WO2021039882A1

WO2021039882A1 - Method for culturing tie2-positive stem/progenitor cell-containing cell population using culture substrate, and utilization thereof

Info

Publication number: WO2021039882A1
Application number: PCT/JP2020/032302
Authority: WO
Inventors: 酒井　大輔; 嘉彦中村; 枝利香松下
Original assignee: 学校法人東海大学; 日本臓器製薬株式会社
Priority date: 2019-08-28
Filing date: 2020-08-27
Publication date: 2021-03-04
Also published as: JPWO2021039882A1

Abstract

The present invention provides a means for efficiently preparing a cell population that contains abundant cells having a specific characteristic corresponding to the purpose of use (for example, type II collagen-positive nucleus pulposus cells) from a cell population containing Tie2-positive stem/progenitor cells (for example, nucleus pulposus stem/progenitor cells). The culture method according to the present invention comprises culturing a cell population containing Tie2-positive stem/progenitor cells in a culture substrate in which: a plurality of indentations forming compartments, said compartments being for culturing the material to be cultured therein, and embankments positioned between indentations adjacent to each other are provided on the upper surface of the culture substrate having a sheet shape; the embankments and indentations adjacent to each other together form a continuous curved surface; the plurality of indentations are formed on the upper surface of the culture substrate and densely arranged thereon; and at least the inner surface of the indentations is coated with a cell adhesion inhibitor. The diameter of the opening of the indentations is 400-1000 μm and the depth of the indentations is 50-500 μm.

Description

Method for culturing a cell population containing Tie2-positive stem / progenitor cells using a culture substrate and its use

The present invention refers to stem cells and / or progenitor cells (referred to herein as "Tie2-positive stem / progenitor cells") that are positive for the expression of the cell surface marker Tie2 (tyrosine kinase with Ig and EGF homology domain-2). The present invention relates to a method for culturing a cell population containing the cells. More specifically, the present invention is carried out using a specific culture medium, and is used in a step of inducing differentiation of Tie2-positive stem / progenitor cells into cells having a predetermined trait (for example, type II collagen-expressing nucleus pulposus cells). The present invention relates to a method for culturing a cell population containing Tie2-positive stem / progenitor cells.

Back pain ranks second in the complaint rate and is a common illness that two-thirds of the adult population experiences once, contributing to work-related injuries and social problems in the health economics. Disc damage, which is said to be 20% of the causes of low back pain, is a serious problem that can induce disc herniated disk, degenerative spondylosis, spinal canal stenosis, spondylolisthesis, etc., but it is an irreversible change in disc tissue and is a disease. Physically, it is a condition called intervertebral disc degeneration. The intervertebral disc is a donut-shaped cartilage organ, with the central nucleus pulposus (Nucleus Pulposus; NP), the fibrous cartilage that surrounds it (Annulus Fibrosus; AF), and the adjacent vertebrae. It is formed of cartilage (Cartilaginous Endplate; EP) that connects the above and below. Gelatin-like NP is an avascular organ and contains a large amount of extracellular matrix (ECM) composed of large proteoglycans and collagen secreted from spinal cord-derived nucleus pulposus cells contained in NP. .. Notochord-derived nucleus pulposus cells have been reported to disappear early in life in some vertebrates, including humans, and after their disappearance, their origin has not yet been determined, but morphologically chondrocytes. Chondrocytes similar to the above form the nucleus pulposus. It is considered that such conversion of cell traits affects the ECM composition, causes aging and degeneration of the intervertebral disc such as a decrease in water content and fibrosis, and is ultimately greatly related to low back pain and lumbar degenerative diseases. Many animal species such as mice, rats, rabbits, and pigs retain notochord-derived nucleus pulposus cells for life, and almost no intervertebral disc degeneration is observed. Therefore, the control mechanism of notochord-derived nucleus pulposus cells and other nucleus pulposus cells. Is considered to be different from humans.

As an example of a method for preventing or treating intervertebral disc degeneration, research and development of allogeneic disc cell preparations, that is, cell preparations containing allogeneic nucleus pulposus cells, ECM, etc. for administration to intervertebral disc tissue are underway. A certain amount of allogeneic nucleus pulposus cells is required for the production of such cell preparations. For example, the intervertebral disc nucleus pulposus tissue excised by surgery in a patient with herniated disc can be used as a source of nucleus pulposus cells, but the amount of nucleus pulposus tissue that can be collected in this way, that is, the nucleus pulposus cells contained therein. The number is small. However, the mixed use of nucleus pulposus cells derived from multiple herniated disc patients (donors) to secure the number of nucleus pulposus cells cannot completely rule out the risk of viral infection and should be avoided. Is desirable. Therefore, rare stem or progenitor cells that are contained in a small amount of disc tissue from a single donor (medullary nucleus, annulus fibrosus, etc.) and capable of inducing differentiation into mature nucleus pulposus cells are cultured and treated. It is important to prepare a cell population containing a sufficient number of nucleus pulposus cells for this purpose.

In Patent Document 1, a nucleus pulposus cell (a cell population derived from the nucleus pulposus of the intervertebral disc) is cultured under "conditions that interfere with cell attachment" (preferably cultured in a serum-free medium) to obtain the cell population. It is disclosed to make a "discosphere" composed of contained stem cells and progenitor cells. That is, Patent Document 1 describes (a) a step of growing nucleus pulposus cells in a medium under "conditions that interfere with cell attachment", and (b) disc stem cells and disc progenitor cells (disc). A method of producing a disc stem cell population, which comprises a step of concentrating progenitor cells) or a combination thereof, and (c) producing a disc bulb containing nucleus pulposus cells, thereby producing a disc stem cell population. "(Claim 3 etc.). Regarding the "disc sphere", a single disc stem cell, which is an in vitro free floating circular-spherical structure containing a disc stem cell, a disc progenitor cell, or a combination thereof, is a single disc stem cell. A nucleus pulposus containing a disk sphere, including floating nucleus pulposus stem cells and nucleus pulposus progenitor cells arranged in a circular-spherical structure, which is a cell sphere that gives rise to its own clone and progenitor cells. It is explained that the cells are attached to each other (paragraphs 0024 and 0039). Patent Document 1 further describes an "isolated disc stem cell population" (claimed) enriched for disc stem cells, disc precursor cells or combinations thereof, cultured under "conditions that interfere with cell attachment". 1st grade); "isolated disc sphere" which is an in vitro floating sphere structure containing disc stem cells, disc precursor cells, or a mixture thereof concentrated from nucleus pulposus cells (claim 10 etc.) An "artificial disk replacement device" containing a disk scaffold and a disk sphere containing a disk stem cell, a disk precursor cell, or a nucleus pulposus enriched for a combination thereof (claim 11 et al.); A method for producing a disk artificial replacement device, which comprises growing a disk sphere containing disk stem cells, disk precursor cells or a mixture thereof concentrated from cells in a disk scaffold (claim 12, etc.). A step of culturing nucleus pulposus cells plated at a predetermined low density under "conditions that interfere with cell attachment" and an in vitro floating sphere structure containing disc stem cells, disc precursor cells or a mixture thereof. A "method of producing a concentrated cell population" that comprises the steps of selecting and thereby producing a concentrated cell population (such as claim 17); a disc containing disc stem cells, disc precursor cells or a combination thereof. The step of dissociating the sphere into one or more dissociated disc sphere cells and the one or more dissociated disc sphere cells interfering with cell attachment and a predetermined additive (eg, FGF2, "Methods for expanding the population of concentrated disc stem cells, disc precursor cells or combinations thereof" including the step of culturing in a medium containing EGF) (claim 18 and the like) are also described. The "disk" in Patent Document 1 is a literal translation of the original word "disc", but is considered to mean "intervertebral disc".

Patent Document 1 relates to a culture vessel or a medium for culturing an inhomogeneous cell population (medullary nucleus-derived cell population) including disc stem cells, disc progenitor cells, disc cells, etc. derived from disc nucleus pulposus tissue. The matters are described as follows.

In Patent Document 1, as a culture under "conditions that interfere with cell adhesion", the cells are plated and cultured at a low cell density in a serum-free medium containing a substance that interferes with cell adhesion (methyl cellulose), or are ultra-low. An embodiment is disclosed in which a disc sphere, which is a floating sphere structure, is formed from a nucleus pulposus-derived cell population (stem cells and the like contained therein) by culturing in an attachment plate (paragraph 0156, Example). 1: See paragraphs 0170 to 0181, corresponding to the inventions of claims 3, 17 and the like). More specifically, in the examples of Patent Document 1, a cell / medium suspension containing methyl cellulose and an anti-adhesion substance (poly2-hydroxyethyl methacrylate) precoated as an ultra-low adhesion plate 6 It is described that the well plate was used in combination (paragraph 0179). Further, in the examples of Patent Document 1, it is described that a culture dish having a diameter of 35 mm was used (paragraph 0050). However, in Patent Document 1, a plate having a shape feature on the surface of the base material (bottom surface of the well) is used as an ultra-low adhesion plate, a medium to which methyl cellulose is not added at that time is used, or the surface of the base material is used. There is no description or suggestion of using a (well bottom surface) having a shape feature.

Further, in Patent Document 2 and Non-Patent Document 1, among the cells contained in the intervertebral disc tissue (nucleus pulposus), the cells positive for Tie2 and / or GD2 as cell surface markers are the stem cells of the nucleus pulposus cells or Cells that should be called progenitor cells, especially cells that are positive for both Tie2 and GD2 (active nucleus pulposus stem cells), form spherical colonies and finally matured nucleus pulposus through a series of differentiation cascades. By having the ability to differentiate into cells (and also the ability to differentiate into fat cells, bone cells, chondrocytes and nerve cells), and by transplanting nucleus pulposus stem / precursor cells into the intervertebral disc (nucleus nucleus) It is described that it is possible to produce an extracellular matrix such as type II collagen in a tissue, maintain or reconstruct the disc tissue, and prevent or treat disc degeneration.

As a more specific embodiment, in Patent Document 2 and Non-Patent Document 1, spherical colonies (along with adherent colonies) are formed by suspension-culturing a cell population contained in a disc tissue (nucleus pulposus) in a methylcellulose medium. The formation, such spherical colonies are derived from the above-mentioned Tie2-positive (and GD2-positive) cells, and the spherical colonies (some of the cells) express type II collagen and proteoglycan. It is described (see, for example, Examples of Patent Document 2, paragraphs 0067, 0070, etc.). However, neither Patent Document 2 nor Non-Patent Document 1 describes or suggests the use of a substrate surface (bottom surface of a well) having a shape feature.

On the other hand, in Patent Document 3, there is a high probability that spheroids that are uniformly aggregated three-dimensionally are formed from non-cultures such as cells and tissue pieces, and as a culture substrate capable of efficiently culturing spheroids, A plurality of recesses forming a separate chamber in which the culture medium is cultured and a bank portion interposed between the adjacent recesses are located on the upper surface of the plate-shaped culture base material, and the adjacent bank portions and the recessed portions are present. It has been proposed that and is a continuous curved surface. Further, in Patent Document 3, a plurality of the recessed portions may be formed on the upper surface of the culture medium and arranged densely, or at least the inner surface of the recessed portions may be covered with a cell adhesion inhibitor. That is also described.

However, Patent Document 3 does not describe culturing Tie2-positive nucleus pulposus cells (intervertebral plate nucleus pulposus stem / progenitor cells) on the culture substrate, and at that time, a medium to which methyl cellulose is not added is used. It is also not stated that it can be used.

Japanese Patent No. 5509073 (corresponding to WO2009 / 00902020) Japanese Patent No. 5863639 (corresponding to WO2011 / 12261) Patent No. 5921437 (corresponding to WO2002 / 036011)

As mentioned above, in order to produce allogeneic disc cell preparations for the prevention or treatment of degenerative disc disease, functional nucleus pulposus cells that produce extracellular matrix such as type II collagen and proteoglycan are gathered to some extent. Needed in quantity. For that purpose, for example, Tie2-positive cells, which are considered to be stem cells and / or progenitor cells of nucleus pulposus cells, contained in the intervertebral tissue (such as the nucleus pulposus) excised from the affected part of a herniated disk patient are efficiently used. It is necessary to proliferate and differentiate into a large amount of functional nucleus pulposus cells. In particular, in order to enhance the therapeutic effect when a cell preparation is administered to a patient with disc degeneration, etc., when culturing Tie2-positive cells (marrow nucleus stem / progenitor cells) contained in the disc and inducing differentiation, simply It is important not to differentiate into nucleus pulposus cells, but to differentiate into functional nucleus pulposus cells that produce a large amount of extracellular matrix such as type II collagen as efficiently as possible. That is, functional nucleus pulposus cells in the cell population that are finally prepared and administered by efficient amplification and differentiation induction from the cell population containing a certain number of Tie2-positive cells (medullary nucleus stem / progenitor cells). There is a need for practical means to make it more abundant.

Further, in a typical embodiment of the prior art as described in Patent Documents 1 and 2 described above, methyl cellulose was added to a cell population containing a nucleus pulposus stem / progenitor cell contained in the intervertebral disc tissue. By culturing in a medium, spherical colonies (disc spheres, spheroids) were formed and then differentiated into nucleus pulposus cells. However, since methylcellulose is a highly viscous substance, it is difficult or very difficult to efficiently recover the cell population (useful functional nucleus pulposus cells contained therein) generated from the medium to which it is added. Due to the labor involved and the high cost of commercially available methylcellulose products, it has been a hindrance to the efficient production of cell preparations.

In the present invention, from a cell population containing stem cells and / or progenitor cells positive for Tie2 expression (for example, a cell population containing nucleus pulposus stem / progenitor cells derived from intervertebral disc), cells having a predetermined trait according to the application (for example, An object of the present invention is to provide a means for efficiently preparing a cell population rich in (functional nucleus pulposus cells that produce an extracellular matrix such as type II collagen).

When culturing a cell population containing Tie2-positive cells (medullary nucleus stem / progenitor cells) contained in the nucleus pulposus tissue of the intervertebral disc, the present inventors have a depression and a bank as described in Patent Document 3. By using a culture substrate having a portion in which the opening and depth of the recess are within a specific range, the nucleus pulposus stem / progenitor cells can be efficiently amplified without adding methyl cellulose to the medium. It was found that a large amount of functional nucleus pulposus cells can be obtained by inducing differentiation from the cells after amplification.

That is, the present invention can be expressed as, for example, an invention including at least the following matters.

[Item 1]
A plurality of recesses forming a separate chamber in which the culture medium is cultured and a bank portion interposed between the adjacent recesses are located on the upper surface of the plate-shaped culture base material, and the adjacent bank portions and the recessed portions are present. Is a continuous curved surface, and a plurality of the recessed portions are formed on the upper surface of the culture substrate and are densely arranged, and at least the inner surface of the recessed portions is coated with a cell adhesion inhibitor. In,
A method for culturing a cell population containing stem cells and / or progenitor cells (hereinafter referred to as "Tie2-positive stem / progenitor cells") that are positive for expression of Tie2 (tyrosine kinase with Ig and EGF homology domain-2).
A culture method in which the diameter of the opening of the recess is 400 to 1000 μm and the depth of the recess is 50 to 500 μm.
[Item 2]
Item 2. The culture method according to Item 1, wherein the diameter of the opening of the recess is 600 to 1000 μm, and the depth of the recess is 350 to 450 μm.
[Item 3]
Item 2. The culture method according to Item 1, wherein the diameter of the opening of the recess is 400 to 600 μm, and the depth of the recess is 150 to 250 μm.
[Item 4]
Item 8. The culture method according to any one of Items 1 to 3, wherein the medium of the cell population does not contain a substance that interferes with cell adhesion.
[Item 5]
Item 4. The culture method according to Item 4, wherein the substance that interferes with cell adhesion is methyl cellulose.
[Item 6]
Item 8. The culture method according to any one of Items 1 to 5, wherein the Tie2-positive stem / progenitor cell is a Tie2-positive stem / progenitor cell derived from the nucleus pulposus tissue of the intervertebral disc.
[Item 7]
A method for preparing a cell population containing target cells differentiated from the Tie2-positive stem / progenitor cell from a cell population containing the Tie2-positive stem / progenitor cell.
A culturing step for inducing differentiation of Tie2-positive stem / progenitor cells into target cells, which comprises the step of carrying out the culturing method according to any one of Items 1 to 6 (hereinafter referred to as "differentiation culture step").
Preparation method, including.
[Item 8]
Item 6. The preparation method according to Item 7, wherein the target cell is a cell expressing at least type II collagen.
[Item 9]
Item 8. The preparation method according to Item 8, wherein the cell expressing at least type II collagen is a nucleus pulposus cell.
[Item 10]
Item 7. Any one of Items 7 to 9, further comprising a culture step for amplifying Tie2-positive stem / progenitor cells in the cell population (hereinafter referred to as "amplification culture step") prior to the differentiation culture step. The preparation method described.
[Item 11]
Item 8. The preparation method according to any one of Items 7 to 10, wherein a cell population in which Tie2-positive stem / progenitor cells also remain is obtained by the differentiation culture step.
[Item 12]
A cell population obtained by the culture method according to any one of Items 1 to 6 or the preparation method according to any one of Items 7 to 11.
[Item 13]
A culture system comprising the culture base material defined in the culture method according to any one of Items 1 to 6 and a cell population housed in a recessed portion of the culture base material.
[Item 14]
Item 3. A composition for cell therapy containing the cell population according to Item 13.
[Item 15]
Item 4. The cell therapeutic composition according to Item 14, which is for treating or preventing a disease in which a disorder, degeneration or hernia of an intervertebral disc appears as a symptom.

The method for culturing Tie2-positive stem / progenitor cells of the present invention preferably is a differentiation culture step whose main purpose is to induce differentiation of Tie2-positive stem / progenitor cells in which the culture method is carried out into mature cells having a predetermined trait. It is possible to prepare a cell population rich in target cells by adopting a preparation method including a combination of the above-mentioned and the amplification culture step whose main purpose is to amplify Tie2-positive stem / progenitor cells. it can. By utilizing the cell population obtained based on the culture method and preparation method of the present invention, it is possible to efficiently produce an effective cell preparation for the treatment or prevention of a predetermined disease. Become.

Further, in the present invention, since it is not necessary to add a highly viscous component such as methyl cellulose used in the prior art described in the above-mentioned Patent Documents 1 and 2 to the medium, the Tie2-positive stem / It becomes possible to recover a cell population containing progenitor cells or target cells induced to differentiate from them from the medium without waste.

According to a typical embodiment of the present invention, a Tie2-positive stem / progenitor cell (nuclear nucleus stem cell) contained therein is used by using an intervertebral disc (nucleus pulposus) that can be collected only in a small amount by surgery of a herniated disk patient. Etc.), which are expected to have a high therapeutic effect when transplanted by efficiently proliferating and differentiating), and are rich in (and somewhat) functional nucleus pulposus cells with high extracellular matrix-producing ability such as type II collagen. It is possible to easily and inexpensively obtain a large amount of cell populations (where Tie2-positive stem / progenitor cells also remain) with high efficiency and reproducibility. Conventionally, it has not been possible to prepare such a suitable cell population from a living human-derived intervertebral disc (medullary nucleus), but since it is possible by the present invention, administration of the cell population (cell preparation containing) It will be much easier to regenerate the intervertebral disc, and industrialization will be realistic.

FIG. 1 is an optical micrograph of the surface of the culture substrate on the 7th day of culture using the culture container 4. FIG. 2 is a graph showing the results of the spherical colony formation rate (A) and the cell growth rate (B) in Example 1. FIG. 3 is a graph showing the results of mRNA expression levels of agrecan (Acan), angiopoietin 1 (AngPT1), type I collagen A2 (COL1A2) and type II collagen A1 (COL2A1) in Example 1. FIG. 4 is a graph showing the result of ΔDHI (DHI 3 months after transplantation-DHI 1 month after transplantation) in Example 2.

-the term-
"Stem cell" is a term that refers to cells that have self-renewal and differentiation potential (totipotent, pluripotent, multipotent or unipotent). "Progenitor cells" do not have self-renewal ability in a strict sense because they eventually become all terminally differentiated cells, but they have the ability to differentiate into predetermined cells while proliferating relatively actively. It is a term that refers to a cell having. Cells commonly understood (specified) by those skilled in the art by names that include "stem cells" or "progenitor cells" fall under "stem cells" or "progenitor cells" herein.

In the present specification, "stem cell and / or progenitor cell" is a notation that includes stem cell, progenitor cell, or both, and may be referred to as "stem / progenitor cell". Further, in the present specification, a cell population containing stem cells and / or progenitor cells is referred to as a "stem / progenitor cell population", and a cell population containing mature cells (terminal differentiated cells) differentiated from stem cells and / or progenitor cells. May be referred to as "mature cell population".

"Stem cells" and "progenitor cells" are generally distinguished from other cells by the positive or negative expression of one or more specific genes (marker genes, cell markers). Can be done. That is, "stem cells" and "progenitor cells" having self-renewal ability and / or differentiation ability as described above can also be defined as terms referring to cells in which the expression of a specific marker gene is positive or negative, respectively.

Whether the expression of a marker gene (cell marker) is "positive" or "negative" depends on the expression of mRNA transcribed from the gene (genome) or protein translated from the mRNA according to a general method. The amount is measured quantitatively or qualitatively, and it is judged as positive when the expression level is above a certain level (or above a certain level) and negative when the expression level is below a certain level (or below a certain level). can do. The expression level of a protein can be measured quantitatively or qualitatively by an immunological assay using an antibody and a labeling agent specific to the protein, such as flow cytometry, immunostaining, and ELISA. The Tie2 protein is a protein expressed on the cell surface, and Col2 is a protein expressed inside the cell, which is suitable as a method (immunofluorescent staining method, etc.) for detecting the protein existing on the cell surface and inside the cell, respectively. Should be used. The expression level of mRNA is quantitatively or qualitatively measured by an assay using (complementary) nucleic acid specific to the mRNA such as RT-PCR, microarray, biochip, and a labeling agent or nucleic acid amplification method (means). Can be measured. The ratio (positive rate or negative rate) of cells in the cell population in which the expression of a predetermined marker gene (cell marker) is positive or negative is determined by the number of all cells in the cell population and the method as described above. Alternatively, the number of cells determined to be negative can be calculated by various methods as described above, for example, by measurement in flow cytometry.

In the present specification, "stem cells and / or progenitor cells positive for Tie2 expression", that is, "Tie2-positive stem / progenitor cells" is known as one of the cell markers, Tie2 (tyrosine kinase with Ig and EGF homology). A cell having a trait as a stem cell and / or a progenitor cell whose expression of domain-2) is determined to be positive for expression as a protein by, for example, flow cytometry. Representative Tie2-positive stem / progenitor cells in the present invention are Tie2-positive stem / progenitor cells "derived from the nucleus pulposus tissue of the intervertebral disc", that is, Tie2-positive (recoverable from the nucleus pulposus) present in the nucleus pulposus of the intervertebral disc. It is a Tie2-positive stem / progenitor cell obtained by subculturing the stem / progenitor cell or its Tie2-positive stem / progenitor cell, and is a cell corresponding to the "nucleus-nucleus stem / progenitor cell" described below.

In the present specification, the "target cell" is a cell having functionality according to a use obtained from a Tie2-positive stem / progenitor cell by a predetermined differentiation induction, and more specifically, a predetermined gene (cell marker). Refers to cells whose expression is determined to be positive or negative, for example by flow cytometry. A typical target cell in the present invention is one of the "medullary nucleus cells" described below, which is positive for the expression of extracellular matrix (ECM) genes such as type II collagen (Col2) and agrecan.

In the present invention, "medullary nucleus cell" refers to a mature cell that has reached terminal differentiation, or a cultured cell having a trait equivalent thereto, which occupies the majority of the cell population in the intervertebral disc (medullary nucleus). Specifically, the nucleus pulposus cells are negative for Tie2 and GD2 (and usually positive for CD24) as marker genes, and are positive for at least type II collagen in the extracellular matrix (more usually). It can be defined as a cell (which is also positive for proteoglycan (aggrecan)). For example, cells determined by flow cytometry to be negative for Tie2 and GD2 (and positive for CD24) and positive for type II collagen (and positive for agrecan) as proteins (cell markers) are the present invention. Corresponds to the nucleus pulposus cells in. For extracellular matrices such as type II collagen and agrecan, the amount of protein produced may be measured by flow cytometry, and the amount of expression of their mRNA may be measured by real-time PCR or the like.

In the present invention, the "medullary nucleus stem / progenitor cell" is a progenitor cell (progenitor cell) that occupies a part of the cell population in the nucleus pulposus tissue of the intervertebral disc and has at least the ability to differentiate into the nucleus pulposus cell. Stem cells (nucleus stem cells) that have the ability to differentiate into and self-renewal, or cultured cells that have similar traits. Nucleus stem / progenitor cells can be specifically defined as Tie2 and / or GD2 positive cells as marker genes. For example, a cell determined by flow cytometry to be either Tie2 positive and GD2 negative, Tie2 positive and GD2 positive, or Tie2 negative and GD2 positive as a protein (cell marker). , Corresponds to the nucleus pulposus stem / progenitor cell in the present invention.

In the above-mentioned Patent Document 2, based on the expression state of Tie2 and GD2 as cell markers of the nucleus pulposus-derived cells, the cells positive for Tie2 are referred to as "intervertebral nucleus nucleus stem cells" (of which, the cells negative for GD2 are dormant. Those in a state, those in which GD2 is positive are in an active state), cells in which Tie2 is negative and GD2 are positive are "disc precursor cells", and cells in which Tie2 is negative and GD2 is negative are "differentiated". Classified as "finished, mature intervertebral nucleus pulposus cells" (paragraphs 0024, 0025, 0032). Further, in Patent Document 2, regarding the cells appearing in the differentiation hierarchy of nucleus pulposus cells, (i) Tie2 positive and GD2 negative (further CD24 negative, CD44 positive / negative, CD271 positive, Flt1 positive) cells, (ii) Tie2. Cells that are positive and GD2 positive (further CD24 negative, CD44 positive, CD271 positive, Flt1 positive), (iii) Tie2 negative and GD2 positive (further CD24 negative, CD44 positive, CD271 positive / negative, Flt1 positive / negative). Cells, (iv) Tie2 negative and GD2 positive (further CD24 positive, CD44 positive, CD271 negative, Flt1 negative), (v) Tie2 negative and GD2 negative (further CD24 positive, CD44 positive, CD271 negative, Flt1 negative) The above (i) to (iii) are classified into "cells" (NP stem / progenitor cells), and the above (iii) to (v) are classified as "cells". The notation "NP committed cells" is used (see Fig. 7-2). Although the notation is different, the “medullary nucleus medullary stem cells” and “intervertebral medullary nucleus progenitor cells” of Patent Document 2, that is, the cells (i) to (iv) above are the “medullary nucleus stem / progenitor cells” in the present invention. It corresponds to a "cell", and the "mature intervertebral disc nucleus pulposus cell that has completed differentiation" in Patent Document 2, that is, the cell of (v) above corresponds to the "marrow nucleus pulposus cell" in the present invention. If necessary, the cells in the present invention are defined as those according to the definition described in Patent Document 2 (particularly, the definition of positive or negative for one or more cell markers other than Tie2 and GD2 such as CD24). Can be replaced with.

In the present invention, a "spherical colony" is a spherical cell aggregate that includes stem cells and / or progenitor cells, and may further contain cells differentiated from them. The "spherical colony" is also generally referred to by those skilled in the art as "sphere", "spheroid", etc., and is a "discosphere" or a "floating sphere structure" (free floating) in Patent Document 2 above. circular-spherical structure) also corresponds to a "spherical colony".

-Culture method-
The method for culturing a cell population containing Tie2-positive stem / precursor cells according to the present invention is "a bank portion interposed between a plurality of recesses forming a separable chamber in which a culture medium is cultured and adjacent recesses. Is on the upper surface of the plate-shaped culture substrate, and the adjacent bank portion and the recessed portion are continuous curved surfaces, and a plurality of the recessed portions are formed on the upper surface of the culture substrate and are densely arranged. , At least the inner surface of the recess is a culture medium in which the inner surface of the recess is coated with a cell adhesion inhibitor ”(a culture vessel provided with the same), and the diameter and depth of the opening of the recess are within a predetermined range. It is in.

In a typical embodiment of the present invention, the cell population containing Tie2-positive stem / progenitor cells is a cell population derived from the nucleus pulposus tissue of the intervertebral disc. In a exemplary embodiment of the invention, cells differentiated from Tie2-positive stem / progenitor cells produce and secrete more extracellular matrix than common cells, eg, extracellular matrix in intervertebral disc nucleus tissue. It is a nucleus pulposus cell that plays a role of producing and secreting. Mature nucleus pulposus cells express at least type II collagen as an extracellular matrix, and also express an extracellular matrix such as proteoglycan (aggrecan). In a preferred embodiment of the present invention, cells expressing extracellular matrix such as type II collagen and proteoglycan (agrecan) from Tie2-positive stem / precursor cells, particularly type II collagen, are expressed not only as mRNA but also as protein. It is intended to differentiate functional collagen cells with excellent amount (production amount).

<Culture medium>
In the culture method of the present invention, or the step of the amplification culture step in which the method is carried out, on a culture substrate having a predetermined recess and a bank portion as described above, or on the surface of a culture container provided with the culture substrate. In, a predetermined cell population is cultured. Such a culture substrate and a culture container provided with the same are known, and for details thereof, basically the above-mentioned Patent Document 3 can be referred to, and "EZSPHERE (registered trademark)" (AGC Techno Glass Co., Ltd.) ) Can be purchased.

In the present invention, the diameter (X) of the opening of the recess is 400 to 1000 μm, and the depth (Y) of the recess is 50 to 500 μm. For example, the X / Y combination can be about 500 μm / about 100 μm, about 500 μm / about 200 μm, about 800 μm / about 400 μm, about 800 μm / about 300 μm, and the like. In addition, "about" is a range in which the difference (variation) between the catalog value (standard value) for "EZSPHERE (registered trademark)" (AGC Techno Glass Co., Ltd.) and the value in the actual product can be accommodated. For example, ± 50 μm, ± 25 μm, ± 20 μm, ± 10 μm, ± 5 μm. Further, when the recessed portion has a substantially elliptical shape, its "major axis" is considered to correspond to the above "diameter".

In a preferred embodiment of the present invention, the diameter (X) of the opening of the recess is 400-600 μm and the depth (Y) of the recess is 150-250 μm, for example, the combination of X / Y is about. It is 500 μm / about 200 μm.

In a particularly preferred embodiment of the present invention, the diameter (X) of the opening of the recess is 600 to 1000 μm and the depth (Y) of the recess is 350 to 450 μm, for example, the combination of X / Y. It is about 800 μm / about 400 μm.

The "opening diameter" and "depth" of the recessed portion in the present invention are synonymous with those in Patent Document 3 mentioned above, and are displayed in the catalog of EZSPHERE (registered trademark) (AGC Techno Glass Co., Ltd.). The "caliber" and "depth" of the "well" that is present can be considered to correspond to them.

A culture container provided with a culture substrate having a predetermined recess and a bank can take the form of a dish, a microplate, or the like. The dish has a diameter of, for example, 35 mm, 100 mm, or the like. In one aspect of the invention, the dish diameter is preferably 20-50 mm, for example 35 mm. The microplate has, for example, 6 wells, 24 wells, 96 wells, etc., and the bottom surface of each well is a culture medium having a predetermined recess and a bank (a plurality of densely arranged recesses). Is formed).

For the cell suspension added to the culture vessel provided with the culture substrate, consider the number of depressions on the culture substrate and the area of the region where the depressions are formed (the density of the depressions calculated from them). However, it can be prepared as a suspension containing cells at an appropriate density. The cell density in the cell suspension can usually be adjusted in the range of ^{1 × 10 3} to 1 × 10 ^{5 cells / ml.} For example, when a cell suspension is added to a 35 mm dish provided with a culture substrate in which a recess having the predetermined opening diameter and depth is formed, a cell suspension containing about 10,000 cells / ml cells is added. A liquid can be used.

Examples of the cell adhesion inhibitor that covers at least the inner surface of the recessed portion include a lipid phosphate polymer, polyhydroxyethyl methacrylate, polyethylene glycol, and the like. Examples of the material of the culture base material include a transparent synthetic resin such as polystyrene.

On the other hand, the step of the amplification culture step, which is performed as necessary in the preparation method described later, is performed using a general or known culture container (base material), culture device, or the like, depending on the embodiment. Can be done. As the culture vessel, a flask, a dish, a plate, a bag, or the like having a general shape can be used, and a well having a well for accommodating cells may be formed. As the culture container, one made of a general material such as glass, plastic, or resin can be used. The surface of the culture vessel (base material) may be untreated, or may have been subjected to a treatment related to cell adhesion or other treatment. The size (area, volume) of the culture vessel, and if the culture vessel has wells, the size (caliber, depth) and number of the wells can be appropriately selected. If necessary, the cell population may be cultured while shaking or rotating the culture vessel and stirring the medium.

-Preparation method (culture stage)-
The method for preparing a cell population containing a target cell differentiated from a Tie2-positive stem / progenitor cell from a cell population containing a Tie2-positive stem / progenitor cell according to the present invention includes at least the following differentiation culture steps, preferably. Includes both amplification and differentiation culture stages (amplification culture stage first, differentiation culture stage later):
Amplification culture stage: A culture stage for enhancing Tie2 expression in Tie2-positive stem / progenitor cells and amplifying Tie2-positive stem / progenitor cells in a cell population;
Differentiation culture stage: A culture stage for inducing differentiation of Tie2-positive stem / progenitor cells into target cells.

-Steps related to the differentiation culture stage In the preparation method of the present invention, the culture method of the present invention is carried out in the step of the differentiation culture stage. The main purpose of the "step of differentiation culture stage" is to differentiate Tie2-positive stem / progenitor cells into predetermined cells by culturing according to predetermined conditions, and the action and effect for that purpose (more than other action and effects). It means a process that is played (relatively strongly). That is, if the number and / or ratio of the target cells is higher in the post-cultured cell population than in the pre-cultured cell population, the culturing step can be said to be a “differentiation culture step”.

-Steps relating to the amplification culture step In a preferred embodiment of the present invention, the steps of the amplification culture step are carried out before the step of the differentiation culture step. The main purpose of the "step of amplification culture stage" is to amplify Tie2-positive stem / progenitor cells by culturing according to predetermined conditions, and the action and effect for that purpose (relatively stronger than other action and effects). ) Means the process to be performed. In other words, if the number and / or ratio of Tie2-positive stem / progenitor cells is higher in the post-cultured cell population than in the pre-cultured cell population, the culturing step is the "amplification culturing step". It is permissible for Tie2-positive stem / progenitor cells to differentiate into other cells (target cells) within that limit.

Examples of the step of the amplification culture step include a step of culturing a cell population containing Tie2-positive stem / progenitor cells in a medium to which a growth factor having a Tie2 expression enhancing action is added. Growth factors having a Tie2 expression-enhancing effect include, for example, FGF and / or EGF.

<Cell population>
In a cell population containing Tie2-positive stem / progenitor cells (collectively referred to as "pre-culture cell population" in the present specification) used in the culture method or culture method (steps of each step contained therein) of the present invention. The ratio and / or number of Tie2-positive stem / progenitor cells and other cells (cells differentiated from Tie2-positive stems / progenitor cells, etc.) are basically arbitrary, and Tie2-positive stem cells and Tie2-positive progenitor cells The ratio of is basically arbitrary. The composition of the pre-culture cell population can be appropriately adjusted according to the embodiment of the invention, taking into consideration the culture method of the present invention, the action and effect in each culture step, and the like.

The pre-cultured cell population can be prepared or prepared according to a conventional method. For example, when using a cell population contained in the intervertebral disc nucleus pulposus tissue collected from the body as a pre-cultured cell population, the nucleus pulposus tissue is first chopped to an appropriate size using an instrument such as a scissors (eg: After mincing a few millimeters square), the cells are then treated with a proteolytic enzyme such as collagenase to disperse the cells, and if necessary, treatments such as filtration, centrifugation, and washing are performed to form the nucleus pulposus tissue. The contained cell population can be isolated and recovered.

The cell population separated from the tissue prepared as described above can be cryopreserved according to a conventional method until it is subjected to the next culturing method or culturing step. The cryopreserved cell population can be thawed according to a conventional method when starting the next culture method or culture step. Treatments preferred for the cell population or tissue may be combined during cryopreservation and thawing. For example, a cryoprotectant (DMSO or the like) may be added during cryopreservation, in which case the cryoprotectant may be removed under appropriate conditions during thawing.

In a cell population containing Tie2-positive stem / progenitor cells (collectively referred to as "post-cultured cell population" in the present specification) obtained by the culture method or preparation method of the present invention (steps contained therein), Tie2 The ratio and / or number of positive stem / progenitor cells and other cells (cells differentiated from Tie2-positive stems / progenitor cells, etc.) is basically arbitrary, and Tie2-positive stem cells and Tie2-positive The proportion of progenitor cells is also basically arbitrary. The composition of the cell population after culturing can be appropriately adjusted according to the embodiment of the invention, taking into consideration the use of the cell population obtained by the culturing method or the culturing step of the present invention.

After culturing, the cell population can be recovered from the medium according to a conventional method and subjected to the next culturing method or culturing step, or can be subjected to other methods or steps such as preparation of cell preparations.

In a cell population containing Tie2-positive stem / progenitor cells (collectively referred to as "cultivated cell population" in the present specification) during the culture method or preparation method (steps of each step contained therein) of the present invention, Tie2 The ratio of positive stem / progenitor cells to other cells (cells differentiated from Tie2-positive stems / progenitor cells, etc.) is basically arbitrary, and the ratio of Tie2-positive stem cells to Tie2-positive progenitor cells is also basically arbitrary. It is optional. The composition of the culturing cell population is a composition in the process of transition from the pre-cultured cell population to the post-culturing cell population. For example, the ratio of Tie2-positive stem / progenitor cells in the culturing cell population (in the present specification, "Tie2-positive stem /". The "progenitor cell ratio") is usually a numerical value included in the range between the Tie2-positive stem / progenitor cell ratio of the pre-cultured cell population and the Tie2-positive stem / progenitor cell ratio of the post-cultured cell population. It is permissible that the value temporarily deviates from the relevant range. The composition of the cell population during culturing varies depending on the embodiment of the invention, the culturing method of the present invention, the number of days in each culturing step, the number of passages, and the like.

The "human or other animal" (donor) from which each of the above cell populations is derived is the use of the cell population finally obtained by the method for culturing Tie2-positive stem / progenitor cells of the present invention, or each of the methods included in the method. It can be selected in consideration of the culture method or the use of the cell population obtained by each culture step. In a typical embodiment of the present invention, when a cell population for producing a prophylactic or therapeutic cell preparation for a predetermined disease, symptom, etc. is prepared, "human or other animal" is the cell preparation. It is an organism of the same species as the administration target (recipient) of, preferably human.

-Cell Population Concerning Amplification Culture Stage In the present invention, the cell population (collectively referred to as "pre-amplification culture cell population" in the present specification) used for the steps in the amplification culture stage is typically human or other. It is a cell population (primary cultured cell population) contained in a tissue (intervertebral disc) collected from the body of an animal or a cell population obtained by subculturing the primary cultured cell population (passaged cultured cell population).

When a cell population contained in an intervertebral disc collected from a human is used as the pre-amplification culture cell population, the Tie2-positive stem / progenitor cell ratio generally tends to be high and the niche tends to be good, up to the 20s. It is preferable that the cell population is contained in the intervertebral disc collected from humans, and more preferably from humans in their teens. Further, the cell population before amplification culture is preferably a cell population having a Tie2-positive stem / progenitor cell ratio as high as possible, for example, 30% or more, 40% or more, 50% or more, 60% or more.

In addition, as the pre-amplification culture cell population, depending on the embodiment, a cell population that is not a cell population contained in a tissue collected from the body of a human or other animal, for example, a cell of a human or other animal is used. It may be a cell population containing Tie2-positive stem / progenitor cells obtained by inducing differentiation of pluripotent or pluripotent cells such as iPS cells or ES cells prepared in the above.

In the present invention, the cell population obtained by the step in the amplification culture step (collectively referred to as "the cell population after amplification culture" in the present specification) is a cell subjected to the step in the differentiation culture step (the culture method of the present invention). Used as a group. In the cell population after amplification culture in such an embodiment (use), it is preferable that the ratio of Tie2-positive stems / progenitor cells and / or the number of cells is as high as possible. The Tie2-positive stem / progenitor cell ratio in the post-amplification culture cell population varies depending on individual differences in the pre-amplification culture cell population and the nucleus pulposus tissue from which it is derived, and therefore cannot be unequivocally determined, but is preferably 5% or more, for example. Is 7% or more, 9% or more, 11% or more, 13% or more, and 15% or more. The number of Tie2-positive stems / progenitor cells in the post-amplification culture cell population varies depending on individual differences in the pre-amplification culture cell population and the nucleus pulposus tissue from which it is derived, and therefore cannot be unequivocally stated. Compared with the number of cells, for example, it is 5 times or more, preferably 10 times or more, 15 times or more, 20 times or more, 25 times or more, and 30 times or more.

-Cell population related to the differentiation culture stage In the present invention, the cell population (referred to as "pre-differentiation culture cell population" in the present specification) subjected to the step in the differentiation culture stage (the culture method of the present invention) is Tie2 positive in advance. It is preferably a cell population enriched with stem / progenitor cells. The Tie2-positive stem / progenitor cell ratio in the pre-differentiation culture cell population varies depending on the individual differences in the pre-amplification culture or post-amplification culture cell population and the nucleus pulposus tissue from which it is derived, and thus cannot be unequivocally stated. % Or more, preferably 7% or more, 9% or more, 11% or more, 13% or more, 15% or more. In one embodiment of the present invention, the pre-differentiation culture cell population differentiates the cell population obtained by the amplification culture step (post-amplification culture cell population), for example, a cell population containing amplified Tie2-positive stem / progenitor cells. It is a cell population divided so as to have an appropriate number of cells according to the embodiment of the culture stage (type, size, etc. of the culture vessel).

In addition, the pre-differentiation culture cell population is contained in a cell population (for example, a tissue (intervertebral disc) collected from the body of a human or other animal) that was not obtained by the amplification culture step depending on the embodiment. Cell population ((primary cultured cell population) or cell population obtained by subculturing the primary cultured cell population (passaged cultured cell population), or iPS cells or ES prepared using human or other animal cells It may be a cell population containing Tie2-positive stems / precursor cells obtained (via Tie2-positive stems / precursor cells) by inducing differentiation of pluripotent or pluripotent cells such as cells.

In the present invention, the use of the cell population obtained by the step in the differentiation culture stage (collectively referred to as "post-differentiation culture cell population" in the present specification) is not particularly limited, and the composition of the obtained cell population and the like are not particularly limited. It can be adjusted as appropriate according to the application. For example, for a cell population used to produce a cell preparation for transplantation, a target cell having functionality useful for producing a therapeutic or prophylactic effect by transplantation (for example, a nucleus pulposus cell producing II collagen: Col2). It is preferable that the cell population contains as much positive medullary nuclei cells as possible, and at the same time, contains some Tie2-positive stems / progenitor cells (for example, medullary nucleus stems / progenitor cells) that have the ability to produce such target cells. ..

The Col2-positive (medullary nucleus) cell rate in the post-differentiation culture cell population varies depending on the individual differences in the pre-differentiation culture cell population and the nucleus pulposus tissue from which it is derived, and therefore cannot be unequivocally stated. It is preferably 10% or more, 15% or more, 20% or more, 25% or more, and 30% or more.

The Tie2-positive (medullary nucleus) stem / progenitor cell ratio in the post-differentiation culture cell population varies depending on the individual difference in the pre-differentiation culture cell population and the nucleus pulposus tissue from which it is derived, and therefore cannot be unequivocally stated. % Or more, preferably 2% or more, 4% or more, 6% or more, 8% or more, 10% or more.

The number of cells contained in the cell population usually increases even in the differentiation culture step. The number of cells in the post-differentiation culture cell population (Col2-positive cells, Tie2-positive stem / precursor cells, etc.) varies depending on the pre-differentiation culture cell population and individual differences in the nucleus pulposus tissue from which it is derived. However, it is, for example, 2 times or more, 5 times or more, 10 times or more, 20 times or more, 50 times or more, and 100 times or more as compared with the number of cells in the cell population before differentiation and culture.

<Medium>
The medium used in the culturing method or preparation method (steps contained therein) of the present invention may be any medium suitable for culturing Tie2-positive stem / precursor cells and cells that differentiate from them, and the culturing method or culturing step. Appropriate basal medium and additive components can be selected in consideration of the purpose of the above. Additive components are suitable for amplification culture of Tie2-positive stem / progenitor cells if the culture step is in the amplification culture stage, and from Tie2-positive stem / progenitor cells if the culture step is in the differentiation culture stage. Those suitable for inducing differentiation into target cells are selected.

In a typical embodiment of the present invention, when the nucleus pulposus stem / progenitor cells and the nucleus pulposus cells differentiated from them are cultured, the mediums for each step of the amplification culture step and the differentiation culture step are based on, for example, as follows. It can be prepared by using an appropriate amount of each of the medium, additive components, growth factors, and other components.

As the basal medium, for example, DMEM (Dulbecco modified Eagle medium, with or without glucose addition), αMEM (Eagle's minimum essential medium α modified type), Ham'sF-10 medium, Ham'sF-12 medium, or a mixture thereof. Can be mentioned.

Examples of additive components for amplification culture or differentiation culture include FBS (fetal bovine serum), BSA (bovine serum albumin), L-ascorbic acid (as L-ascorbic acid phosphate magnesium salt, etc.), and selenous acid (as selenite). Examples include insulin-transferrin-sodium selenite (ITS: Insulin-Transferrin-Selenium) and the like) and 2-mercaptoethanol. If necessary, antibiotics such as penicillin and streptomycin and other components may be added to the medium.

Examples of growth factors include FGF (fibroblast growth factor), EGF (Epidermal Growth Factor), PDGF (platelet-derived growth factor), and Ang-1 (angiopoetin). -1) can be mentioned. In one embodiment of the present invention, it is preferable to use at least FGF as the growth factor to be added to the medium, more preferably to use both FGF and EGF, and to use Ang-1 in addition to them as necessary. It is also preferable.

As the FGF, for example, bFGF (basic fibroblast growth factor: a basic fibroblast growth factor, sometimes called FGF-2) can be used. The concentration of FGF in the medium can be usually in the range of 1-50 ng / mL, preferably in the range of 5-15 ng / mL, for example about 10 ng / mL.

Ang-1 is preferably added in a serum-free medium. Further, as Ang-1, those solubilized in water (Solute Ang-1, Recombinant Ang-1) are preferable. The concentration of Ang-1 (preferably soluble Ang-1) in the medium can usually be in the range of 100-1000 ng / mL, for example about 500 ng / mL.

In one aspect of the present invention, the culture method of the present invention or the medium used in the differentiation culture step in which it is carried out does not contain substances that interfere with cell adhesion. In the present invention, by using a culture substrate (equipped with a culture substrate) having a predetermined recess and bank, the cell growth rate can be increased without adding a substance such as methyl cellulose that interferes with cell adhesion to the medium. It is possible to obtain a cell population that is excellent and is rich in functional nucleus pulposus cells that are highly capable of producing extracellular matrix such as type II collagen and agrecan.

In one aspect of the present invention, the culture medium used in the culture method of the present invention or the differentiation culture step in which it is carried out contains Ham's F-10 medium and DMEM as basal medium (for example, in a ratio of 40:60) and is added. FBS (for example, 30%) as a component, BSA (e.g., 1%), selenious acid (e.g. 0.01%), 2-mercaptoethanol (e.g. 5 × ^{10 -5} M) and L- ascorbic acid (e.g. 0.075mg / Ml) and contains bFGF (eg 10 ng / ml) and EGF (eg 100 ng / ml) as growth factors. By using a medium having such a composition (not containing a substance that interferes with cell adhesion such as methyl cellulose) with a culture substrate (with a culture substrate) having a predetermined depression and bank, the cell growth rate Can be further improved.

<Culture period and other conditions>
_{The duration and other conditions (eg, pH, CO 2} concentration, O ₂ concentration, etc.) of the culturing method and culturing method (steps contained therein) of the present invention are basically the objectives of the culturing method and preparation method. Therefore, it can be appropriately adjusted so as to obtain a cell population having a desired cell composition (type and number / ratio). The pH can be weakly alkaline (eg, about 7.15). The CO ₂ concentration can be, for example, about 5%. The O ₂ concentration can be 5% or less (for example, about 2%). During the culturing method and culturing step (each step) of the present invention, the medium is replaced with a fresh one at predetermined days as needed, or components are added or components are added after a predetermined number of days. The medium may be changed or the atmosphere may be changed by increasing or decreasing the concentration or pH.

The period of the amplification culture stage is usually about 1 to 3 weeks. For example, the period of the culture step using the FGF-added medium can be about one week, and such a culture step can be repeated a plurality of times, for example, twice in the amplification culture step. The amplification culture step may be terminated when the desired post-amplification culture cell population is obtained.

The period of the differentiation culture stage (the culture method of the present invention) is usually about 1 to 3 weeks, for example, about 2 weeks. In addition, the duration of other steps that can optionally be included in the differentiation culture step of the present invention is about the same. The differentiation culture step may be terminated when the desired post-differentiation culture cell population is obtained.

-Cell therapy composition-
The cell therapeutic composition of the present invention contains the cell population obtained by the culture method or preparation method of the present invention as described above, and optionally contains other pharmaceutically acceptable components. Can be done.

In a representative embodiment of the present invention, the cell therapeutic composition comprises Col2-positive nucleus pulposus cells differentiated from the nucleus pulposus stem / progenitor cells (preferably also including Tie2-positive stems / progenitor cells). Is. The target of application of the cell therapeutic composition in the embodiment, that is, the disease that can be prevented or treated by administering the composition, is a disease in which a disorder, degeneration, herniated disk, etc. of the intervertebral disc (medullary nucleus) appears as a symptom. For example, lumbar or cervical spinal disc disease, herniated disk, cervical spondylotic myelopathy, radiculopathy, spondylolisthesis / spondylolisthesis, lumbar spinal canal stenosis, lumbar degenerative spondylolisthesis, lumbar degenerative scoliosis and the like can be mentioned.

The dosage form of the cell therapy composition of the present invention may be any one that can be transplanted or delivered to a site targeting a cell population (for example, the nucleus pulposus of an intervertebral disc), and for example, an injection, preferably an intervertebral disc (the nucleus pulposus). Alternatively, it can be an injection for local administration to the vicinity thereof, or an injection for vascular administration that can be targeted.

Pharmaceutically acceptable components include, for example, water for injection or physiological saline when prepared as an injection, a culture solution for a cell population, other suitable solvent / dispersion medium, other additives, and the like. ..

The cell therapy composition of the present invention may be administered in an amount effective for exerting a desired therapeutic or preventive effect. Such an effective amount is determined by taking into consideration the components, dosage form, administration target, administration route, other embodiments, etc. of the cell therapy composition, and the dose per administration, the number of administrations, and the administration interval (for a certain period of time). It can be adjusted as appropriate depending on the number of administrations) and the like. The cell therapeutic compositions of the present invention can be applied to humans and non-human vertebrates.

[Example 1]
Primary human nucleus pulposus cells (n = 3) collected and cryopreserved were used with the consent of the person undergoing the operation at the time of herniated disc surgery. The nucleus pulposus cells were suspended in αMEM medium (Nacalai Tesque) supplemented with 10% FBS at 10,000 cells / ml. 2 ml of this cell suspension was seeded on each of the six types of "EZSPHERE" (registered trademark, IWAKI) products shown in Table 1. The cells were cultured at 37 ° C., 5% CO ₂ , 5% O _{2 for} 14 days, and the spherical colony formation rate (well occupancy rate) and cell growth rate were measured. The spherical colony formation rate was calculated as the ratio of the number of wells in which spherical colonies were formed to the total number of wells contained in the field of view by taking an optical micrograph of the surface of the culture medium. FIG. 1 shows an optical micrograph of the surface of the culture substrate on the 7th day of culture using the culture vessel 4.

In addition, the expression level of mRNA of extracellular matrix and the like (aggrecan, type I collagen (COL1A2), type II collagen (COL2A1) and angiopoietin 1) in the cells after the above culture was measured by a real-time PCR method. As a control, a methyl cellulose medium (“Methocult” manufactured by Stem Technology) was used instead of the above “10% FBS-added αMEM medium”, and a normal 35 mm dish (without low adhesive coating) was used instead of “EZSPHERE” as a culture container. ) Was used, the primary human nucleus pulposus cells were cultured in the same manner, and the expression levels were compared.

The results of the spherical colony formation rate and the cell growth rate are shown in FIG. Regarding the spherical colony formation rate (A), the result of the culture vessel 4 (903) was the best (about 45%), which was significantly higher than that of any of the other five types. In addition, the results of the culture vessel 3 (902) and the culture vessel 5 (904) were also relatively high. Regarding the cell growth rate (B), the culture container 4 (903) is excellent (about 7.5 times), and the culture container 3 (902), the culture container 2 (900), and the culture container 4 (903) are also the same. It was an excellent result.

Regarding the cell growth rate, in the culture vessel 4, instead of the above-mentioned "10% FBS-added αMEM medium", a medium obtained by adding 100 ng / mL EGF and 100 ng / mL PDGF to the medium was used as a primary human. When the nucleus pulposus cells were cultured, the cell growth rate increased to 62.5 times (not shown).

The results of the expression level of extracellular matrix, etc. are shown in FIG. The expression level of type II collagen (COL2A1) mRNA, which is important as the extracellular matrix of nucleus pulposus cells, was extremely high (at least several hundred times) compared with the control (methyl cellulose medium / normal dish) in each set. ..

[Example 2]
As the cultured human nucleus pulposus cells for transplantation, a cell population obtained in the same manner as the culture method using the culture vessel 4 of Example 1 and the “αMEM medium containing 10% FBS” was used. ^{1 × 10 5} cultured human nucleus pulposus cells were mixed with 25 μL of hyaluronic acid (the product of the present invention) and transplanted into the caudal disc of a rat model of intervertebral disc degeneration by injection. In addition, PBS alone, a mixture of human fibroblasts and hyaluronic acid, and a mixture of cell population and hyaluronic acid obtained in the same manner as the culture method using the usual 35 mm dish and methylcellulose medium of Example 1 (conventional). Each of the products) was also transplanted by injection into the caudal disc of a rat model of disc degeneration.

Immediately after transplantation, and one month, two months, and three months after transplantation, X-rays were taken for each of the degenerated model in which each of the above was transplanted and the degenerative model in which nothing was transplanted, and the intervertebral disc height at the transplantation site ( DHI) was measured. The DHI measurement method was based on the literature of Hu et al. (J Orthoped Res. 2018 Jan; 36 (1): 202-211. Doi: 10.1002 / jor.23628. Epub 2017 Jul 9.). The difference between DHI immediately after transplantation and DHI 1 month after transplantation is "DHI1", the difference between DHI immediately after transplantation and DHI 2 months after transplantation is "DHI2", DHI immediately after transplantation and DHI 3 months after transplantation. The difference between the two was defined as "DHI3", and the value of DHI3-DHI1 (referred to as ΔDHI) was used as an index for evaluating the regeneration effect by transplantation.

The results are shown in Fig. 4. The ΔDHI of the product of the present invention was significantly higher than that of the degenerated model ΔDHI that had not undergone transplantation surgery (P <0.05, Unpaired t-test), and a regeneration effect by transplantation was observed.

Claims

A plurality of recesses forming a separate chamber in which the culture medium is cultured and a bank portion interposed between the adjacent recesses are located on the upper surface of the plate-shaped culture base material, and the adjacent bank portions and the recessed portions are present. Is a continuous curved surface, and a plurality of the recessed portions are formed on the upper surface of the culture substrate and are densely arranged, and at least the inner surface of the recessed portions is coated with a cell adhesion inhibitor. In,
A method for culturing a cell population containing stem cells and / or progenitor cells (hereinafter referred to as "Tie2-positive stem / progenitor cells") that are positive for expression of Tie2 (tyrosine kinase with Ig and EGF homology domain-2).
A culture method in which the diameter of the opening of the recess is 400 to 1000 μm and the depth of the recess is 50 to 500 μm.
The culture method according to claim 1, wherein the diameter of the opening of the recess is 600 to 1000 μm, and the depth of the recess is 350 to 450 μm.
The culture method according to claim 1, wherein the diameter of the opening of the recess is 400 to 600 μm, and the depth of the recess is 150 to 250 μm.
The culture method according to any one of claims 1 to 3, wherein the medium of the cell population does not contain a substance that interferes with cell adhesion.
The culture method according to claim 4, wherein the substance that interferes with the cell adhesion is methyl cellulose.
The culture method according to any one of claims 1 to 5, wherein the Tie2-positive stem / progenitor cell is a Tie2-positive stem / progenitor cell derived from the nucleus pulposus tissue of the intervertebral disc.
A method for preparing a cell population containing target cells differentiated from the Tie2-positive stem / progenitor cell from a cell population containing the Tie2-positive stem / progenitor cell.
A culture step for inducing differentiation of Tie2-positive stem / progenitor cells into target cells (hereinafter referred to as "differentiation culture step"), which comprises the step of carrying out the culture method according to any one of claims 1 to 6. )
Preparation method, including.
The preparation method according to claim 7, wherein the target cell is a cell expressing at least type II collagen.
The preparation method according to claim 8, wherein the cell expressing at least type II collagen is a nucleus pulposus cell.
Any one of claims 7 to 9, further comprising a culture step for amplifying Tie2-positive stem / progenitor cells in the cell population (hereinafter referred to as "amplification culture step") prior to the differentiation culture step. The preparation method described in.
The preparation method according to any one of claims 7 to 10, wherein a cell population in which Tie2-positive stem / progenitor cells also remain is obtained by the differentiation culture step.
A cell population obtained by the culture method according to any one of claims 1 to 6 or the preparation method according to any one of claims 7 to 11.
A culture system comprising the culture substrate specified in the culture method according to any one of claims 1 to 6 and a cell population housed in a recessed portion of the culture substrate.
A composition for cell therapy containing the cell population according to claim 13.
The cell therapeutic composition according to claim 14, which is for treating or preventing a disease in which a disorder, degeneration or hernia of an intervertebral disc appears as a symptom.