cell expresses at least one gene specific for stromal cells, more specifically for mesenchymal stromal cells. Preferably, the cell displays at least one surface marker specific for stromal cells, more specifically for mesenchymal stromal cells, on its surface.

In one embodiment, at least one surface marker specific for stromal cells, more specifically for mesenchymal stromal cells, is selected from a list including, but not limited to, membrane cofactor protein (CD46), decay accelerating factor or CD55, and MAC- inhibitory protein (CD59). All three markers, as previously described, are associated with the complement cascade. As shown in Figure 7B, the cells described herein exhibit these markers on their surface. Without wishing to be bound by any particular theory, one or more of these surface markers are believed to play a role in protecting cells from complement-mediated cell lysis in vivo. Preferably, the cell expresses at least one of CD46, CD55 and CD59, more preferably at least any two of CD46, CD55 and CD59, and most preferably all of CD46, CD55 and CD59. To the inventors' knowledge, CD46, CD55, and CD59 have not previously been described in specific PC-derived MSCs, although it certainly cannot be ruled out that some cells do express one or more of these surface molecules.

Preferably, the cell of the present invention expresses CD73. CD73 is also known as ecto-5'-nucleotidase and is a mesenchymal stromal cell (MSC) marker, see Dominici et al., 2006 (Cytotherapy, vol. 8, p. 315-317).

Preferably, the cell of the present invention expresses CD105. CD105 (also known as endoglin) is also a mesenchymal stromal cell (MSC) marker, see Dominici et al., 2006 (Cytotherapy, vol. 8, p. 315-317).

Preferably, the cell of the present invention expresses CD90. CD90 (also known as Thy-1) is also a mesenchymal stromal cell (MSC) marker, see Dominici et al., 2006 (Cytotherapy, vol. 8, p. 315-317).

In the most preferred embodiment, the cell of the present invention expresses both CD105 and CD73. In addition to the general methods described above, these clusters of differentiation can be detected, for example, using SH2 and SH3 antibodies (Horwitz et al., Curr. Opin. Hematol., 2006, vol. 13, p. 419-425). Typically, the mesenchymal stromal cell of the present invention expresses both CD105 and CD73, and preferably also CD90. Thus, expression of CD105 and/or CD73 and/or CD73 may be preferred as a feature to distinguish the cells of the invention from certain other cells. More preferably, the cell expresses, in addition to any one or more of CD105 and/or CD73 and/or CD73, also at least one of CD46, CD55 and CD59, more preferably at least any two of CD46, CD55 and CD59 , and most preferably all of CD46, CD55 and CD59.

In a preferred embodiment, the cell also expresses PPARG. An example is shown in Figure 8. Without wishing to be bound by any particular theory, it is believed that the PPARG gene product is capable of maintaining a multipotent phenotype in cells, in particular MSCs, in particular to prevent differentiation into bone tissue. For details see, for example, Xu et al. (Curr. Stem Cell Res. Ther., 2016, vol. 11, p. 247-254).

In another, and non-mutually exclusive, preferred embodiment, the cell also expresses the FLVCR2 gene. An example is shown in figure 8. The abbreviation FLVCR2 means a member of the 2 family of cell receptors subgroup C of the feline leukemia virus. The FLVCR2 encoded protein is a transmembrane protein, a member of the membrane transporter superfamily, that acts as a calcium carrier. In living organisms, the encoded protein, as previously described, plays a role in the development of cerebrovascular endothelial cells.

In a preferred embodiment, the cell does not express CD117. CD117 is a cell surface marker specific for certain types of hematopoietic (blood) progenitor cells, such as those found in the bone marrow. The cell of the invention is not a blood cell, and preferably also is not a bone marrow cell. CD117 is also associated with some cancers; however, the cell of the invention is usually not a cancer cell. In other words, cancer cells are not referred to herein as the cells of the invention.

The cell of the invention preferably expresses MHC I. In the case of a human mesenchymal stromal cell, preferably the human cell expresses at least one, preferably at least any combination of the two, and most preferably all of the human leukocyte antigens HLA-A, HLA-B and HLA-C.

In some embodiments, the cell of the invention does not express MHC II, or expresses MHC II at low levels. "Low levels" is a comparative term indicating that expression levels are lower than those in antigen presenting cells from the same individual, eg, dendritic cells, mononuclear phagocytes, and B cells from the same individual. In one embodiment, the cell does not express MHC II substantially. In particular, in the case of a human mesenchymal stromal cell, it is preferred if the cell does not express any one or more of the human leukocyte antigens HLA-DP and/or HLA-DQ. Optionally, HLA-DR is also not expressed; alternatively, HLA-DR is expressed at low levels. The lack of expression of these human leukocyte antigens (HLA), or expression at low levels, renders the cell of the invention immunologically privileged, allowing, for example, co-culture with cells from HLA mismatched individuals, and even cells from other species, including cells of the immune system. systems. Expression of these human leukocyte antigens can be tested, for example, by surface staining with specific antibodies, or by qRT-PCR.

In one embodiment, the cell of the invention is negative for CD34 and/or CD45 surface markers. This means that the cell does not display CD34 and/or CD45.

In one embodiment, the cell of the invention produces prostaglandin E2 (PGE2). As is known, prostaglandin E2 (PGE2), also known as dinoprostone, is a natural prostaglandin described as an activator of the Wnt signaling pathway (Goessling et al., Cell, 2009, vol. 136, p. 1136-1147). Several commercial products, in particular ELISA kits, are available for determining PGE2 production. Detection can be performed using either the cell supernatant or cell lysate, or a combination thereof, as a sample.

In one embodiment, the cell of the invention expresses one or more stem cell markers. Such stem cell markers may include, in general, markers selected from, but not limited to, Oct-4 and Nanog.

Cell size

In addition to other features and embodiments described herein, the cell of the present invention preferably also has a relatively small size. Size can be described, for example, in terms of cell volume, cell diameter, or cell surface area, or in terms of a parameter that correlates with cell volume, cell diameter, or cell surface area. The size can also be described based on a combination of any of the listed parameters. "Relatively" means in comparison with other cells, in particular, in comparison with mesenchymal stromal cells from other sources and/or isolated in other ways. "Other sources" means other anatomical regions, such as, for example, other organs, such as the bone marrow, but also other sections of the umbilical cord, such as the perivascular zone, as well as any perivascular Wharton's jelly.

Without wishing to be bound by any particular theory, it is believed that in some embodiments, smaller cells (eg, cells with a smaller diameter and/or cells with a smaller surface area) are earlier in the cell cycle. Methods for determining the state of the cell cycle of MSCs have been published (eg, Achille et al., J. Cell. Biochem., 2011, vol. 112, p. 1817-1821) and can be applied to the cells of the present invention, for example, for analytical purposes.

In accordance with the present invention, the value of forward scatter (FSC), determined for an individual cell by flow cytometry, correlates with cell volume. Larger cells, ie in particular cells with a larger volume, are characterized by higher FSC values and vice versa. As is known, the FSC value is a dimensionless number. To determine FSC, one or more cells are analyzed by flow cytometry. As a rule, FSC values determined by flow cytometry depend on the instrument used, in particular, the characteristics of the laser and the power of photomultipliers in different instruments. Therefore, there is no absolute value for FSC, the value generally depends on the instrument and laser used. Thus, in order to ensure reproducibility, the following details, in combination, are preferred in the context of the present invention.

In the present invention, when determining the FSC value by flow cytometry, the excitation wavelength is preferably in the range of 470-500 nm, most preferably 488 nm, and forward scatter (FSC) is determined. The cell itself does not need to be fluorescent for FSC detection. For this determination by flow cytometry, as a rule, use a commercial instrument for flow cytometry. In a preferred embodiment, a FACSAria III flow cytometer (BD Biosciences) with an excitation wavelength of 488 nm is used. All specific FSC numerical values given below preferably refer to the specified conditions, i.e., the specified cytometer and the specified wavelength. Preferably, the cell of the invention has an FSC value of 60 or less, such as 59 or less, such as 58 or less, such as 57 or less, such as 56 or less, such as 55 or less, such as 54 or less, eg 53 or less, eg 52 or less, eg 51 or less, or even 50 or less. In some embodiments, a cell of the invention has an FSC value of 32-58, such as 33-57, such as 34-56, such as 35-55, such as 36-54, such as 37-53, such as 38- 52, such as 39-51, such as 40-50, such as 41-49, such as 42-48, such as 43-47, such as 44-46. In a preferred embodiment, the FSC value is about 45. These specific FSC numbers refer to determination using a FACSAria III flow cytometer under the conditions described above.

Preferably, the cell of the invention has an FSC value that is less than the FSC value of the OCBC-derived cell. Preferably, the FSC value of the cell of the invention is significantly less than the FSC value of the OCBC-derived cell, see for example Example 4.

Preferably, the cell according to the invention has a smaller cellular volume than the volume of the cell derived from OCBC. More preferably, the cell according to the invention has a significantly smaller cellular volume than that of a cell derived from OCBC. Cellular volume can be indirectly determined by determining the FSC value by flow cytometry (see above for details on determining the FSC value). Then, for comparative purposes, a bead of known size is optionally subjected to flow cytometry under identical conditions; thus it is possible to determine the cell volume based on the experimentally determined FSC value for the cell and the experimentally determined FSC value for the granule, as well as the known volume of the granule. The bead used preferably has a refractive index similar or substantially identical to that of the MSC. A commercially available program for calculating cell volume based on an experimentally determined FSC value. Preferred cell volume tools are FACS ARIA III and FACS DIVA (both from Becton Dickinson, Franklin Lakes, NJ, USA). Data analysis is performed using the FACS DIVA version 6.0 program, which is usually attached to FACS ARIA III. Use standard FACS DIVA configuration. To compare multiple samples, or cells, based on FSC values, you must always use the same instrument and always with the same FSC photomultiplier settings.

FSC, and thus cell volume, can be determined both for freshly isolated cells and for cells cultured and separated with an enzyme, eg trypsin. However, FSC and cell volume are preferably determined for non-attached cells. For flow cytometry, any adhering cell must be separated from the surface of the culture vessel to which it is attached, usually by enzymatic treatment, more particularly by trypsinization, before cell volume is determined. More specifically, FSC is typically determined by subjecting non-attached cells to flow cytometry and by forward scatter (FSC) determination. A commercially available program is for determining cell volume based on an experimentally determined FSC value. The preferred commercial cell volume software of the present invention is BD FACSDiva (Becton Dickinson, Franklin Lakes, NJ, USA).

Although the inventors have shown that the cells of the present invention are relatively homogeneous, as a rule, there is some variation, as with all biological products, including in relation to cell volume. Because of this, the cell volume can also be described as the average cell volume. In a population of multiple cells, the average cell volume is preferably a cell volume corresponding to the cell volume of the cells obtained as described in Example 1 and Example 2A for experimental determination of FSC. Example 4 describes an illustrative determination of FSC of multiple cells by flow cytometry and forward light scatter. "Average volume" is the arithmetic mean of the experimentally determined volumes of individual cells. Typically, "average volume" is the arithmetic mean of the volumes of at least 1,000 individual events, e.g., at least 5,000 individual events, e.g., at least 10,000 individual events, in some embodiments, 100,000,000 individual events. Each event, determined by flow cytometry, usually corresponds to one cell. Since cell volume determination by flow cytometry is a high throughput method, the average value for such numbers of individual cells can usually be determined without undue time and effort.

In one embodiment, not the cell volume itself is determined, but the FSC value is determined. Typically, when a particular cell has an FSC value lower than the FSC value of a reference cell, then that particular cell will also have a smaller cell volume than that of the reference cell; thus, the determination of FSC allows one to indirectly draw a conclusion about the volume of the cell.

Preferably, the cell according to the invention has a surface area of not more than 3500 µm ² . Preferably, the cell has a surface area of not more than 3000 µm ² . Preferably, the cell has a surface area of not more than 2500 µm ² . Preferably, the cell has a surface area of not more than 2100 µm ² . In some embodiments, the implementation of the cell has a surface area of not more than 2000 μm ² . "Surface area" means the 2-dimensional surface area of a cell.

In the context of the present invention, the 2-dimensional cell surface area is determined when the cell is in adherent culture, ie, the cell is attached to the culture plate. It is important that the cells are in a state of low confluence. Subconfluence is considered to be the preferred state for reproducible assay results, since subconfluent cells are not considered to substantially inhibit each other through contact inhibition. To determine the surface area, one or more cells are photographed or otherwise visually observed or recorded, usually with a microscope, from a position perpendicular (i.e., 90°) to the surface of the culture plate to which the cell or cells are attached. The boundaries of the region are determined manually on a graphic image (ie, photograph) of the cell. It is preferable to avoid agitation of the plates before obtaining the specified graphic image, or keep it to a minimum. Methods and programs for determining surface area are not particularly limited, and the corresponding program is available from several commercial vendors, such as Carl Zeiss (Jena, Germany). For the determination results shown in Figure 6, Axiovision LE 4.8 from Carl Zeiss (Jena, Germany) was used. As an example, methods for determining the surface area of cells growing in a culture dish are described in Agley et al., 2012, J. Histochem. Cytochem., vol. 60, p. 428-438, among others.

Although the inventors have shown that the cells of the present invention are relatively homogeneous, as a rule, there is some variation, as with all biological products, including in relation to cell surface area. Thus, the cell surface area can also be described as the average cell surface area. In this case, preferably, the cell has an average surface area of not more than 3000 μm ² . Preferably, the cell has an average surface area of not more than 2500 µm ² . Preferably, the cell has an average surface area of not more than 2100 µm ² . In some embodiments, the implementation of the cell has a surface area of not more than 2000 μm ² . In a population of many cells, the average surface area is preferably within the range specified herein. "Average area" is the arithmetic mean of the experimentally determined areas of individual cells. Typically, "average area" is the arithmetic mean of the areas of at least 35 individual cells, such as at least 50 individual cells, such as at least 100 individual cells, in some embodiments 35-200 individual cells.

As shown in Figure 6, the cell of the present invention preferably has a surface area that is significantly less than that of bone marrow-derived MSCs. In some embodiments, the cell of the present invention also has a surface area that is less than the surface area of perivascular Wharton's jelly (PVC) derived mesenchymal stromal cells, such as those described in WO 2004/072273 A and Sarugaser et al., 2005 , Stem Cells, vol. 23, p. 220-229. Perivascular Wharton's jelly (PVC)-derived mesenchymal stromal cells may also be referred to as "perivascular-derived mesenchymal stromal cells".

Preferably, the average volume and/or surface area of the cells of the present invention is less than the average volume and/or surface area of bone marrow-derived MSCs (Figure 6) and less than that of umbilical cord-derived MSCs (Figure 5B). In the literature, a smaller cell volume of MSCs is considered to be preferable for various reasons (see, for example, Ge et al., Stem Cell Rev., 2014, vol. 10, p. 295-303). Thanks to the present invention, cells with these preferred properties are now readily available.

Preferably, the intrinsic complexity of the cells of the present invention is lower than the intrinsic complexity of OSVS-derived MSCs. Internal complexity is typically determined in a side scatter (SSC) analysis. Typically, cells with more complex internal structures appear brighter/lighter in side scatter analysis due to internal structures, see example 4A. Without being tied to any particular theory, low intrinsic complexity may be related to stemness. To compare multiple samples based on SSC values, you must always use the same instrument and always with the same settings for FSC and SSC photomultipliers, because SSC values, like FSC values determined by flow cytometry, are instrument dependent, depending on in particular, the characteristics of the laser and the power of photomultipliers in different devices. The flow cytometry instrument, laser and settings, and programs used in the present invention for determining SSC values are preferably the same as those described above for determining FSC values.

In one embodiment, the CBC-derived cells of the present invention have lower intrinsic complexity (as shown by SSC) than the CBC-derived cells.

In a preferred embodiment, said CBC-derived cells have both lower cell volume (as shown by FSC) and lower intrinsic complexity (as shown by SSC) than OCBC-derived cells. OSVS-derived cells can be obtained, for example, as described in the Comparative Examples herein.

Biological properties of the cell: differentiation potential, aging and immortality

As a rule, the differentiation potential, aging and immortality can be collectively referred to as the "biological properties of the cell". Of course, the cell according to the invention has additional biological properties of the cell, which can be analyzed by a person skilled in the art. As used herein, the term "biological properties of a cell" is an open term that also includes additional properties of a cell that can be determined in a biological assay, even if those additional properties are not explicitly stated herein.

The cell of the invention is a mesenchymal cell, in particular a mesenchymal stromal cell (MSC). The specified MSC is usually clonogenic, unless the context clearly indicates otherwise. Thus, even a single cell according to the invention is a colony forming unit (CFU) giving rise to many daughter cells.

The cell of the invention may also be referred to as a "stromal progenitor cell". In general, a stromal progenitor cell is generally capable of giving rise to phenotypically and genotypically identical daughter cells ("self-renewal") as well as cells of at least one definitive cell type. In one embodiment, the cell of the present invention is not committed.

Thus, preferably, the cell is not terminally differentiated. In one embodiment, the cell does not differentiate further, remaining a mesenchymal cell. Preferably, the cell is capable of differentiating into different cell types. In some embodiments, differentiation into one or more of these different cell types is induced, for example, by external stimuli and/or certain culture conditions.

In a preferred embodiment, the cell is at least multipotent. Multipotency can be tested by demonstrating that a cell is capable of differentiating into several, at least two, different cell types. There are no particular restrictions on assays suitable for such a demonstration. However, as a rule, the demonstration is carried out in vitro. Thus, in a preferred embodiment, the MSC of the invention is a cell having a multipotent ability to differentiate in vitro. Examples of different types of differentiation are given in examples 12, 13 and 14. For example, the osteogenic potential of differentiation can be determined as described in example 12, although alternative known methods can be used. The osteogenic differentiation potential assay described in Example 12 was specifically designed in the context of the present invention. Compared to existing assays for osteogenic potential, many of which have been developed with bone marrow-derived MSCs, the assay in Example 12 allows relatively slow cell expansion; this, in turn, allows the (umbilical cord-derived) cells to attach to the culture vessel, which is usually a prerequisite for osteogenic potential analysis.

For example, adipogenic differentiation potential can be determined as described in Example 13, although other known methods can be used. For example, chondrogenic differentiation potential can be determined as described in Example 14, although other known methods can be used. "Differentiation potential" means that a particular cell is capable of differentiating into a cell of a particular lineage.

Preferably, the cell is capable of differentiating into a cell of the osteogenic lineage. Preferably, the cell is capable of differentiating into a cell of the chondrogenic lineage. Preferably, the cell is capable of differentiating into a cell of an adipogenic lineage. Preferably, the cell is capable of differentiating into an osteogenic cell and a chondrogenic cell. Preferably, the cell is capable of differentiating into an osteogenic cell and an adipogenic cell. Preferably, the cell is capable of differentiating into an osteogenic cell and an adipogenic cell. Most preferably, the cell is capable of differentiating into both an osteogenic lineage cell and a chondrogenic lineage cell and an adipogenic lineage cell. If a cell is capable of differentiating into a cell of a particular lineage, this should mean that the cell will differentiate appropriately if it is under the appropriate conditions. For example, particularly preferred conditions for differentiation into an osteogenic lineage cell are shown in Example 12. Alternatively, a commercial kit suitable for differentiating a mesenchymal stromal cell into an osteogenic lineage cell can be used, provided that the kit allows attachment of cells to a culture vessel, for testing whether any particular cell (mesenchymal stromal cell) is capable of differentiating into an osteogenic lineage cell; one kit described for such an assay is the BulletKit™ hMSC osteogenic differentiation kit for mesenchymal stem cells from Lonza (Wakersville, MD, USA). For example, appropriate conditions for differentiation into a chondrogenic lineage cell are described in Example 14. Alternatively, a commercial kit suitable for differentiating a mesenchymal stromal cell into a chondrogenic lineage cell can be used to test whether any particular cell (mesenchymal stromal cell) is capable of ) differentiate into a cell of the chondrogenic line of differentiation; one suitable kit is the BulletKit™ chondrogenic differentiation kit for hMSC mesenchymal stem cells from Lonza (Wakersville, MD, USA). For example, appropriate conditions for differentiation into a chondrogenic lineage cell are described in Example 14. Alternatively, a commercial kit suitable for differentiating a mesenchymal stromal cell into a chondrogenic lineage cell can be used to test whether any particular cell (mesenchymal stromal cell) is capable of ) differentiate into a cell of the chondrogenic line of differentiation; one suitable kit is the BulletKit™ chondrogenic differentiation kit for hMSC mesenchymal stem cells from Lonza. For example, appropriate conditions for differentiation into an adipogenic lineage cell are described in Example 13. Alternatively, a commercial kit suitable for differentiating a mesenchymal stromal cell into an adipogenic lineage cell can be used to test whether any particular cell (mesenchymal stromal cell) is capable of ) differentiate into a cell of an adipogenic line of differentiation; one suitable kit is the BulletKit™ hMSC adipogenic differentiation kit from Lonza.

Optionally, the cell is even pluripotent, but it is not totipotent. Differentiation potential can be determined by exposing the cell to certain conditions. Transdifferentiation is described in the art and thus one can test whether any particular cell, such as MSC, is capable of differentiating into a particular ectodermal cell or endodermal cell.

Optionally, the cell has additional differentiation potentials. Such additional differentiation potentials are selected from differentiation potentials typical of mesenchymal stromal cells. However, as is typical for mesenchymal stromal cells, the cell of the invention is generally not totipotent.

In a preferred embodiment, the cell of the invention is a mesenchymal stem cell. Thus, in such an embodiment, the mesenchymal stromal cell is a mesenchymal stem cell. In other words, it is preferable if the cell has properties similar to those of stem cells. Stem cell-like properties and their markers are described, for example, in Melton, in: Chapter 2 of Lanza and Atala (eds.), Essentials of Stem Cell, Biology, ^3rd ed., Elsevier, 2014.

Preferably, the cell of the present invention does not exhibit abnormally high levels of telomerase activity. "Telomerase activity" means the enzymatic activity of the telomerase enzyme. Telomerase is known to be an enzyme that lengthens telomeres in DNA strands, thereby allowing senescent cells that would otherwise become post-mitotic and undergo apoptosis to overcome the Hayflick limit and, in some cases, become immortal. In some alternative embodiments, the implementation of the cell according to the invention shows telomerase activity, however, at low levels. Telomerase activity is usually determined by determining enzymatic activity; more specifically, it is generally determined by determining the enzymatic activity of telomerase reverse transcriptase (abbreviated as TERT, or hTERT in humans), which is the catalytic subunit of the telomerase enzyme. An example is described in Example 10. Telomerase activity can be determined using the TeloTAGGG Telomerase PCR ELISA PLUS kit (Roche Diagnostics) photometric enzyme immunoassay to quantify telomerase activity. For illustration, see, for example, Example 10. Telomerase activity is said to be low if, when the kit is used according to the manufacturer's instructions, the analyzed cells exhibit 10% or less, for example, preferably 5% or less, telomerase activity compared to the activity of the positive control. provided in the specified TeloTAGGG Telomerase PCR ELISA PLUS kit (taken as 100%). The percentage, that is, 10% or less, for example, preferably 5% or less, refers to the average number of cells, that is, it means that the analyzed cells show an average of 10% or less, for example, preferably 5% or less, telomerase activity in comparison with the specified control. Several alternative reagents and kits for determining telomerase activity are commercially available and may alternatively be used in the context of the present invention.

Alternatively, the cell of the invention does not express telomerase at all. In other words, in this embodiment, the cell of the present invention does not exhibit any telomerase activity. More precisely, such a cell is characterized by the absence of detectable expression of telomerase. Gene expression can be determined directly or indirectly by detecting the protein transcribed from the expression product. The absence of any detectable telomerase at enzymatic activity level, protein level, or gene expression level may be appropriate for specific further characterization of the cells of the invention, also relative to other MSCs described in the literature (e.g., US 2004/0136967 A1). "Not expressing telomerase at all" means that telomerase expression cannot be detected in the cell by current methods available as of the effective date of this document, such as, in particular, qRT-PCR. This generally means that TERT activity (hTERT activity in the case of human cells) cannot be detected. To determine the expression of the gene encoding telomerase, for example, PCR primers specific for the hTERT gene can be used (Nekani et al., 2010, Stem Cell Res., vol. 3, p. 244-254).

When cells lack telomerase expression and/or telomerase activity, they cannot be maintained permanently in culture; in such embodiments, the implementation of the cell according to the invention is not immortal; however, the cell of the invention is suitable for cultivation under the conditions described in the experimental examples for at least 12 passages, or the equivalent number of population doublings.

In a preferred embodiment, the cell of the invention is not immortal. For example, as shown in Example 10, telomerase activity in the cells of the invention is relatively low. The cells in example 10 are thus not considered immortal.

The cell of the invention, present in an appropriate growth medium under appropriate conditions, is preferably mitotically active. More preferably, the cell is in an exponential growth phase, this is applicable when the cell is in an appropriate growth medium under appropriate conditions. It is clear that the frozen cell is not mitotically active; however, when the frozen cells of the invention are thawed and placed in an appropriate growth medium under appropriate conditions, they can become mitotically active and preferably grow in an exponential growth phase. For example, when frozen cells from a primary cell bank (described in detail below) are thawed and placed in an appropriate growth medium under appropriate conditions, they become mitotically active and give rise to multiple daughter cells, which in turn can optionally be used to generate secondary cell bank.

The cell of the invention is not an epithelial cell such as an amniotic epithelial cell.

The cell of the invention is not an endothelial cell.

The cell of the invention, despite its excellent proliferative properties, is not a cancer cell.

The cell claimed in the present invention is not an embryonic cell. In particular, the claimed cell is not an embryonic stem cell. Damage or destruction of embryos (including human embryos) is not intended by the present invention and was not used in the study underlying the present invention.

In a preferred embodiment, the mesenchymal stromal cell of the invention is substantially free of blood or its derivatives, in particular cord blood. Although some MSCs are a component of the bone marrow stromal scaffold that provides physical and functional support during the process of hematopoiesis, the cell of the present invention is not a hematopoietic cell. In particular, the cell according to the invention is not a blood cell. More specifically, the cell is neither a hematopoietic stem cell (HSC), nor a multipotent hematopoietic progenitor cell (MPP), nor a conventional myeloid progenitor cell (CMP).

In one embodiment, the cell of the invention is non-senescent. In another embodiment, the cell according to the invention is characterized by a low level of aging. The enzyme β-galactosidase is known to be particularly active in senescent cells, where it catalyzes the hydrolysis of β-galactosides into monosaccharides. Without wishing to be bound by any particular theory, it is believed that β-galactosidase activity is mediated by endogenous lysosomal β-galactosidase, particularly in senescent cells. Without being linked to any particular theory, its activity itself is not considered necessary for aging. However, β-galactosidase activity can easily be detected by a reliable method, both in situ and in vitro. In the present invention, β-galactosidase activity is determined by methods conventional in the art (see, for example, Lee et al., Aging Cell, vol. 5, p. 187-195. A synonym for the term "aging cell" is "withering cell".

In one embodiment, the cell of the invention is a cell that has been grown to a P11 passage or less, more preferably to a P10 passage or less, more preferably to a P9 passage or less, more preferably to a P8 passage or less, more preferably to a P7 passage, or less, more preferably up to passage P6 or less, more preferably up to passage P5 or less, and most preferably up to passage P4 or less. As shown in Example 9, the cells of the invention have excellent proliferative properties over many passages, and as shown in Example 11, the cells of the invention typically do not, or only minimally, age over many cumulative population doublings.

In one embodiment, a cell of the invention remains multipotent for at least 16 cumulative population doublings, eg, at least 18 cumulative population doublings, at least 20 cumulative population doublings, after isolation. In one embodiment, the cell of the invention remains multipotent for at least 12 passages after isolation.

In one embodiment, the cell of the invention remains undifferentiated for at least 16 cumulative population doublings after isolation. In one embodiment, the cell of the invention remains undifferentiated for at least 12 passages after isolation.

Cells obtained by the method according to the invention

The cell according to the second aspect of the invention can also be characterized by the way in which it can be obtained. This is possible because the cell of the present invention has properties that are different from those of cells obtained by the previously described methods (see Examples and Comparative Examples in this document), and thus, the method for producing a cell of the present invention makes it possible to obtain a cell with unique properties. In particular, the cell according to the second aspect can be characterized as a cell obtained by the method according to the first aspect of the invention.

In another, and not mutually exclusive, embodiment, the cell according to the second aspect can be obtained by the method according to the first aspect of the invention. In this embodiment, the method according to the first aspect of the invention contributes to cell definition by imparting certain properties to the cell, such as, for example, umbilical cord production from stromal Wharton jelly and/or viability and/or volume and surface area associated with the isolation procedure. and reproduction, as just a few features for illustrative purposes.

The preferred embodiments of the first aspect of the invention may be combined with the preferred aspects of the second aspect of the invention in each and in any combination, unless the context clearly dictates otherwise. Thus, the method according to the first aspect of the invention also determines, in some embodiments, the product directly obtained by this method. Thus, in some embodiments, the implementation of the cell according to the present invention can be described as a cell obtained by the method according to the first aspect of the invention. The features of the product, characterized by the method of its production, which are a consequence of the method according to the first aspect of the present invention, as a rule, can be combined with the features of the product according to the second and third aspects of the present invention.

The present invention also relates to a cell obtained by the method according to the second aspect of the invention. In this regard, the method according to the second aspect defines and characterizes the cell obtained by this method. Preferably, the mesenchymal stromal cell can be obtained by isolation and optionally, but preferably, propagation, as described herein.

Cell derived from mesenchymal stromal cell

In addition, due to its differentiation potential, the cell of the invention can be used to produce mature cells or cell lines. A mature cell is a cell that does not have stem cell-like properties. Differentiation of mesenchymal stromal cells into mature cells can be initiated by certain conditions, such as the addition of certain exogenous growth factors to the culture medium, see examples 12, 13 and 14 for illustration.

In one embodiment, the invention relates to a cell derived from the mesenchymal cell of the invention. In one embodiment, the derived cell is unipotent or terminally differentiated. In one embodiment, the derived cell is not immortal. Preferred derived cell types include those of the osteogenic lineage, the adipogenic lineage, and the chondrogenic lineage.

Cell population

In a third aspect, the invention relates to a population of cells (cell population). The cell population contains at least one cell according to the second aspect of the invention.

In one embodiment, the cell population of the invention is a cell population directly obtained by isolating cells from cord stromal Wharton's jelly as described herein. Most preferably, however, the cell population of the invention is a cell population obtained by ex vivo propagation of cells that have previously been isolated from umbilical cord stromal Wharton's jelly as described herein.

Some features and embodiments of the cell population of the present invention, described below, are associated with the physical characteristics of the cell population, in particular, the cells contained in the population. Solely by way of illustration, and notwithstanding the details disclosed elsewhere in this document, physical characteristics include those characteristics that can be directly determined by physical methods, such as gene expression, volume or surface area, shape, and so on, cells, ratio, and the distribution of any of these properties in a cell population. Some features and embodiments of the cell population, which are described below, are related to the way in which the cell population can be obtained or has been obtained. Solely by way of illustration, and notwithstanding the details disclosed elsewhere herein, cell population characteristics related to the manner in which the cell population can be or has been obtained may include (a) the anatomical origin of the cells, in particular the umbilical cord and Wharton's stromal jelly. as its preferred section, (b) the conditions used to isolate the cells, and (c) the conditions used to propagate the cells to obtain a cell population.

The cell population according to the invention contains a plurality of cells. Thus, a cell population contains at least two cells, but will typically contain much more than two cells. Although there is no clear upper limit on the number of cells contained in a cell population, a natural upper limit in a typical embodiment is the total number of cells produced in a full run, unless more than one umbilical cord is used as starting material. A full-scale cycle starting with a human umbilical cord, including step (c) and continuing until passage P2, typically results in at least 1x10 ⁹ cells, more preferably at least 2x10 ⁹ cells, more preferably at least 3x10 ⁹ cells, more preferably at least 4x10 ⁹ cells, more preferably at least 5x10 ⁹ cells, more preferably at least 6x10 ⁹ cells, more preferably at least 7x10 ⁹ cells, more preferably at least 8x10 ⁹ cells, more preferably at least 9x10 ⁹ cells, and in some embodiments at least 10x10 ⁹ cells. More cells can be obtained in later passages.

The cell population contains at least one cell expressing APCDD1. Typically, this can be confirmed by demonstrating that a sample of the cell population is positive for APCDD1 expression. In other words, the cell population is characterized by the expression of the APCDD1 gene. In one embodiment, the majority of cells in the cell population express the APCDD1 gene. Preferably at least 80% (by number of cells), preferably at least 90% (by number of cells), more preferably at least 95%, e.g. at least 96%, at least 97%, at least 98%, at least 99% (in each case by number of cells) of the cells contained in the cell population express APCDD1.

Optionally, the cell includes at least one additional cell, that is, one or more additional cells that differ from the cell according to the invention, in particular the cells according to the second aspect of the invention. In particular, in this embodiment, at least one additional cell differs in at least one feature, mandatory or optional (preferred), from the cell according to the second aspect of the invention. Preferably, however, at least one additional cell is syngeneic, more preferably autologous, to the cell of the invention.

In one embodiment, all cells of the cell population are MSCs. The cells of this embodiment can generally be obtained by culturing after the initial passage ("P0"). While, in some embodiments, cells originally isolated from the umbilical cord include MSCs as well as other cells (e.g., erythrocytes), there will be little or no other cells present after the P0 passage because (a) non-adherent cells are removed along with the growth medium at the end of P0, (b) the growth medium used is preferably particularly suitable for propagating MSCs, and (c) cells with proliferative properties inferior to those of MSCs will not substantially proliferate as well as MSCs, and thus over several cumulative population doublings, the cell population becomes enriched in MSCs.

In another embodiment, the cell population contains MSCs as well as non-MSCs. The cells of this embodiment can generally be obtained immediately after isolation, for example, before or during the initial passage ("P0"), and/or by combining at least one cell of the invention with at least one another cell.

Preferably, all cells in a cell population are from the same species. More preferably, all cells in the cell population are human cells. Preferably, all cells in the cell population are syngeneic to each other; more preferably, all cells contained in the cell population are autologous to each other. Even more preferably, all cells in a cell population are from the same individual. Such a cell population can be obtained using one umbilical cord, or part of it, as a starting material.

Preferably, the cell population does not contain MSCs derived from tissues other than the umbilical cord. More preferably, the cell population does not contain detectable amounts of MSCs derived from sections of the umbilical cord other than stromal Wharton's jelly. A cell population conforms to this feature when an endothelial cell-specific marker (eg, 3G5) and an epithelial cell-specific marker (eg, EpCAM) cannot be detected in the cell population.

Preferably, the cell population contains a plurality of cells, of which at least 80% (by number of cells), preferably at least 90% (by number of cells), more preferably at least 95%, for example, at least 96%, at least 97%, at least 98%, at least 99% (in each case by number of cells) are cells according to the second aspect. All preferred embodiments of the second aspect also apply to the third aspect of the invention. Preferably at least 70%, preferably at least 80%, more preferably at least 90%, such as at least 95%, at least 96%, at least 97%, at least 99%, or at least at least 99%, most preferably 100%, of the total number of cells are cells according to the second aspect and optionally one or more of its preferred embodiments, alone or in combination. In one embodiment, the cell population consists primarily of cells according to the second aspect. In one embodiment, the cell population consists solely of cells according to the second aspect.

Preferably, the population is an isolated population of cells. In one embodiment, the cell population is outside the human or animal body. An isolated cell population is typically a cell population outside of its physiological in vivo environment. Preferably, the cell population is an ex vivo cell population. In particular, the ex vivo cell population is preferably located outside the umbilical cord. In particular, the ex vivo cell population is preferably free of blood vessels. In one ex vivo embodiment, the cell population is in a culture vessel. In one ex vivo embodiment, the cell population is in a suitable storage container, such as a vial. In one embodiment, ex vivo, the cell population is at a temperature of about 35°C - 39°C, preferably about 36°C - 38°C, for example, about 37°C. In one non-mutually exclusive embodiment, the ex vivo cell population is in a culture vessel or in a suitable storage container. Preferably, a suitable storage container is suitable for transporting cells within the temperature ranges indicated above. In one ex vivo embodiment, the cell population is in a suitable cryopreservation container, such as a cryo vial, preferably at -180° C. or less. In one ex vivo embodiment, the cell population is in a culture vessel. In one embodiment, the ex vivo cell population is a metabolically active, preferably proliferating, cell population.

Some of the characteristics of a cell population can be described in terms of physical properties. To determine the physical properties, as a rule, you can take a sample of the cell population and determine the physical properties of all or part of the cells contained in the sample of the cell population. Since cell populations of the present invention are generally homogeneous and/or characterized by the fact that the individual cells that make them up are similar or very similar, results for specific properties such as, for example, gene expression, volume or surface area, the shape, and so on, of the cells contained in a sample will generally also apply to the rest of the cells in that population, unless the context clearly dictates otherwise and/or unless otherwise indicates that a particular cell population contains cells that are clearly genotypically and/or phenotypically different from each other. "Homogeneous" in this context means that substantially all of the cells are very similar or similar.

The cell population is preferably also characterized in that all cells have substantially uniform physical characteristics. "Substantially uniform" means that at least 90% of the cells, such as at least 91% of the cells, such as at least 92% of the cells, such as at least 93% of the cells, such as at least 94% of the cells, e.g., at least 95% of the cells, e.g., at least 96% of the cells, e.g., at least 97% of the cells, e.g., at least 98% of the cells, and preferably at least 91% of the cells, have one or more of the desired physical characteristics. It is particularly desirable if the cell volume, which correlates with the forward scatter (FSC) scores in the flow cytometry assay, as described above, is a physical characteristic that is substantially uniform across the cell population of the invention.

Preferably, the average volume of cells contained in the population of the present invention is less than the average volume of MSCs derived from SSBC. OSVS-derived MSCs can be obtained, for example, as described in the Comparative Examples herein. After determining the cell volume of an individual cell, the average cell volume can be determined arithmetically. Methods for determining cell volume are not particularly limited, although the FACS-based method, in particular detailed above for a single cell, is preferred.

The cell population is also preferably characterized in that the volumes and/or surface areas of the cells in the cell population are similar, or alternatively, in that a high percentage of cells have a low volume and/or surface area.

Preferably, the cell population of the invention is characterized by an average FSC value of 60 or less, or preferred values in the specified range described above for a single cell. For example, a cell population may have an average FSC value of 55 or less, such as 50 or less. In a specific embodiment, at least 90% of the cells in the cell population have an FSC value of 45 ± 10. In a more specific embodiment, the FSC value of all cells in the population is 45 ± 10. In a preferred embodiment, an average FSC value of at least 90% cells in the cell population is about 45. In a more preferred embodiment, the average FSC value of all cells in the population is about 45. Preferably, the cell of the invention has an FSC value that is less than the FSC value of the OCBC-derived cell.

Preferably, the average surface area of the cells in the cell population is not more than 3000 μm ² . Preferably, the cell has a surface area of not more than 2500 µm ² . Preferably, the cell has a surface area of not more than 2100 µm ² . In some embodiments, the implementation of the cell has a surface area of not more than 2000 μm ² . In a population of many cells, the average surface area is preferably within the range specified herein. In some embodiments, the average cell surface area in the cell population is preferably 1000-300 µm ² , preferably 1100-2700 µm ² , preferably 1200-2500 µm ² , preferably 1300-2200 µm ² , preferably 1400-2000 µm ² , preferably 1500- 1900 µm ² , more preferably 1600-1800 µm ² and most preferably about 1700 µm ² .

Preferably, the cell population is also characterized in that at least 70% of the cells (by cell number), preferably at least 80% (by cell number), preferably at least 90% (by cell number), more preferably at least 95%, eg at least 96%, at least 97%, at least 98%, at least 99% (in each case by cell number) are viable. The term "viable" refers to a cell that is not apoptotic. Thus, in turn, the cell population contains apoptotic cells in an amount of 30% or less of the total cells, preferably 20% or less of the total cells, more preferably 10% or less of the total cells, and most preferably 5% or less than the total number of cells. In one embodiment, the cell population contains no apoptotic cells at all. A suitable marker for apoptotic cells is 7AAD (see eg example 3). In one embodiment, viability is determined immediately after cell isolation, that is, before propagation. High viability, as a percentage, is an advantage compared to previously described MSC populations.

Preferably, the cell population is also characterized in that at least 60% of the cells (by cell number), at least 70% of the cells (by cell number), preferably at least 80% (by cell number), preferably at least 90 % (by cell number), more preferably at least 95%, e.g. at least 96%, at least 97%, at least 98%, at least 99% (in each case by cell number) are multipotent .

In one embodiment, the cell population contains mast cells in an amount of 2% or less of the total number of cells, preferably 1% or less of the total number of cells. Most preferably, the cell population does not contain mast cells.

In one embodiment, the cell population contains fibroblasts in an amount of 2% or less of the total number of cells, preferably 1% or less of the total number of cells. Most preferably, the cell population does not contain fibroblasts. In this embodiment, the term "fibroblasts" should be understood in a narrow sense, as referring to cells that have already differentiated into fibroblasts, and not referring to cells that are multipotent and simply have the ability to differentiate into fibroblasts.

In one embodiment, the cell population contains cells of the hematopoietic line of differentiation in an amount of 2% or less of the total number of cells, preferably 1% or less of the total number of cells. Most preferably, the cell population does not contain cells of the hematopoietic lineage. In a preferred embodiment, the cells do not include blood cells, such as cord blood cells. Several markers are known for blood cells and thus compliance with this preferred embodiment can be easily tested.

In one embodiment, the cell population contains perivascular cells in an amount of 2% or less of the total number of cells, preferably 1% or less of the total number of cells. Most preferably, the cell population does not contain perivascular cells. The marker for perivascular cells is 3G5. Thus, this embodiment may alternatively be characterized in that the cell population does not contain cells that express the 3G5 marker and/or cells that exhibit 3G5 on their surface. In one embodiment, the cell population is not contaminated with perivascular cells. 3G5 expression has also been described for bone marrow-derived MSCs (Khan et al., J. Orthop. Res., 2010, vol. 28, p. 834-40). Thus, the absence of 3G5 expression may also be a suitable feature to distinguish the cell population of the present invention from bone marrow-derived MSC populations. In one embodiment, the cell population is not contaminated with perivascular cells. In one embodiment, the cell population does not contain bone marrow-derived MSCs.

In one embodiment, the cell population contains epithelial cells in an amount of 2% or less of the total number of cells, preferably 1% or less of the total number of cells. Most preferably, the cell population does not contain epithelial cells. The marker for epithelial cells is the epithelial cell adhesion molecule (EpCAM). Thus, this embodiment may alternatively be characterized in that the cell population does not contain cells that express the EpCAM marker and/or cells that exhibit EpCAM on their surface. In one embodiment, the cell population is not contaminated with epithelial cells.

In one embodiment, the cell population contains endothelial cells in an amount of 2% or less of the total number of cells, preferably 1% or less of the total number of cells. Most preferably, the cell population does not contain endothelial cells. The endothelial cell marker is CD31. Thus, this embodiment may alternatively be characterized in that the cell population does not contain cells that express the CD31 marker and/or that exhibit CD31 on their surface. In one embodiment, the cell population is not contaminated with endothelial cells.

In one embodiment, the cell population contains erythrocytes in an amount of 2% or less of the total number of cells, preferably 1% or less of the total number of cells. Most preferably, the cell population does not contain erythrocytes. RBC markers are globin and BGP1. Thus, this embodiment may alternatively be characterized in that the cell population does not contain cells that express BGP1 and/or globin. In one embodiment, the cell population is not contaminated with erythrocytes.

Preferably less than 10% of the cells contained in the cell population exhibit telomerase activity, more preferably less than 5% of the cells contained in the cell population exhibit telomerase activity, and most preferably less than 2.5% of the cells contained in the cell population exhibit telomerase activity. The definition of telomerase activity and aspects related to it are described above and are also applicable to this third aspect of the invention. The respective cell populations can be distinguished from other cell populations previously described (eg US 2004/0136967 A1).

In one embodiment, the cell population is also characterized in that none of the cells it contains, or more particularly and preferably none of the MSCs it contains, exhibit MHC II on its surface, or express MHC II. Thus, preferably, none of the MSCs of the cell population expresses MHC II. In one embodiment, the expression of MHC II may be a suitable feature to distinguish the cell population of the present invention from other cell populations containing MSCs (see, for example, Sarugaser et al., 2005, Stem Cells, vol. 23, p. 220-229) .

Preferably, the cell population can be obtained by the method according to the first aspect of the invention, in particular, with the inclusion of step (c), that is, the step of reproduction. Thus, the method of the present invention, including step (c), can result in a population of mesenchymal stromal cells. The features of the product, characterized by the method of its production, which are a consequence of the method according to the first aspect of the present invention, as a rule, can be combined with the features of the product according to the second and third aspects of the present invention. Alternatively or additionally, the cell population of the present invention may thus be described as the cell population obtainable by the method of the first aspect of the invention comprising step (c).

Preferably, the stromal cell population of the present invention is substantially free of contaminants. A substantially contaminant-free stromal cell population is defined herein as a cell population substantially free of infectious agents such as viruses (encapsulated and unencapsulated), parasites, bacteria, and endotoxins, i.e., a cell population containing such infectious agents. agents in amounts that are not detectable by culture, serology, or molecular identification systems, or by the LAL test (endotoxins).

Preferably, the cell population of the invention is substantially free of antibiotics. A cell population substantially free of antibiotics can be obtained by growing the cells for at least one passage without antibiotics. For example, even if antibiotics are present in the first passage after cell isolation ("P0"), antibiotics are preferably stopped for all passages after P0; in other words, for example, growth medium MSCBM CD with 5% hTL can be used without the addition of antibiotics at P1 and all subsequent passages.

In one ex vivo embodiment, the cell population is frozen. Frozen cells are generally stable when stored at temperatures of -180° C. or less, although any conditions suitable for cell storage are suitable in this embodiment. Cells may be frozen, preferably flash frozen in liquid nitrogen. A cryoprotectant may be added before freezing. In one embodiment, the cell population is stored in liquid nitrogen. In a preferred embodiment, the frozen cell population is a cell bank. This embodiment is described in more detail below.

cell bank

As shown, for example, in example 6, the cells of the present invention are consistently characterized by a certain phenotype in different allocations from individual umbilical cords, and retain a certain phenotype over several cumulative population doublings. Thus, cells obtained according to the invention, or otherwise related to the present invention, consistently have a certain phenotype and retain this phenotype when propagated over many passages. Thus, the cells of the invention are particularly suitable for establishing a cell bank.

Indeed, preferably, the cell population of the present invention is a cell bank. Thus, the cell population of the invention may be in the form of a cell bank. The cell bank contains SHS-derived cells. Unless specifically stated otherwise, the cell population included in the cell bank, as a whole, corresponds to the cell population described above. Preferably, the cell bank contains a plurality of cells, of which at least 70%, preferably at least 80%, more preferably at least 90%, such as at least 95%, at least 96%, at least 97% , at least 99% or at least 99%, most preferably 100%, of the total number of cells are cells described in the second aspect of the invention, and optionally one or more of its preferred embodiments, alone or in combination.

The cell bank of the invention may be prepared by a method comprising propagating the SHS-derived cells of the invention. Alternatively, a cell bank of freshly isolated cells (non-expanded cells) can be maintained, from which expanded cells can optionally be obtained at a later stage. Preferably, however, the cell bank contains expanded cells, that is, cells obtained by the process of the invention comprising step (c).

The cell bank preferably contains non-adherent cells. Such a cell bank can be prepared in a manner involving separation with an enzyme, for example by trypsinizing the adhering cells prior to the creation of the cell bank. Enzymatic separation of cells can be performed by conventional methods.

In one embodiment, a cell bank is obtained after cells have been grown for 5-30 cumulative population doublings (excluding cumulative population doublings in the P0 process), e.g., 8-25 cumulative population doublings (excluding cumulative population doublings in the P0 process). ).

The cell bank is maintained such that some or all of the cells can be selected from the cell bank and/or used, eg for subsequent culturing, at a desired point in time, eg at a point in time in the future.

When a cell population is "maintained", this can be done under any conditions that do not interfere with future culturing of the cell population; cell populations can be maintained in frozen and non-frozen form; for the cell bank of the invention, however, keeping in frozen form is preferred.

Preferably, the cell bank of the present invention is a frozen cell bank. Frozen cells are generally stable when stored at temperatures of -180° C. or less, although any conditions suitable for cell storage are suitable in this embodiment. Cells may be frozen, preferably flash frozen in liquid nitrogen. A cryoprotectant may be added before freezing. Cells can be stored under any suitable conditions, although freezing at temperatures of -180°C or less is most typical.

A cell bank typically contains cells in multiple individual aliquots, such as multiple individual vials. Preferably, each vial contains a cell suspension. Any liquid suitable for suspending mesenchymal stromal cells, in particular a growth medium, and preferably a growth medium that is suitable for the present invention, is suitable for resuspension of the cells of the present invention. In a preferred embodiment, the cell bank contains a liquid containing a minimal mesenchymal stromal cell culture medium, such as, for example, MSCBM CD, or a minimal medium substantially equivalent thereto. Preferably, the liquid in which the cells of the cell bank are suspended also contains a platelet lysate, for example 5% (v/v) platelet lysate (TL), and in the case of human cells, human platelet lysate (hTL).

Cell banks, or aliquots thereof, can be used in the future, for example, for subculture inoculation, for example, in a production bioreactor.

Exemplary embodiments of the cell bank of the invention include a primary cell bank (PCB) and a secondary cell bank (SCB). Thus, the cell bank of the invention is preferably selected from a primary cell bank (PCB) and a secondary cell bank (SCB).

In the broadest sense, the term "primary cell bank" refers to the first cell bank, more preferably the first frozen cell bank, created after the isolation of cells from the umbilical cord.

In one embodiment, a primary cell bank is established after the cells have grown for 5-14 cumulative population doublings (not counting the cumulative population doublings in the P0 process).

Primary cell bank (PCB) is usually in aliquots. Aliquots are prepared after the appropriate pre-passage (eg P2) and enzymatic separation of the cells, eg by trypsinization, centrifugation and resuspension. Preferably, the cells for the primary cell bank are divided into aliquots of 100-300 vials, eg 150-200 vials, eg 160-180 vials, or more specifically 165-175 vials.

Preferably, each primary cell bank vial contains from 1x10 ⁶ cells to 1x10 ⁷ cells, preferably 5x10 ⁶ cells to 500x10 ⁶ cells, preferably from 10x10 ⁶ cells to 100x10 ⁶ cells, preferably from 15x10 ⁶ cells to 50x10 ⁶ cells, preferably 18x10 ⁶ cells to 30x10 ⁶ cells, for example about 20x10 ⁶ cells.

Preferably, the volume contained in each vial is 1-100 ml, eg 2-20 ml, preferably 3-10 ml, and most preferably about 4 ml.

In one exemplary embodiment, the primary cell bank obtained after a full-scale cycle consists of 150-200 vials, each containing 20×10 ⁶ cells, and each containing 4 ml of (frozen) fluid containing cells.

It is also possible to place the cell suspension intended to create a secondary cell bank in vials or containers of different sizes or containing different numbers of cells or cells at different concentrations. However, the above quantities are indicative of what can generally be obtained in a full scale run and can be converted to vials or containers of different sizes or containing different numbers of cells or cells at different concentrations.

In its broadest sense, the term "secondary cell bank" refers to a second cell bank, more preferably a second frozen cell bank, created after cell isolation from the umbilical cord; in other words, to the first cell bank, more preferably the first frozen cell bank, created from all or part of the cells contained in the primary cell bank.

In one embodiment, a secondary cell bank is established after the cells have grown for 14-25 cumulative population doublings (excluding cumulative population doublings in the P0 process).

Since a primary cell bank typically contains multiple aliquots of cells that can be used at the same or different time points for further culturing, multiple secondary cell banks can be created, simultaneously or sequentially, from a single primary cell bank.

For example, one secondary cell bank can be generated from each vial in a primary cell bank. For example, starting from one primary cell bank, 100-300 secondary cell banks, e.g. 150-200 secondary cell banks, e.g. 160-180 secondary cell banks, or more specifically 165-175 secondary cell banks can be created.

Secondary Cell Bank (SCB) is usually in aliquots. Aliquots are prepared after the appropriate pre-passage (eg P4) and enzymatic separation of the cells, eg by trypsinization, centrifugation and resuspension. Preferably, cells for secondary cell banking are aliquoted into 100-300 vials, eg 150-200 vials, eg 160-180 vials, or more specifically 165-175 vials.

Preferably, each secondary cell bank vial contains 1x10 ⁶ cells to 1x10 ⁷ cells, preferably 5x10 ⁶ cells to 500x10 ⁶ cells, preferably 10x10 ⁶ cells to 100x10 ⁶ cells, preferably 15x10 ⁶ cells to 50x10 ⁶ cells, preferably 18x10 ⁶ cells to 30x10 ⁶ cells, eg about 20x10 ⁶ cells.

Preferably, the volume contained in each secondary cell bank vial is 1-100 ml, eg 2-20 ml, preferably 3-10 ml, and most preferably about 4 ml.

It is also possible to place the cell suspension intended to create a secondary cell bank in vials or containers of different sizes or containing different numbers of cells or cells at different concentrations. However, the above quantities are indicative of what can generally be obtained in a full scale run and can be converted to vials or containers of different sizes or containing different numbers of cells or cells at different concentrations.

Industrial Applicability

Mammalian cells with proliferative properties and associated cell populations are of value to non-commercial and commercial activities such as research. The cells of the present invention are particularly attractive for such purposes, in particular because of the good reproducibility of the method, excellent proliferative properties and high cell yield, among other things. The cell populations of the invention may be suitable for the production of cell products, for example for research or commercial use. For example, a primary cell bank or a secondary cell bank can be produced commercially and used commercially, such a cell bank, or more specifically individual aliquots thereof, and sold for use in research such as, for example, in vitro cell assays.

In addition, it is possible to apply the whole method, or part thereof, according to the invention in automatic mode, using devices, including robots, to carry out one or more steps of the method. Thus, the method according to the invention can also find industrial application.

In some embodiments, the cells of the invention can be cultured and propagated in vitro, for example, for research or production purposes. For example, the cell of the invention, or cells derived from it, can be used to screen for efficacy and/or cytotoxicity and/or the mechanism of action of pharmaceutical compounds and compositions, and/or to study the mechanism of certain diseases, and/or for in vitro transmission genes, and/or for the production of biomolecules such as proteins, including therapeutically active proteins.

The cells of the invention can be cultured as a pure culture, or co-cultured with other cell lines, primary or secondary.

In addition, due to their differentiation potential, the cells can be used to produce mature cells or cell lines. Differentiation of mesenchymal stromal cells into mature cells can be initiated using certain conditions, such as the addition of certain exogenous growth factors to the culture medium.

The following examples and figures are for illustrative purposes only and are not intended to limit the invention as defined by the appended claims.

EXAMPLES

Example 0: Materials and Methods Common to Various Aspects of the Invention

In all experimental examples, unless otherwise indicated, MSCBM CD or a minimal environment nearly equivalent is used. Exemplary environments are described below.

MSCBM CD Minimal Medium and Growth Medium Containing It

The MSCBM CD minimal media is commercially available from Lonza (Wakersville, MD, USA; cat. no. 95062-688). According to the manufacturer, it is a serum-free medium optimized for multi-passage expansion of all types of human mesenchymal stem cells, supporting multilineage differentiation, and requiring no attachment matrix for cell seeding. Minimal environments almost equivalent to MSCBM CD are also suitable.

"MSCBM CD/GA" is prepared as follows:

- 500 ml MSCBM CD (Lonza)

- 0.5 ml GA1000 (Lonza; according to the manufacturer, GA1000 contains 30 mg/ml gentamicin and 15 µg/ml amphotericin)

"MSCBM CD/hTL" - unless otherwise specified in amounts or ratios - contains 5% (v/v) human platelet lysate (hTL) and 2 U/ml heparin, both of which are added to MSCBM CD (i.e., no antibiotics) gentamicin and amphotericin). hTL is preferably prepared from a composition containing from 3.8×10 ⁸ platelets/ml to 1.2×10 ⁹ platelets/ml, averaging about 7.9×10 ⁸ platelets/ml); such a platelet lysate can be obtained, for example, from the blood bank of a polyclinic in Modena, Italy, or from any other supplier.

"MSCBM CD/GA/hTL" - unless otherwise specified in amounts or ratios - contains 5% (v/v) human platelet lysate (hTL) and 2 U/ml heparin, both of which are added to MSCBM CD/GA (then yes, with antibiotics gentamicin and amphotericin).

Growth factors (other than those ultimately found in hTL and/or minimal medium) are not added unless specifically stated otherwise.

The above media are particularly suitable for propagating the cells of the present invention.

Cell count

To determine the number of cells, unless otherwise indicated, cells are counted using automatic cell counters, such as, in particular, NucleoCounter ^® . If necessary, the cells are separated with the help of an enzyme, for example by treatment with trypsin, before counting the cells. If erythrocyte count is not desired, cell count is performed after the cell population has been treated with ammonium chloride; such treatment is known to result in lysis of erythrocytes. As a result, it is possible to determine the total number of cells in the population, excluding erythrocytes. For this purpose, an ammonium chloride solution recommended for erythrocyte lysis is used, which is commercially available from many vendors (eg, STEMCELL Technologies). Such a solution may be added and used in accordance with the manufacturer's instructions.

Example 1: Segmentation and division into sections of the umbilical cord, and obtaining crushed stromal Wharton's jelly

A human umbilical cord is obtained after the birth of a child at term. The umbilical cord is used if its total weight is 40 g or more. The umbilical cord is segmented by cutting it in a perpendicular direction into segments about 2.5 cm long using a sharp sterile scalpel. Flush the umbilical cord segments with HEPES-buffered saline (HBSS) + 300 µg/ml gentamicin and 0.15 µg/ml amphotericin B for a total of 10 minutes. Shake mechanically to flush any remaining blood from the tissue.

From each segment obtained in this way, continue to obtain a section of stromal Wharton's jelly as follows: cut the umbilical cord in the longitudinal direction, remove the vessels with the matrix surrounding them, as a rule, at a distance of 3 mm (vascular-perivascular zone, also called vascular-perivascular Wharton's jelly - OSVS) and discarded. Removal should be done with care in the sense that vascular rupture and/or loss of vascular or perivascular cells should be avoided. Avoid pulling the umbilical cord matrix.

Prior to harvesting the matrix tissue, visually inspect each tissue segment to ensure that the RBC has been completely removed. Neither vessels nor vessel fragments should remain in the segment.

After removing the SSBC from the umbilical cord segments, cut pieces of matrix tissue from the umbilical cord segments using sharp scissors. The inner surface of the umbilical cord segments is scraped with a sharp scalpel to obtain the remaining matrix tissue. The thin amniotic epithelial membrane remaining after scraping is very carefully removed and discarded. The subamniotic (or mesenchymal) layer of the lining of the umbilical cord is included in the BC, which is isolated. This is achieved by carefully scraping tissue from the amniotic epithelium; it is important to be careful not to include pieces of the amniotic epithelial membrane when collecting said tissue. The tissue thus obtained is pure Whartonian stromal jelly (SHS).

Carefully grind the thus obtained (trimmed and scraped) SHS with sharp scissors and/or a scalpel. Add HEPES buffered saline (HBSS) to ground tissue. Combine the ground tissue in a Petri dish.

When extracting CBC, precautions are taken to avoid extraction of blood PC cells, epithelial or endothelial cells, and cells from vascular structures. As a control (optional, but recommended, especially when playing this example for the first time), the marker for endothelial cells is CD31; the marker for epithelial cells is the epithelial cell adhesion molecule (EpCAM), the marker for perivascular cells is 3G5.

The steps of Example 1 are shown in Figure 3.

The method of the present invention as described in Example 1 results in pure Wharton's jelly by completely removing and eliminating non-IC components of the umbilical cord (such as cord lining/amniotic membrane, vessels and blood) from sections, avoiding extraction of blood PK cells, epithelial or endothelial cells and cells from vascular structures. It allows you to get a clean tissue of Wharton's jelly (BC tissue). Due to its origin, this particular VS tissue is a stromal VS tissue.

Example 2A: Enzymatic Treatment

Materials and Methods: BC tissue prepared as described in Example 1 was used to isolate BC-derived primary cells as follows: ground tissue/HBSS mixture was suspended in digestion buffer (see Table) at a ratio of 100 mg of ground tissue/HBSS mixture per 1 ml of digestion buffer (see Table below) in a 250 ml conical tube. The digestion buffer contains an enzyme suitable for releasing MSCs from isolated tissue.

Table: Formulation of 100 ml of digestion buffer (all ingredients are sterile)

- 1 ml hTL, for example, from the blood bank of the polyclinic in Modena, Italy (obtained from a composition containing from 3.8×10 ⁸ platelets/ml to 1.2×10 ⁹ platelets/ml, averaging about 7.9×10 ⁸ platelets/ml)

- heparin up to a final concentration of 2 U / ml

- 1 ml sodium pyruvate (from 100 mM stock)

- 36 µl 1M CaCl ₂

- NB4 or NB6 collagenase preparation (both from SERVA) to a final activity of 0.18 Wunsch units/ml digestion buffer

- minimum medium (commercial mammalian cell growth medium such as DMEM or MSCBM CD) - up to 100 ml

A collagenase preparation from SERVA (NB4, NB6), typically NB4, was used. According to the manufacturer, the NB4/NB6 Collagenase preparation is a natural product derived from a collagenase producing strain of Clostridium histolyticum and is a collagenase preparation that is non-toxic and contains collagenase and other proteases. According to the manufacturer, standard grade NB4 (17454.02 and 17454.01 (both from SERVA)) is designed to dissociate different tissues to isolate different cell types, e.g. chondrocytes, muscle cells, epithelial cells, skin fibroblasts, hepatocytes, adipocytes, which use for research purposes. According to the manufacturer, collagenase NB4 can also be used as a cheaper alternative to collagenase NB6 in method development. According to the manufacturer, NB6 GMP grade collagenase (NB6 GMP grade, 17454.02 (SERVA)) is specifically designed to isolate cells for some specific transplant cases. All experiments described herein used NB4 unless specifically stated otherwise. According to information from SERVA, the manufacturer of the collagenase preparation used in this experiment, SERVA reports collagenase activity in Wunsch PZ units (PZ-U) (synonymous with "Wunsch units" herein).

Heparin is known to be a suitable additive for liquid compositions containing platelet lysate to avoid sticking (eg, Lohmann et al., 2012, PLoS ONE, vol. 7e37839).

Cleavage buffers with higher collagenase activity (final concentration above 0.18 Wunsch U/mL) were also initially tested based on collagenase activity previously described for enzymatic digestion of adipose tissue, however digestion in the presence of higher collagenase activity was not beneficial. (data not shown).

The crushed tissue (example 1) was placed in sterile tubes. Digestion buffer was added (see above). These tubes containing ground tissue and digestion buffer (hydrolysate) were incubated at 37±1° C. for a digestion time interval of 3 hours with constant gentle agitation at 75 rpm on an orbital digestion shaker. "Hydrolyzate" means the combination of degradable tissue and degradable buffer.

At the end of the digestion time interval, the hydrolyzate was diluted by adding 2 volumes of MSCBM CD containing 1% (v/v) human platelet lysate (hTL) and passed through two layers of one sterile gauze pad to separate from non-cleaved tissue/partially digested tissue. The filtrate was called the "split mixture".

The digested mixture was centrifuged at 680 x g for 15 minutes in 250 ml centrifuge tubes. At the end of centrifugation, the supernatant was removed and the pellet (containing freshly isolated mesenchymal stromal cells from umbilical cord Wharton's jelly) was resuspended in 1-2 ml MSCBM CD/GA/hTL (see example 0).

Then the total number of cells was determined. Most often, not all of the cells contained in the digested mixture are MSCs.

Example 2B: First culture (P0 and optionally P0+)

The resuspended cells from Example 2A were plated in T-225 culture flasks ( ^Falcon® , brand of Corning, Corning, NY, USA) in MSCBM CD/GA with 5% hTL (see Example 0; 50 ml medium per T-225 flask) for initiation of the first in vitro culture of cells derived from stromal Wharton's jelly. This cell culture is called "P0". The day the P0 culture was initiated is called "day 0" and the days are counted in ascending order. The cell density at the beginning of P0 is 8000 cells/cm ² or more. The ^cm2 dimension refers to the bottom surface of the vial covered with growth medium during cell culture in P0.

Feed the cells every 3 days starting from day 5 with fresh MSCBM CD/GA/hTL medium. Alternatively, it is also possible to culture the cells in the P0 process in bags instead of flasks: in this case, use 150 ml of MSCBM CD/GA/hTL per 1x1 bag to initiate the first cell culture; however, unless specifically stated otherwise, P0 cultures are performed in T-225 culture flasks.

Cells are cultured in P0 as described until 80-90% cell confluence is reached. This usually happens after 5-12 days. The cells are then treated with trypsin. Cells are counted after trypsin treatment at the end of the passage.

The following acceptance criteria are evaluated: 1 - yield, 2 - viability, 3 - confluence, 4 - morphology. Determination of acceptance criteria 1 and 2 produced after treatment of cells with trypsin. Details are given below.

Acceptance criterion After P0 and P0+ Test 1 Exit At least 8 million cells Cell count after trypsin treatment 2 Viability More than 80% viable cells 7-amino-actinomycin D (7-AAD) staining 3 Confluence At least 80%, preferably 80-90% confluence Determination under a microscope 4 Morphology Most cells are small, fusiform and in distinct colonies Determination under a microscope

In this experimental example, all four acceptance criteria must be met. In addition, when inspecting morphology, it is also visually checked whether the sizes and shapes of the cells are relatively uniform. Preferably, checking confirms this.

In the event that any one or more of criteria 2, 3 or 4 is not met, the entire cell culture is discarded. The inventors of the present invention have found that criteria 2, 3 and 4 are generally met, and therefore discarding is not necessary. However, the ability to discard a culture if the criteria are not met ensures that subsequent passages are only carried out if the acceptance criteria are met.

In the event that the total number of cells after P0 exceeds 8 million cells (provided that criteria 2, 3 and 4 are also met), continue as in example 2B.

In the event that the total number of cells after P0 does not exceed 8 million cells (however, subject to criteria 2, 3 and 4), an additional subculture step called "P0+" or "P0 plus" is carried out. (For the avoidance of doubt, the text of this document specifically states if some cells are subcultured in the "P0+" step; in other words, in the absence of such a specific indication, cells are transferred from P0 directly to P1 (Example 2B)). The P0 conditions are exactly the same as the P0 conditions described above, except that the resuspended cells are subjected to P0+ after P0, not directly from Example 2A.

Cells are cultured in P0+ until reaching 80-90% cell confluence. The cells are then treated with trypsin. The above acceptance criteria are evaluated: 1 - yield, 2 - viability, 3 - confluence, 4 - morphology. If all criteria are met, continue as in Example 2B. If one or more of the criteria are not met (with the exception of exit), the cells are discarded. In particular, cells are discarded if the total number of cells after P0+ is not at least 8 million cells.

The total cell numbers in this example are based on whole human umbilical cord as starting material (full scale cycle). If the amount of starting material is different (eg, only half of the human umbilical cord), then the total number of cells for acceptance criterion 1 is arithmetically adjusted accordingly.

Depending, in particular, on cell yield, which varies to some extent in different full-scale cycles, and also due to variations in biological material from different individuals, the cumulative population doublings in the P0 process are also subject to variation.

Example 2C: Further propagation (P1 and subsequent passages)

Trypsin-treated cells from P0 (or P0+, but only if specifically indicated) in Example 2B described herein are prepared and subcultured for one or more passages as follows.

The first subculture is called "P1". After completion of P1, the cells are again trypsinized and subcultured. This subculture/trypsin cycle is preferably performed for 4 passages (P1 is considered the "first passage"), although in some cases it is performed for up to 12 passages. Each passage is numbered sequentially, that is, P1 follows P0, P2 follows P1, and so on. Unless specifically stated otherwise, cells are grown and subcultured to passage 4 (P4).

For each passage from P1 onwards, all culture plates have the same defined size. Specifically, for each passage, the culture vessel consisted of a stack of CS5, CS10, or CS20 culture plates (Corning, Corning, NY, USA). For each passage from P1 onwards, cells are grown in MSCBM CD with 5% hTL without antibiotics (see example 0; 50 ml MSCBM CD/hTL per T-225 vial). Unless specifically stated otherwise, antibiotics are stopped in this example at all passages after P0; in other words, MSCBM CD/hTL is used in this example without the addition of antibiotics at P1 and all subsequent passages.

The cell density at the beginning of each passage is 2500 cells/cm ² .

After each passage, the cells are treated with trypsin. Cells are counted at the end of each passage after trypsin treatment.

Based on the cell density at the time of inoculation, the bottom surface of the cell culture vessel (which collectively determines the total number of cells at the beginning of the passage), and the total number of cells after the end of the passage, the number of cumulative population doublings can be arithmetically determined.

In the experience of the inventors, the number of cumulative population doublings during P1 and P2 (collectively P1 plus P2, but not including P0) is most often typically in the range of 9-13.

In the experience of the inventors, the number of cumulative population doublings during P3 and P4 (collectively P3 plus P4, but not including P0, P1 and P2), most often, as a rule, is in the range of 6-10. Therefore, the number of cumulative population doublings during P1, P2, P3, and P4 (collectively P1 plus P2 plus P3 plus P4, but not including P0) is most often typically in the range of 15-23.

Optionally - unless specifically stated herein - cells are frozen after a specific passage, eg P2. Cells can be frozen by standard methods, such as flash freezing in liquid nitrogen. It is quite possible to thaw such frozen cells and, for example, subject them to cultivation in subsequent passages after thawing. Cells can be frozen either as a whole batch or as aliquots. Freezing in aliquots, preferably in vials, is more preferred and was generally used in this example unless specifically noted otherwise.

When cells are frozen and thawed after a certain passage, the passage numbering continues after thawing. For example, if cells are frozen after P2, the next passage after thawing becomes P3. Preferably, unless otherwise indicated, counting the number of cumulative population doublings continues after thawing.

In a preferred embodiment, cells are aliquoted and frozen after P2. The frozen aliquots thus obtained are available for thawing individually at the desired time point. Frozen cells, preferably in aliquots, may be referred to as a primary cell bank (PCB). In other words, it is not required - and generally undesirable - to thaw all PCB aliquots at the same time. In contrast, aliquoting and individually freezing aliquots has the advantage that individual aliquots can be recovered after freezing, thawed and further cultured (subcultured, further expanded) at separate, and desired, time points.

In a specific example of a full scale run, 168 flasks (each containing 20×10 ⁶ cells in a 4 ml volume) were obtained after P2, starting with one human umbilical cord as starting material. Each of the vials can be frozen and individual vials can be thawed at separate time points for further subculture, eg P3 onwards. In one specific example of a full run, one vial obtained and frozen after P2 is thawed and the cells contained therein are subjected to P3 and P4. In this particular example, after P4, 168 vials were obtained (each containing 20×10 ⁶ cells in a volume of 4 ml).

Cells are cultured at each passage as described until 80-90% cell confluency is reached. This usually happens after about 3-4 days. The following acceptance criteria are then preferably evaluated: 1 - yield, 2 - viability, 3 - confluence, 4 - morphology. Determination of acceptance criteria 1 and 2 produced after treatment of cells with trypsin. Details are given below.

Acceptance criterion After P1 and all passages following P1 Test 1 Exit (full-scale cycle) After P1: at least 225×10 ⁶ cells Cell count after trypsin treatment After P2: at least 7×10 ⁹ cells (*) After P3: at least 250×10 ⁶ cells (*) After P4: at least 7×10 ⁹ cells (*) 2 Viability More than 80% viable cells 7-amino-actinomycin D (7-AAD) staining 3 Confluence At least 80%, preferably 80-90% confluence Determination under a microscope 4 Morphology Most cells are small, fusiform and in distinct colonies Determination under a microscope

(*) After P2, cells can be frozen in aliquots to create a primary cell bank (PCB). The number of cells for P3 and P4 are given based on the scenario where P3 is started with a single flask of PCB that contains 20×10 ⁶ cells.

It is important to note that the cell yields achieved by propagation according to the present invention are very high compared to previously known methods for obtaining and expanding mesenchymal stem cells.

In this example, all four acceptance criteria must be met: if one of the criteria is not met, the entire cell culture from the corresponding passage is discarded. The inventors of the present invention have found that all of criteria 1, 2, 3 and 4 are generally met and therefore no discard is necessary. However, the ability to discard a culture if the criteria are not met ensures that subsequent passages are only carried out if the acceptance criteria are met.

In summary, Example 2 shows that Wharton's stromal jelly contains competent cells for reproduction. Cells from Wharton's stromal jelly have been identified as a convenient source of potentially multipotent stromal cells that can be maintained and expanded in culture.

Comparative examples

For comparison purposes, cells were isolated as described in WO 2004/072273 A and Sarugaser et al., 2005, Stem Cells, vol. 23, p. 220-229, in particular as described in the following comparative examples 1, 2A, 2B and 2C.

Comparative Example 1

Vessels were isolated from the human umbilical cord. The cut vessels were sutured to create a loop blocking the flow of fluid into or out of the vessel.

Comparative Example 2A

The loop (comparative example 1) was immediately placed in a 50 ml tube containing a 1 mg/ml solution of Sigma C-0130 collagenase in PBS (without Mg ²⁺ and Ca ²⁺ ) at 37°C. According to the literature, Sigma C-0130 collagenase has a specific activity of 0.25-1.0 FALGPA units/mg solid. For information, FALGPA units cannot be directly arithmetically converted to Wunsch units because the corresponding units refer to different assays with different substrates; however, there is some general agreement in the art as to how the Wunsch units relate to the FLGPA units. Based on this general opinion, it can be concluded that the total activity of the collagenase used in Comparative Example 2 is much higher than the total activity of the collagenase used in Example 2.

After 18-20 hours of incubation with collagenase, digestion in solution was stopped by adding 2 volumes of PBS and 1 ml of fetal bovine serum (FBS). Thus, the total duration of collagenase treatment in Comparative Example 2 is much longer than the total duration of collagenase treatment used in Example 2. Cleaved samples were filtered to remove undigested tissue and centrifuged at 1800 rpm (Beckham Coulter Allegra™ 25R) for 15 minutes . Cells were resuspended in 75% α-MEM medium, 15% defined EBS, 10% antibiotics. Blood cells were lysed with a solution of ammonium chloride (NH ₄ Cl), potassium bicarbonate (KHCO ₃ ) and EDTA at a ratio of 1:3 v/v with cell suspension for 5-10 minutes on ice with gentle stirring. After centrifugation at 500 xg for 10 minutes, cells were resuspended and counted with trypan blue dye (0.4%).

Comparative example 2B

Perivascular Wharton's jelly (PWJ) derived cells, obtained as described in Comparative Example 2A, were seeded at a density of 6000-8000 cells/cm ² and incubated at 37° C. in α-MEM (nucleoside-free, Life Technologies, Waltham, MA, USA, no. 22561-021) supplemented with 15% defined EBS (HyClone, GE Healthcare, USA, no. SH30070.03) and antibiotics in a 5% CO ₂ , 95% humidity incubator, initiating a "P0" culture.

The medium was replaced for the first time after 5-6 days, then renewed every 2 days. After 6-10 days of cultivation, the cells reached confluence. At the end of P0, after 6-10 days, the cells reached confluence. Then they were treated with trypsin. The total number of cells was determined using trypan blue dye (0.4%).

Comparative example 2C

The trypsin-treated cells obtained in Comparative Example 1B were obtained and subcultured as follows. From culture passage 1 (“P1”) onwards, cells were seeded at a density of 3500-4000 cells/cm ² in α-MEM with 15% defined FBS. Antibiotics should be absent after P0; in other words, the growth medium is used without the addition of antibiotics. The cells are referred to as perivascular Wharton's jelly-derived cells ("PRCC"). Perivascular Wharton's jelly (PVC)-derived mesenchymal stromal cells may also be referred to as "perivascular-derived mesenchymal stromal cells". Unless specifically stated otherwise, cells were grown and subcultured to passage 4 (P4).

Optionally - but only when specifically indicated - cells were frozen after a certain passage, eg P2. It is quite possible to thaw (thaw) such frozen cells and, for example, subject them to cultivation in subsequent passages after thawing.

Example 3 Analysis of cells after digestion

Cells obtained as described in Example 2A (ie, after the digestion step, with or without the addition of platelet lysate (PL)), are referred to as stromal Wharton's jelly-derived cells, or "SBC". They are compared to SSBC-derived control cells (“SSBC”, Comparative Example 2A). SBC and OCBC were stained with 7-amino-actinomycin D (7AAD) (Via Probe; BD Biosciences; used according to manufacturer's instructions) to detect apoptosis in a FACS assay. The unstained sample was used to detect the intrinsic fluorescence of the sample and photomultiplier tubes (PMT; FACS sensors) were installed for all required channels. Compensation settings were established by using single color tubes. The data collected was analyzed using the Diva program (BD Biosciences). Student's t-test was used for statistical analysis. The results are shown in Figure 4A.

FACS analysis of freshly isolated samples showed that for both digest protocols using the NB4 collagenase preparation (with and without the addition of platelet lysate (TL), see Figure 4A), the inventors determined that viable cells constituted more than 97% of the total cell population. In particular, after digestion with NB4 collagenase preparation without TL (3-hour digestion), they observed an apoptosis rate of 1.5% ± 1.3%, and after digestion with NB4 collagenase preparation with 1% TL (3-hour cleavage), apoptotic cells accounted for only 1.45% ± 0.3% of the total number of isolated cells. However, 30.6% ± 9.6% of the total isolated according to Comparative Example 1 OSBC were stained with 7AAD (FIG. 4A, taller shaded bar on the right). Thus, it can be seen that the samples treated with the NB4 collagenase (SERVA) preparation for 3 hours were more viable than the cells isolated as described in Comparative Example 1. This indicates that the protocol used in Comparative Example 1 , more harmful to cells than the protocol of the present invention; that is, while the protocol used in Comparative Example 1 results in the isolation of more cells, most of them are apoptotic cells. Apoptotic cells are not suitable for reproduction.

In addition, by comparing the use of platelet lysate (yes/no) and not seeing a significant difference in the percentage of AAD7-positive cells (Figure 4A), the inventors were able to demonstrate that platelet lysate does not negatively affect collagenase activity to release cells and their viability. . Surprisingly, the presence of platelet lysate (usually containing plasma) did not adversely affect cleavage, or more specifically cell yield, although platelet lysate has previously been reported to adversely affect the activity of several enzymes, including proteases (Chesney et al., J. Clin Invest., 1974, vol.53, pp. 1647-1654).

Example 4A: Analysis of the physical properties of CBC-derived cells isolated according to the present invention compared to CBC-derived cells

The inventors then decided to evaluate the total cell number and physical properties of the SHS-derived cells isolated according to the present invention (Example 1 and Example 2A) compared to the SHS-derived cells obtained using the protocol in Comparative Example 1 and Comparative Example 2A. ). This comparative study consisted in the evaluation of collagenase efficiency and physical parameters (by FACS method) of the corresponding freshly isolated cells.

First, with regard to the total number of freshly isolated cells, the inventors obtained more CRBC-derived cells by the method used in Comparative Example 1 than they obtained CBC-derived cells by the method of the present invention described in Example 1.

Secondly, the analysis of physical parameters by flow cytometry, namely, by determining the forward light scatter (FSC) and side light scatter (SSC), showed that cells derived from SHS have lower FSC and SSC values compared to cells derived from FSC, isolated in the manner described in Comparative Example 1. The results are shown in the following table and visualized in Figure 5A.

Table. Experimentally determined SSC and FSC values

Cells FSC SSC OCBC-derived cells according to Comparative Example 2A Average of six samples 75.42166667 64.65666667 ST-DEV 18.66791951 15.1647779 SEM 7.75 6.19 CBC-derived cells according to Comparative Example 2A Average of four samples 46.1925 50.795 ST-DEV 9.660766986 21.28367273 SEM 5.58 12.29 Student's criterion 0.025231608 0.296671217

On average, CBC-derived cells have a smaller cell volume (determined from FSC) with lower intrinsic complexity (determined from SSC) than OCBC-derived cells. The differences in FSC scores are statistically significant. In particular, CBC-derived cells (n=4 of the present invention) have lower FSC and SSC values compared to CBC-derived cells isolated by the method described in Comparative Examples 1 and 2A (CBC, n=6). These flow cytometry results indicate that the SHS-derived cells isolated by the method of the present invention are more uniform small cells with low cytoplasmic complexity, while the SHS-derived cells are a heterogeneous population with a large average volume and greater cytoplasmic complexity. "Homogeneous" in this context means that substantially all of the cells are very similar or similar. Less volume and lower intrinsic complexity are features indicative of stemness and efficiency (see, for example, Wagner et al., 2008, PLoS One, 2008, 3: e2213). Typically, cells with more complex internal structures appear brighter/lighter in side scatter analysis due to internal structures.

Example 4B Morphological Phenotype of Freshly Isolated Cells: Cells isolated from OSBC are different from cells isolated according to the present invention

The morphology of cells isolated from SHS and cells isolated from OSHS was compared. To ensure a high degree of comparability, one umbilical cord was divided into two parts: one part was processed in the manner described in examples 1, 2A and 2B, the other part was processed in the manner described in comparative examples 1, 2A and 2B, however, in both cases, the cells were analyzed exactly through two or three days after sowing the culture in passage 1 (P1), (original magnification 100X).

The inventors found that the cells derived from the pure stromal Wharton's jelly of the present invention (SHS) are homogeneous, small and fusiform (Figure 5B, left), while the cells derived from the perivascular Wharton's jelly (PRW) are large and have a prominent cytoskeleton (Figure 5B, right). Perivascular Wharton's jelly (PVC)-derived mesenchymal stromal cells may also be referred to as "perivascular-derived mesenchymal stromal cells".

Example 4C: Colony formation from freshly isolated cells: cells isolated from OCBC differ from cells isolated according to the present invention

The colony-forming potential of the cells of the present invention was then studied. This was done as follows: SBC was prepared as described in Examples 1 and 2A, SSBC was prepared as described in Comparative Example 1. Then, using a sterile funnel, the cell-containing extract was filtered through two layers of one sterile gauze pad to remove uncleaved tissue. Centrifuged at 680 x g for 15 minutes in 250 ml centrifuge tubes. The supernatant was discarded (drained or aspirated using low vacuum). The pellet was resuspended in 1-2 ml MSCBM CD/GA/hTL (see example 0). Resuspended cells were seeded in 3x T225 flasks or 1x1 pouch in MSCBM CD/GA/hTL medium (see example 0; 50 ml medium per T-225 flask or 150 ml per 1x1 pouch) to initiate P0 culture. The P0 crop must not be moved during the first 5 days after seeding. The medium was changed after 5 days. Thereafter, every 3 days was fed with medium MSCBM CD/GA/hTL. P0 cells were harvested on day 12 using 3 U/ml trypsin and neutralized with MSCBM+1% hTL. The results are shown in Figure 5C. These results show that the cell isolation protocol of the present invention results in a more homogeneous cell population capable of forming more compact colonies after initial seeding (passage 0).

Example 5: Comparison of SHS-derived cells of the present invention with bone marrow-derived cells (BM).

Bone marrow-derived MSCs (KM-MSCs obtained as described in Grisendi et al., Cytotherapy, 2010, vol., 12, p. 466-477) were seeded at a density of 6000 cells/cm ² . The SHS-derived cells of the present invention (examples 1, 2A, 2B and 2C, in particular those cultured to passage 5 (P5) were plated at a density of 2800 cells/cm ² . After 2-3 days of culture, the cells were subconfluent and five representative images of both types were obtained.In particular, MSCs attached to the plastic were visualized using an Axioskop-Observer-1 (Zeiss) microscope with a 10x objective equipped with an Axiocam camera.The images were obtained and processed using AxioVision 4.8.2 software to estimate cell area (Moore et al., Exp. Eye Res. 2013, vol. 115, p. 178-188; Zeilbeck et al., PLoS One., 2014; vol. 9(4), e95546).

The results are shown in Figure 6. The SHS-derived cells of the present invention were found to have a lower mean surface area than BM-derived MSCs.

Example 6: Cell surface markers of freshly isolated cells

Materials and Methods: Three umbilical cords were treated in two different ways (modern method described in Comparative Examples 1 and 2A, "CBC" vs. cell preparation method of the present invention, see Examples 1 and 2A, "CBC").

Immediately after isolation of OCBC/SBC-derived cells were counted and resuspended in 1 ml 1X PBS in polypropylene tubes for analysis by fluorescence activated cell sorting (FACS) (200,000 cells/tube). Cells were incubated in 100 µl of blocking buffer containing Dulbecco's Modified Eagle's Medium (DMEM), 10% fetal bovine serum (EBS), 0.1 M sodium azide, and 66.6 mg/ml human immunoglobulin G for 20 min. ice. They were then washed in 1 ml of 1x PBS and centrifuged at 300 g for 5 minutes.

Samples were then treated for 30 min on ice with primary antibodies in 90 µl PBS with 0.5% bovine serum albumin (BSA; Sigma) and analyzed on a FACSAria III flow cytometer (BD Biosciences) equipped with two air-cooled lasers having wavelengths 488 and 633 nm. For indirect staining (for anti-GD2 only), a secondary rat anti-mouse immunoglobulin allophycocyanin (APC) conjugated antibody (BD Pharmingen) was used. The results of staining with 7-amino-actinomycin D (7AAD) (ViaProbe; BD Biosciences; used according to the manufacturer's instructions) were evaluated by flow cytometry to detect apoptosis. An unstained sample was used to determine the intrinsic fluorescence of the sample and PMT was set for all required channels; the level of fluorescence crosstalk was controlled by compensatory adjustment. Compensation settings were established by using single color tubes. The data collected was analyzed using the Diva program (BD Biosciences).

Results: Immunophenotypic analysis of freshly isolated samples showed that using both methods of isolation, the inventors found CD105, CD73 and CD90, which are typical expressed surface antigens of MSCs. Similarly, platelet growth factor β receptor (CD140b) was detected using both isolation methods. HLA-DR was not detected in any of the samples, however, OCBC-derived cells were found to express 3G5, in contrast to CBC-derived cells, indicating that the method of the present invention did not result in the isolation of perivascular cells, which corresponds to the origin of cells from stromal Wharton's jelly. The relative exposure of some of the surface markers is shown in Figure 7A, left panel. Figure 7A, right panel, shows some of the surface markers that are/are not expressed in most cells.

Cell surface markers were also analyzed after prolonged cell culture. Cell cultures obtained in two independent isolations and with different cumulative population doublings were analyzed. The following was found: there are no significant differences in the exposure of CD105, CD90 and CD73 (positive MSC markers) in different cell isolations. CD105, CD90, and CD73 also had similarly high levels at different cumulative population doublings (CDPs). In contrast, CD45, CD14, CD34, CD19, and HLA-DR (known as MSC negative markers) are expressed at very low levels in cells obtained from both isolations and from various cumulative population doublings (see Table below).

Table: Phenotypic characteristics determined by flow cytometry (% positive cells). cvp=cumulative population doubling

CD45 CD73 HL-DR CD14 CD34 CD90 CD105 CD19 Batch 1 after 8 soups 3.8 98.9 -0.6 0.0 1.1 99.2 99.1 -0.5 Batch 1 after 16 soups 4.1 99.1 -0.7 -0.2 1.7 99.2 99.1 -0.8 Batch 2 after 8 soups 1.4 99.2 -0.3 0.1 2.6 99.2 99.1 -0.2 Batch 2 after 16 soups 0.8 99.1 -0.2 0.7 4.2 99.1 99.10 -0.1

These data demonstrate that the expansion conditions of stromal-derived BC cells in the method of the present invention can result in populations of pure stromal-derived BC cells with high yield and stable phenotype over several cumulative population doublings. Thus, the cells of the invention are suitable for establishing a cell bank.

Example 7 Cell Surface Markers on Expanded Cells

Materials and Methods: In order to characterize surface antigens on SSBC-derived and SVS-derived cells, FACS analysis was performed with cells contained in a cell bank that were obtained from the same umbilical cord in different ways (examples 1 and 2A, 2B and 2C vs. Comparative Examples 1 and 2A, 2B and 2C). Cells were thawed after P2 (Example 2C/Comparative 2C) and expanded to P4 (Example 2C/Comparative 2C) for FACS analysis. After separation with cell separation solution with proteolytic and collagenolytic enzymes (Accutase, Thermo Fisher), the cells were centrifuged at 500 x g for 10 minutes. The pellet was resuspended in culture medium (MSCBM CD/hTL for SHS or α-MEM with 15% EBS determined for SSHS) and counted using a 0.4% trypan blue dye solution. Cells were washed in 1X PBS and centrifuged at 300X g for 5 minutes. Cells were resuspended in BD Pharmingen+EDTA staining buffer and 250,000 cells/well plated in 96-well plates. Flow cytometric screening was performed according to the human cell surface marker panel data sheet (BD Lyoplate™, BD Biosciences) following the manufacturer's instructions. The results are presented in Figure 7B.

Specifically tested whether freshly isolated cells of the present invention (examples 1, 2A) CD46, CD55 and CD59. As shown in Figure 7B, both OCBC-derived and SHS-derived cells express these surface complement regulators. This is consistent with the results previously described, for example, in Ignatius et al., J. Cell. Biochem., 2011, vol. 112, p. 2594-2605 and Schraufstatter et al., World J. Stem Cells, 2015, vol. 7, p. 1090-1108, which showed that bone marrow-derived MSCs express complement regulators on the cell surface, and MSCs genetically modified to increase the expression of CD46, CD55, and CD59 are believed to acquire protection against complement-mediated cell lysis in vitro and in vivo. Figure 7B shows that the cells of the present invention also have this particular expression phenotype.

Example 8: Expression of genes in cells of the present invention as determined by quantitative reverse transcriptase PCR (qRT-PCR).

Materials and methods: Cells obtained in accordance with the present invention (example 2C), in particular after passage 4, were used for this analysis. Total cellular RNA was isolated by a one-step method with TRIzol (Life Technologies) according to the manufacturer's instructions. First strand complementary cDNA was synthesized from 1 μg total RNA using the revertAid H minus first-strand cDNA synthesis kit (Thermo Scientific) according to the manufacturer's instructions. Single strand cDNA was quantified on a spectrophotometer (Beckman Coulter ^DU® 730) to use 10 ng of cDNA per well for real time PCR.

Real-time quantitative PCR was performed using the Applied Biosystems StepOne™ Real-Time PCR System and Fast ^SYBR® Green Master Mix reagent. Quantification of gene expression for each target gene and reference gene was performed in separate tubes. The forward and reverse primers were designed using the IDT ^{PrimerQuest®} (available online at http://eu.idtdna.com/PrimerQuest/Home/Index), taking care to span the intron sequence so that they are mRNA specific and not for genomic DNA. The relative level of expression of the target gene was normalized to the level of expression of the endogenous reference gene β-actin.

The results are shown in Figure 8. Figure 8 shows that, for example, APCDD1, PPARG and FLVCR2 are expressed in CBC-derived cells. Expression was comparable across different SHS-derived cell populations obtained in accordance with the present invention. This general expression pattern is indicative of the stability of the cells of the present invention in terms of gene expression.

It is noteworthy, however, that the expression pattern of these genes is significantly different from that of the OCBC-derived cells isolated by the currently used method. In particular, APCDD1 is not expressed at all in OCBC-derived cells. This conclusion was made based on the fact that amplification of the APCDD1 gene was absent in all samples of OCBC-derived cells. The inventors conclude that APCDD1 expression by real-time PCR can only be detected in CBC-derived cells of the present invention, but is not detectable in CBC-derived cells. Thus, the expression of APCDD1 makes it possible to clearly distinguish the cells of the present invention from the OCBC-derived cells.

While there has been no prior consensus in the literature as to whether non-perivascular MSCs differ from perivascular MSCs (e.g., Davies et al., Stem Cells Translational Medicine, 2017, vol. 6, p. 1620-1630), in this example provided evidence that the cells derived from stromal Wharton's jelly, obtained in accordance with the present invention, differ from perivascular MSCs.

Example 9 Proliferative Potential of Cells of the Invention Compared to Reference Cells

Materials and methods: Cell culture: stromal Wharton's jelly cells (SHS) of the present invention, obtained as described in examples 1, 2A, 2B and 2C, after P1, or perivascular Wharton's jelly cells (PVC), obtained, as described in comparative examples 1, 2A, 2B and 2C, both cell populations were obtained from the same umbilical cord, plated in MSCBM CD medium with 5% hTL (see example 0) without growth factors and placed in an incubator at 37°C, 5% CO ₂ , 95% humidity. Three days after seeding, and every 3 days thereafter, cell cultures were fed. On day 4 or 5 of each passage, cells were trypsinized and counted.

Cell Counting: Cells were counted using a 0.4% trypan blue dye solution in a Burker counting chamber and the cumulative population doubling (CPC) was calculated as described above.

The results are shown in Figure 9. More population doublings (PCs) per passage were observed in SHS-derived cells than in SHS-derived cells (Figure 9A). Thus, UP in the SHS-derived cells of the present invention is faster than in the SHS-derived cells. A statistically significant increase in the number of population doublings occurred in SHS-derived cells and passages 2, 3, and 4 compared to SHS-derived cells (based on Student's t-test).

SHS-derived cells have a higher proliferative potential that is maintained for at least 9 passages under the culture conditions used (growth medium MSCBM CD with 5% hTL). Under these culture conditions, as experimentally determined for two independent isolates of SHS-derived cells, senescence will occur in SHS-derived cells between passage 10 and passage 12, as shown in the following Table.

Table: Growth of SHS Derived Cells of the Invention

Passage Selection 1 Selection 2 1 5.89 4.99 Number of population doublings per passage 2 5.00 5.47 3 5.04 4.76 4 5-02 4.24 5 4.58 5.03 6 4.95 5.30 7 5.40 4.69 8 5.22 4.63 9 4.92 4.54 10 4.83 1.81 eleven 4.58 n/a 12 3.27 n/a Total 58.70 45.44 Cumulative population doubling

This example provides additional evidence that the isolation and expansion conditions of the stromal Wharton's jelly-derived cells (SHS-derived cells) of the present invention allow large cell populations of SHS-derived cells to be obtained. This example also provides additional evidence that SHS-derived cells are different from SHS-derived cells and have a higher proliferative potential.

The high proliferative potential and the increase in population doublings (PCs) per passage result in an increase in cell bank yield and/or a decrease in cell bank generation time, respectively, when compared to reference MSCs such as OCBC-derived cells. This is an advantage in terms of production.

Example 10 Telomerase Activity

For this assay, 200,000-500,000 Wharton's jelly-derived stromal cells (WBCs) of the present invention were aliquoted into 1 ml Eppendorf tubes and then centrifuged at 1200 rpm (Beckham Coulter Allegra™ 25R) for 10 minutes at +4 °C The supernatant was removed and the cells were resuspended in 1X PBS. After the second centrifugation step, the dry pellets were stored at -80°C. The TeloTAGGG Telomerase PCR ELISA PLUS kit (Roche Diagnostics) was used for analysis according to the manufacturer's instructions. The results are shown in Figure 10. An average of 2.4 ± 1.7% telomerase activity was observed in the analyzed samples compared to the positive control provided in the kit (taken as 100%). This confirms that the higher proliferation capacity of SHS-derived cells does not correlate with abnormally high telomerase activity.

Example 11: Aging

Materials and Methods: Wharton's jelly-derived stromal cells (SHS) of the present invention, treated with trypsin (for information about the origin of cells, see examples 1 and 2A; for information about passages, see examples 2B and 2C) after passage 6 (P6), passage 8 (P8) or passage 11 (P11), passage 12 (P12), were seeded in six multiwell plates at a density of 3000 cells/cm ² in 3 ml of culture medium (DMEM with 5% hTL or MSCBM CD with 5% hTL) and cultured in for 4-5 days at 37°C in an incubator with 5% CO ₂ and 95% humidity, in triplicate, unless otherwise indicated. Cells were then washed with 1X phosphate buffered saline (PBS) and fixed for 10 minutes at room temperature in 2% formaldehyde/0.2% glutaraldehyde. The culture plate was washed twice with 1X PBS and 1 ml fresh β-Gal staining solution (1 mg 5-bromo-4-chloro-3-indolyl-βD-galactosidase/100 µl dimethylformamide, 40 mM citric acid/sodium phosphate, pH 6) was added 0.5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl and 2 mM MgCl ₂ ) (used according to manufacturer's instructions). Cells were incubated overnight at 37°C, and the color developed under the light of an inverted microscope, as described in the instructions provided by the manufacturer (Cell Signaling Technology). Positively stained cells had a blue-green color of the cytoplasm. Stained samples were washed with 1X PBS and observed under a 2.5X objective of an inverted microscope (Zeiss). Positively stained cells (in each field) were counted by one observer, and at least 8 visual fields were analyzed for each donor (unless otherwise indicated).

Results: In early culture passages (P6-P8), only a few cells were weakly positive for β-galactosidase staining (data not shown), indicating that even at very low seeding densities, the SHS-derived cells of the present invention tend to slight aging. Without wishing to be bound by theory, it is possible that the platelet lysate contains additives that confer ex vivo cell growth benefits, such as growth factors.

Example 12: Osteogenic differentiation potential

Method Description: SHS-derived cells from passage 5 (Example 2C) were induced to differentiate into cells of the osteogenic lineage. Briefly, SHS-derived cells were seeded at a density of 10,000 cells/cm ² in MSCBM CD medium with 5% hTL in 6-well (6-MW) plates. These 6-MW plates were pre-coated with 0.1% gelatin solution. After equilibration for 3-4 days, the maintenance medium was removed and replaced with osteogenic medium. Osteogenic medium consisted of MSCBM CD with 5% hTL supplemented with 10 mM beta-glycerophosphate, 0.1 mM ascorbic acid 2-phosphate, and 10 nM dexamethasone, plus also 100 ng/mL rhBMP-2 (bone morphogenic protein 2), starting from the 7th day of induction onwards. Osteogenic differentiation was confirmed by von Koss staining (which stains bone matrix dark) in combination with real-time qPCR analysis (described below).

After 2 weeks, cultures in osteogenic medium (see above) and parallel controls (growing in parallel in MSCBM CD plus 5% hTL, i.e., medium that does not induce osteogenesis) were fixed and stained for organic calcium deposits. For von Koss staining, samples were fixed in ice-cold methanol for 2 min, then washed twice in distilled water, and incubated under an ultraviolet (UV) lamp with silver nitrate in water for 30 min. After two additional washes in distilled water, 10 fields were examined at high magnification using 10X magnification (Axioskop-Observer-1 inverted microscope; Zeiss). Digital images were analyzed using the ImageJ program (National Institute of Health, USA), selectively counting dark positively stained areas of organic calcium deposition. The experiments were carried out in three repetitions.

Results: The results are shown in Figure 12. As shown, after 14 days of treatment with osteogenic medium, the morphology of the control samples differed from that of the osteogenic medium-induced samples, and von Koss calcium phosphate staining resulted in dark positive staining (Figure 12A). The difference between positively stained areas in the control and induced samples was statistically significant (based on Student's t-test) (Figure 12B). Molecular real-time PCR analysis was performed to further confirm osteogenic features (FIG. 12C). The relative expression level of the osteogenic biomarker gene was normalized to the expression level of the endogenous reference beta-actin gene. The mRNA induction fold was calculated relative to a culture of SHS-derived cells under standard conditions (MSCBM CD plus 5% hTL), which were defined as non-induced and subconfluent cells. All tested genes were significantly upregulated relative to culture under standard conditions (p≤0.05, based on Student's t-test). The inventors found that alkaline phosphatase (ALPL) and some matrix proteins such as biglycan (BGN), collagen (COL1A1), decorin (DCN) and elastin (ELN) were the most induced biomarkers. The combination of these markers is characteristic of osteogenic lineage cells (Murgia et al., PLoS One. 2016, vol. 11: e0163629). This expression profile confirms the initial osteogenic commitment of the cells.

Example 13: Adipogenic Differentiation Potential

Method Description: For adipogenic differentiation, SHS-derived cells (Examples 1, 2A, 2B, 2C) from passage 5 culture were seeded at a density of 10,000 cells/cm ² in MSCBM CD medium with 5% hTL in 6-well (6-MW) plates , and after 3 days the medium was replaced with adipogenic induction medium. Adipogenic medium consisted of MSCBM CD with 5% hTL supplemented with 1 μM dexamethasone, 60 μM indomethacin, 10 μM insulin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 10% rabbit serum, 5% horse serum, 1% L-glutamine and 1% penicillin/streptomycin mixture. Control cells were cultured in MSCBM CD medium with 5% hTL. Cells were seeded in technical duplicates for each condition.

After 10 days, cells were stained with Oil Red O (a commercially available adipocyte stain) to confirm differentiation as follows: Cells were washed rapidly with 1x PBS, fixed with 37% formaldehyde vapor for 10 minutes, and then washed with water for 2 minutes. Staining was performed by adding a solution of Oil Red O (10 mg/ml Oil Red O in 70% ethanol and acetone) for 3 minutes to the wells. The cells were counterstained with Harris' hematoxylin (Bio-Optica, Milan, Italy) for 3 minutes. After treatment, differentiated cells and controls were analyzed under a microscope (Axio Observer A1 with Axiocam MRC5 color camera and Axiovision 4.82 software; all from Zeiss).

The results are shown in Figure 13. SHS-derived cells after 10 days of treatment were able to produce round lipid droplets within the cells, which confirmed adipose cell commitment. Lipid vacuoles were found in large numbers in induced samples, but not in non-induced controls (figure 13).

Example 14: Chondrogenic Differentiation Potential

Method Description: For chondrogenic differentiation, 200,000-500,000 SHS-derived cells (Examples 1, 2A, 2B, 2C) from passage 5 culture were aliquoted into 15 ml tubes and centrifuged at 1200 rpm (Beckham Coulter Allegra™ 25R) in within 10 minutes. The cells were maintained in the sediment in the growth medium at 37°C with the lid open. After 2 days of incubation, the growth medium was replaced with an induction medium consisting of MSCBM CD with 5% human platelet lysate (hTL) supplemented with: 500 ng/ml rhBMP-6, 10 ng/ml transforming growth factor β, 50 mg/ml ITS+ mixture Premix (containing insulin 6.25 µg/ml, transferrin 6.25 µg/ml, selenic acid 6.25 ng/ml, BSA 1.25 mg/ml and linoleic acid 5.35 µg/ml), 100 nM dexamethasone, 0.2 mM L-ascorbic acid 2-phosphate, 100 µg/ml sodium pyruvate, 40 µg/ml proline. The medium was changed every three days. Before and after each medium change, the tubes were centrifuged at 1200 rpm for 10 minutes as described above. During the incubation period, the cells remained in the sediment with the lids of the tubes open. The induction lasted 21 days, the samples were fixed for 1 hour in 10% formaldehyde solution, and then dehydrated by serial passage through ethanol solutions in increasing concentrations, from 70% (v/v) to 100%. Then the samples were embedded in paraffin blocks, sections were obtained, which were placed on microscope slides for alcian blue staining. Slides were deparaffinized with Histo-C cleansing reagent and rehydrated by passing through ethanol solutions in decreasing concentrations (from 100% ethanol to 70% ethanol) (v/v). Sample sections were then incubated with 0.5 mg/ml hyaluronidase buffered phosphate solution (8 g/l NaCl, 2 g/l NaH ₂ PO ₄ and 0.3 g/l Na ₂ HPO ₄ ) with 10 mg/ml BSA . The slides were washed with water for 5 minutes and then immersed in a 3% (v/v) acetic acid solution for a few seconds. Sections were stained with 10 mg/ml solution of alcian blue (commercial dye) in 3% acetic acid solution (pH 2.5), left in this solution for 30 minutes and, after washing with water, counterstained for 5 minutes with a solution of nuclear fast red (commercial dye).

The results are shown in Figure 14. After 21 days of treatment, SHS-derived cells developed a multi-layered, matrix-rich morphology. The induced SHS-derived cells had a smaller volume of cellular content and a smaller amount of glycosaminoglycans in the extracellular matrix. Control derived from SHS cells had a large amount of cellular content and underdeveloped matrix component.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1:

Cross section of the (human) umbilical cord and schematic representation of sections (anatomical regions) of the (human) umbilical cord. The two outer ellipses in gray (1a and 1b) together form the umbilical cord lining (containing two layers: 1a (dark gray circle on the outside), the amniotic (or epithelial) layer, also called the umbilical cord lining; and 1b (shown in light gray circle inside): subamniotic (or mesenchymal) layer); 2: umbilical vein; 3: umbilical arteries; 4: intervascular area; 5: perivascular region (depicted in gray); 6: illustrative example of the location of Wharton's jelly; 7: cord blood (in the lumen of the vessel). Unless specifically noted otherwise, the subamniotic stroma (1b) is contained within the "stroma" described herein. Unless specifically noted otherwise, subamniotic Wharton's jelly is contained in Wharton's jelly, described herein as "stromal Wharton's jelly".

Figure 2: Immunohistochemical determination of different areas of the human umbilical cord. Sun: Wharton's jelly; MS: multilayer wall; OS: perivascular zone; C: vessel; P: light. Stromal VS: VS at least 3 mm from the outer wall of the umbilical vessels, and inward from the amniotic epithelium.

: Околососудистая область на расстоянии примерно 2,00 мм, включающая ВС+перициты+эндотелиальные клетки.

: Perivascular area at a distance of about 2.00 mm, including BC + pericytes + endothelial cells.

Materials and Methods: A sample of human umbilical cord was fixed in formalin and embedded in paraffin. Fragments were isolated and cut into 5 μm sections for further staining. You can see different staining in different areas representing Wharton's stromal jelly and perivascular region. The perivascular region has a more complex histomorphological pattern with vessels (C), perivascular cells (OC) located on the border of the BC regions. Complexity is also related to the different types of cells present in the perivascular region. In contrast, Whartonian stromal jelly appears histologically as a relatively more uniform area with more uniform cellular elements scattered in the stromal tissue matrix. The isolated cells express markers commonly found in ex vivo MSC populations such as CD10, CD140b, GD2, CD73 (not shown in this figure). The immunohistochemical staining patterns for these markers also differ in the two regions. In Figure 2B, the histological differences are more evident between the umbilical vascular region and the stromal Wharton's jelly. Vessels (C) clearly show the lumen (P) and cells (arrows) representing the endothelial layer (CD31) and subendothelial layer (CD146, CD271, GD2). It has also been found that CD140b is expressed in the intraluminal part of the vessel. CD73-positive staining occurs in the subendothelial layer, with intensity increasing towards the Wharton's jelly matrix (Rasini et al., 2013, Cytotherapy, vol. 15, p. 292-306).

Figure 3: Cutting (sectioning) and extracting cells - see example 1 for details.

Figure 4: The results obtained in example 4, confirming that the method according to the invention leads to a significantly higher cell viability. See example 4 for details.

*OSBC: conventionally prepared cells, see Comparative Examples. CBC: cells isolated in accordance with the present invention.

A: Apoptotic cells: effect of platelet lysate and comparison with conventional method. With regard to the number of freshly isolated cells, the inventors obtained more cells (OSBC) using the protocol described in comparative example 1 (4.15 ± 1.28×10 ⁶ ) than using the protocol of the present invention (1.49 ± 0 .35×10 ⁶ ). The graph represents the means and standard error of the mean (SEM) for apoptotic cells at three splits (n=3). 30.6% ± 9.6% of the total CRBC-derived cells isolated according to Comparative Example 1 were positive for 7AAD (FIG. 4A, taller shaded bar on the right). In contrast, the percentage of SHS-derived cells positive for 7AAD, both in the presence and absence of hTL, is much lower. Thus, the inventors have found that cells obtained in accordance with the present invention have a higher degree of viability than cells obtained by the currently conventional method.

B: FACS analysis of freshly isolated cell samples. 4 FACS graphs represent physical parameters (right) and 7AAD staining (ie, apoptosis, left) of CBC-derived cells (top) and OCBC-derived cells (bottom), respectively. In the graphs on the right, living cells are to the left of the vertical line.

C: Graphical representation of the total number of cells as determined by flow cytometry (figure 4B). The graph represents the means and standard error of the mean (SEM) for apoptotic cells in three splits (n=3 for both cell populations).

D: Graphical representation of the percentage of apoptotic cells as determined by flow cytometry (figure 4B). FACS analysis of freshly isolated cell samples showed that the CBC-derived cells of the present invention had over 79% viable cells in the entire cell population. In contrast, 52.9% ± 2.6% of the total number of cells isolated according to the protocol described in Comparative Example 1 (OCBC-derived cells) were stained positive with 7AAD. C: Efficiency of cell isolation in SBC (n=5) and OSBC (n=9) samples. D: Graph presents the means and standard error of the mean (SEM) for apoptotic cells in three splits (n=3 for both cell populations).

Figure 5: Morphology and colony formation

A: Presented are the physical parameters determined by flow cytometry, i.e., by determination of forward scatter (FSC) and side scatter (SSC), of freshly isolated cells from the umbilical cord (FACS graph on the left). The graph represents mean values and SEM for physical parameters. The isolated CBC-derived cell population (according to Examples 1 and 2A) has significantly lower FSC values than the isolated CBC-derived cell population (according to Comparative Examples 1 and 2A; p=0.02 based on Student's t-test). SSC: SHS-derived cells have lower cytoplasmic complexity than OSBC-derived cells. See example 4A for details.

B: Study of morphology in passage 1 of cultivation (n=3). After expansion, CBC-derived cells (Example 2C) were characterized by small and spindle-shaped cells in the cell population, while CBC-derived cells (according to Comparative Example 2C) were characterized by large cells and a pronounced cytoskeleton. The two images show the morphology of SHS-derived cells (left) compared to SHS-derived cells (right) two or three days after seeding in passage 1 (P1) culture in a representative sample (original magnification 100X).

C: Colony formation by CBC-derived cells and OCBC-derived cells. See example 4C for details. Cells were stained with crystal violet before the image shown below was obtained. Cultures of SHS-derived cell populations consist of cells with low surface area; these cells tend to form colonies, while the cell populations derived from OSBC are composed of cells with different surface areas, also including large cells. Thus, SHS-derived cells look more uniform; "homogeneous" in this context means that the cells look practically very similar or similar. Larger OSBC-derived cells tend to grow relatively slower (data not shown).

Figure 6: Quantification of cell surface area: SHS-derived cells versus bone marrow-derived mesenchymal stromal cells (CM, or CM-MSC). Plastic-attached MSCs from bone marrow and from stromal Wharton's jelly (CM and SHS, respectively) were visualized during passage 5 (P5) of cultivation. Five images were taken for each sample, and 34 KM-MSCs and 33 SHS cells were optically identified. Cells were graphically outlined by hand during visual inspection. BM-derived cells had a larger average cell surface area (4300.5 ± 2486.5 µm ² ) compared to SHS-derived cells (1701.2 ± 1137.3 µm ² ) (p=5.15X10 ^-7 , based on ANOVA). Thus, it was concluded that SHS-derived MSCs of the present invention are smaller in size than CM-derived MSCs.

Figure 7: Results obtained in example 7. The graph on the left shows the mean values and SEM for surface markers in CRBC-derived cells and CBC-derived cells, both freshly isolated from three umbilical cords by two different isolation methods (CBC: according to examples 1 and 2A, OSBC: according to Comparative Examples 1 and 2A). Immunophenotypic analysis of samples freshly isolated by both methods showed the presence of CD105, CD73 and CD90, typical surface antigens exhibited on MSCs. Similarly, the presence of platelet-derived growth factor β receptor (CD140b) on the surface was detected after both methods were used. HLA-DR expression was absent in both samples. However, the presence of 3G5 on the surface was found only in a population of SHS-derived cells, indicating that the method of the present invention is particularly suitable for isolating a population of SHS-derived MSCs that do not contain perivascular cells.

A: The graph on the left shows the mean and SEM values for surface markers in freshly isolated cells from SSBC and SBC derived from three umbilical cords treated with two different cleavage methods (comparative example 1, "SSBC" versus cells of the present invention, "SBC") . The panel on the right shows some of the surface markers that are/are not expressed in most cells.

B: The graph represents the average percentage of cells exhibiting specific surface markers in freshly isolated cells from umbilical cords in two different ways (comparative example 1, "RCBC" versus cells of the present invention, "CBC").

Figure 8: Quantification of gene expression in cells of the present invention by real-time PCR (RT-PCR). Conducted RT-PCR analysis of two independent cell populations isolated according to the present invention; the results for both are shown in Figure 8 compared to the results for the control OCBC-derived cells. The graph shows the differences in expression (delta Ct) between 2 genes (gene of interest vs beta actin gene, housekeeping gene).

APCDD1 expression was not detected in control cells.

Figure 9: Proliferative Potential: Graph presents means and SEM (standard error of the mean) for the number of cumulative doublings of the population of OCBC-derived cells (n=9) and CBC-derived cells (n=7) in the four culture passages analyzed (from P1 to P4). A higher number of population doublings (PCs) per passage was observed in SHS-derived cells compared to RCBC-derived cells. See example 9 for details.

Figure 10: Expression of telomerase in different lots of SHS-derived cells. See example 10 for details.

Figure 11: Aging in different lots of SHS-derived cells. CAKE2 is an abbreviation for Minimal Environment, almost equivalent to MSCBM CD; 5TL is an abbreviation for 5% (v/v) human platelet lysate. See example 11 for details.

Figure 12: Osteogenic potential of BC-derived cell populations. Interruption of the bars indicates a change in the scale. CAKE2 is an abbreviation for Minimal Environment, almost equivalent to MSCBM CD; 5TL is an abbreviation for 5% (v/v) human platelet lysate. See example 12 for details.

Figure 13: Adipogenic potential of BC-derived cell populations. See Example 13 for details. SHS-derived cells produce round lipid droplets within the cells (upper and lower right panels), confirming adipose cell commitment. Lipid vacuoles were found in large numbers in the induced samples, but not in the non-induced controls. CAKE2 is an abbreviation for Minimal Environment, almost equivalent to MSCBM CD; 5TL is an abbreviation for 5% (v/v) human platelet lysate.

Figure 14: Chondrogenic differentiation of SHS-derived cell populations. See Example 14 for details. After 21 days of treatment, non-induced SHS-derived cells (control) developed a stratified matrix-rich morphology (left two panels). The induced SHS-derived cells (two panels on the right) had a lower volume of cellular content and a lower amount of glycosaminoglycans in the extracellular matrix (dark areas corresponding to strongly blue-stained areas in the original color image). CAKE2 is an abbreviation for Minimal Environment, almost equivalent to MSCBM CD; 5TL is an abbreviation for 5% (v/v) human platelet lysate.

Claims (19)

1. A method for producing a mesenchymal stromal cell, including (a) isolation of stromal Wharton's jelly or part thereof from the umbilical cord; (b) enzymatic treatment of the stromal Wharton's jelly, or portion thereof, wherein said enzymatic treatment comprises a collagenase enzyme in the presence of platelet lysate (PL), thereby releasing at least one cell from the stromal Wharton's jelly, or portion thereof; where step (b) is followed by step (c): (c) propagating said at least one cell in a growth medium containing platelet lysate (TL). 2. The method of claim 1 wherein (a) isolating the stromal Wharton's jelly or portion thereof comprises the step of physically removing the vessels and perivascular region from the umbilical cord. 3. The method of claim 2, wherein the physical removal of the vessels and perivascular region consists in separating the vessels and perivascular region from the umbilical cord. 4. The method of any one of the preceding claims, wherein (a) isolating the stromal Wharton's jelly, or portion thereof, also includes the step of physically separating the subamniotic stroma from the umbilical cord lining. 5. The method of claim 4, wherein said separation step comprises separating the Wharton's jelly from the lining of the umbilical cord, in particular at least the epithelial layer of the lining of the umbilical cord. 6. The method according to claim 5, wherein the physical separation of the Wharton's jelly from the lining of the umbilical cord consists in scraping off the Wharton's jelly, preferably without mechanically cutting the lining of the umbilical cord. 7. The method according to any one of paragraphs. 2-6 where the umbilical cord is segmented prior to said step of physically removing the vessels and perivascular region. 8. The method of claim 7, wherein the segmentation is perpendicular to the umbilical cord, and wherein each perpendicularly cut segment preferably has a length of about 1 to about 4 cm, preferably about 2 to about 3 cm, and more preferably about 2.5 cm. 9. The method according to claim 7 or 8, wherein the vascular segment(s) are removed from the umbilical cord segment(s) prior to step (b). 10. The method according to any one of the preceding claims, wherein the enzymatic treatment is carried out by mixing stromal Wharton's jelly or part thereof with a digestion buffer containing collagenase, preferably with a specific activity of not more than 0.5 Wunsch units/ml, more preferably not more than 0.25 U Wunsch/ml and in particular 0.18 Wunsch units/ml. 11. The method according to any one of the preceding claims, wherein steps (a) and (b) are carried out together for a total of less than 3 hours. 12. The method of any one of the preceding claims, wherein in step (b) the platelet lysate is human platelet lysate (hTL). 13. The method according to any one of the preceding claims, wherein in step (c) at least one cell is propagated in a growth medium containing human platelet lysate (hTL). 14. The method according to any one of the preceding claims, wherein the at least one cell is propagated in a growth medium free of any added serum, in particular free of fetal bovine serum (FBS). 15. The method according to any one of the preceding claims, wherein at least one cell is propagated in a growth medium containing minimal MSCBM CD medium.

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