KR20210018345A - Umbilical cord mesenchymal stem cell and method for producing a cell sheet thereof - Google Patents

Abstract

A method for producing umbilical mesenchymal stem cells is disclosed, which can significantly improve cell yield. According to the umbilical mesenchymal stem cell sheet and its manufacturing method, the umbilical mesenchymal stem cell contains umbilical mesenchymal stem cells and all extracellular matrix and growth factors secreted during the sarcoma process thereof; The manufacturing method does not use enzymes and analogs for digestion or does not perform physical stripping, completely maintains the umbilical mesenchymal stem cells and extracellular matrix and growth factors secreted during the breeding process thereof, and separates them from the surface of the culture dish. Thus, a layer-shaped cell sheet can be obtained.

Description

Umbilical cord mesenchymal stem cell and method for producing a cell sheet thereof

This application claims priority to the Chinese patent application of application number CN 201910149006.8 filed on February 28, 2019 under the name "Umbilical Cord Mesenchymal Stem Cells and Method for Manufacturing Cell Sheets thereof". The entire contents of the above Chinese patent application disclosure are incorporated by reference as part of this application.

<Technical field>

The present disclosure relates to the field of regenerative medicine and cell biology, and in particular to methods of producing umbilical mesenchymal stem cells as well as umbilical mesenchymal stem cell sheets and methods of making the same.

Umbilical mesenchymal stem cells (MSCs) are a kind of multifunctional stem cells that exist in neonatal umbilical tissues, which can differentiate into many types of tissue cells and have a wide clinical application prospect. Currently, there are two commonly used methods for umbilical tissue separation, enzymatic digestion and tissue block adhesion; Enzymatic digestion is expensive, complex to work, difficult to control, can cause damage to cells or cause cell mutations, resulting in high risk for clinical application, whereas tissue block adhesion methods are simple to operate and , It is inexpensive, causes less cell damage and is suitable for clinical application. However, the time period for obtaining umbilical mesenchymal stem cells by conventional tissue block adhesion is long, resulting in a small number of primary cells and low purity. The number of cells does not meet international standards for clinical application, resulting in difficulties in clinical application. Therefore, there is a need in the related art for a method of producing umbilical mesenchymal stem cells with high yield.

In addition, protocols of direct injection of a cell suspension of umbilical mesenchymal stem cells or transplantation after combination with tissue engineered scaffold materials are mainly used in current basic research and clinical applications for self-tissue repair, all of which have certain limitations. Have. Whereas direct injection of stem cell suspension results in the loss of large numbers of stem cells, low cell utilization, and limited ability of stem cells to perform tissue repair; Cellular and tissue engineered scaffolds post-combination transplantation solves the problem of cell loss, but the scaffold material can lead to different degrees of inflammation in the body, and the breakdown products of the material change the microenvironment of the local tissue, resulting in more serious lesions. Can lead to

Cell sheet technology is a new technology for stem cell transplantation and application. The cell sheet forms an endogenous scaffold through the extracellular matrix secreted by the cell itself, which is good for interactions between cells, interactions between cells and extracellular matrix, and transfer of genetic information. In a particular sense, cell sheet engineering is capable of maximizing the process of embryonic tissue development with effects and values that far exceed all exogenous biological scaffold materials.

<Summary>

In a first aspect, the present disclosure provides a method of producing umbilical mesenchymal stem cells, which significantly improves cell yield (i.e., the expansion fold for one cell passage) by adding media in batches. Specifically, the method for producing umbilical cord mesenchymal stem cells of the present disclosure comprises the following steps: a. Plating the umbilical cord tissue block in the culture vessel; b. Adding complete medium as a batch feed for cultivation; c. Isolating the cells attached to the culture vessel to obtain umbilical mesenchymal stem cells.

In certain embodiments, complete medium is added to the culture vessel in batches 2-5 in step b, wherein complete medium is added in an amount that keeps the umbilical tissue block wet but does not cover the umbilical tissue block except for the last addition. Become; Complete medium is added in an amount covering the umbilical tissue block at the last addition.

In certain embodiments, complete medium is added to the culture vessel in 3 or 4 batches in step b.

In certain embodiments, the complete medium is added in batches in step b at a time interval of 12-36 hours, such as about 24 hours.

In certain embodiments, step a comprises the following steps:

a1. Separating Wharton jelly from the umbilical cord tissue;

a2. Slicing the Wharton jelly to obtain a tissue block; And

a3. Plating the tissue block in the culture vessel.

In certain embodiments, step c comprises the following steps:

c1. When cells attached to the culture vessel are observed around the tissue block, removing the tissue block and adding an appropriate amount of complete medium to the culture vessel to continue culturing;

c2. Isolating the cells attached to the culture vessel to obtain umbilical mesenchymal stem cells.

In certain embodiments, the method comprises the following steps:

(1) separating Wharton jelly from umbilical cord tissue;

(2) slicing the Wharton jelly to obtain a tissue block;

(3) plating the tissue block in the culture vessel;

(4) adding an appropriate amount of complete medium dropwise to the tissue block in step (3) and performing culture;

(5) adding an appropriate amount of complete medium dropwise to the tissue block in step (4) and continuing the culture;

(6) adding an appropriate amount of complete medium to the culture vessel to cover the tissue block and continue the culture;

(7) when cells adhering to the culture vessel are observed around the tissue block, removing the tissue block and adding an appropriate amount of complete medium to the culture vessel to continue culturing;

(8) Isolating the cells attached to the culture vessel to obtain umbilical cord mesenchymal stem cells.

In certain embodiments, the cells attached to the culture vessel described in step (7) are umbilical umbilical mesenchymal stem cells at a passage number of P0. In certain embodiments, the cells are cultured when the confluent growth of the cells attached to the culture vessel described in step (7) is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%). Isolation from the container can yield umbilical mesenchymal stem cells with a passage number of P0.

In certain embodiments, the complete medium is selected from DMEM/F12, αMEM or DMEM containing 10% fetal bovine serum.

In certain embodiments, the complete medium is a serum-free medium containing a serum substitute.

In certain embodiments, the complete medium comprises serum-free medium Lonza (12-725f) and the serum substitute Pall (15950-017).

In certain embodiments, prior to step (1), the method further comprises the steps of: (i) providing fresh umbilical cord tissue; (ii) washing the umbilical cord tissue to remove blood marks. In certain embodiments, the umbilical cord tissue is washed with PBS buffer or saline. In certain embodiments, the PBS buffer or saline does not contain streptomycin and penicillin.

In certain embodiments, in step (1), the Wharton jelly is separated by removing the umbilical adventitia and blood vessels from the umbilical cord tissue and separating the Wharton jelly.

In certain embodiments, in step (2), the Wharton jelly is shredded into tissue blocks using sterile scissors.

In certain embodiments, the volume of the tissue block is about 1-2 mm 3.

In certain embodiments, in step (3), the culture vessel is a cell culture dish.

In certain embodiments, the culture vessel is a cell culture dish having a diameter of 100 mm.

In certain embodiments, the tissue blocks are evenly plated into the culture vessel at a separation distance of about 2-30 mm.

In certain embodiments, in steps (4)-(7), the culture conditions are 37° C., 5% CO ₂ .

In certain embodiments, in steps (4)-(7), the cultivation is carried out in an incubator at 37° C., 5% CO ₂ .

In certain embodiments, in step (4), the incubation time is about 24 h.

In certain embodiments, in step (4), the amount of complete medium is about 20-100 μl.

In certain embodiments, in step (5), the incubation time is about 24 h.

In certain embodiments, in step (5), the amount of complete medium is about 20-100 μl.

In certain embodiments, in step (6), the incubation time is about 3-5 days.

In certain embodiments, in step (6), the amount of complete medium is about 3 ml.

In certain embodiments, in step (7), the amount of complete medium is about 5 ml.

In certain embodiments, after step (7), the method comprises passing the cells when the confluence of the cells is at least about 85% (e.g., at least about 90%, at least about 95%, or at least about 100%). Includes additionally.

In certain embodiments, the passage is performed at a cell density of about 1×10 ⁶ cells/ml.

Cell passage methods are well known to those skilled in the art. For example, the method may include isolating cells from the culture vessel, uniformly dispersing the cells in the culture medium, and seeding the culture vessel. An appropriate amount of medium is added and an appropriate amount of fresh medium is replaced every 1 to 5 days depending on the growth condition of the cells. The passage operation is repeated until the cells have grown to 70-100% confluence. Each time a cell is passaged, the number of passages increases by 1. Umbilical mesenchymal stem cells grow adhesively, are fibrous and uniform in shape.

In certain embodiments, methods of isolating cells from the culture vessel include, but are not limited to, digestion with pancreatin and analogs using cell scraping or the like.

In certain embodiments, the cells are evenly dispersed in the medium and then seeded by a method such as stirring, vortexing.

Optionally, after culturing and harvesting umbilical cord mesenchymal stem cells, cell growth curves may be determined by MTT method, WST method, DNA content assay, ATP assay, or the like, to evaluate the growth activity of umbilical mesenchymal stem cells. In addition, isolated and cultured umbilical mesenchymal stem cells can be identified by testing cell surface markers through flow cytometry, 3-way differentiation assays, and testing genes expressed by cells through PCR methods.

In certain embodiments, after step (8), the method further comprises cryopreserving the umbilical cord mesenchymal stem cells.

In certain embodiments, cryopreservation is performed at a cell density of about 2×10 ⁶ cells/ml.

In a second aspect, the present disclosure provides a method of making a sheet of umbilical mesenchymal stem cells characterized by: umbilical mesenchymal stem cells as well as extracellular matrix and growth factors secreted from them during proliferation are substantially Retained, which separates from the surface of the Petri dish without the use of enzymes and analogs for digestion to form umbilical mesenchymal stem cell sheets.

Specifically, the method of preparing an umbilical mesenchymal stem cell sheet includes the following steps:

Adding a coating solution to a heat-sensitive Petri dish for incubation, wherein the coating solution contains serum;

Adding umbilical mesenchymal stem cells to a heat-sensitive Petri dish for culture;

Adding a pre-cooled buffer solution to a heat-sensitive Petri dish, and separating the umbilical mesenchymal stem cells and the extracellular matrix secreted by them into layers to obtain an umbilical mesenchymal stem cell sheet.

In certain embodiments, umbilical mesenchymal stem cells are produced by the method of the first aspect of the present disclosure.

In certain embodiments, the umbilical mesenchymal stem cells are umbilical mesenchymal stem cells having a passage number of P0-P20, such as umbilical mesenchymal stem cells having a passage number of P2-P10.

In certain embodiments, a cell suspension of umbilical mesenchymal stem cells is added to a heat-sensitive Petri dish for culture.

In certain embodiments, the umbilical mesenchymal stem cells are umbilical mesenchymal stem cells of passage P0-P20. In certain embodiments, the umbilical mesenchymal stem cells are umbilical mesenchymal stem cells of passage P2-P10.

In certain embodiments, umbilical mesenchymal stem cells are produced by the method described in the first aspect.

The "heat-sensitive Petri dish" described herein refers to a Petri dish coated with a heat-sensitive polymer material in which molecular segments are distinctly stretched at different temperatures to exhibit hydrophilicity or hydrophobicity, so that the hydrophilicity/hydrophobicity of the polymeric material can vary depending on the external temperature. Refers to. When the surface of the heat-sensitive Petri dish is hydrophilic, the adhesion to cells and extracellular matrix secreted by them becomes poor, and the cells fall into layers. In certain embodiments, when the temperature is lowered below the lower limit critical solution temperature (LCST) of the polymeric material, the surface of the heat-sensitive Petri dish becomes hydrophilic, causing the cells to fall into layers.

The present disclosure successfully uses heat-sensitive Petri dishes to achieve separation of layered mesenchymal stem cells from the bottom of heat-sensitive Petri dishes without the use of enzymes and analogs digestion and physical exfoliation to achieve complete connectivity of the extracellular matrix. A retained cell sheet is obtained.

In certain embodiments, the serum is selected from fetal bovine serum (FBS) or serum isolated from human peripheral blood. In certain embodiments, the serum isolated from human peripheral blood is autologous, ie the serum is isolated from autologous peripheral blood. “Self” as used herein means that serum isolated from human peripheral blood is obtained and isolated from a subject, and umbilical mesenchymal stem cell sheets obtained using the serum are administered to the same subject, ie, the donor of the serum is the recipient Means the same as. In this embodiment, without being bound by theory, it is believed that the use of autologous serum would be expected to reduce or eliminate the immune response from the subject.

In certain embodiments, the coating solution is 100% serum. The amount of adhesion factor and coating time contained in the coating solution directly affect the formation of the cell sheet. For example, if the adhesion factor is too low, the cells cannot adhere well, and if the adhesion factor is too high, the growth of cells will be inhibited. Therefore, it is important to control the amount and action time of the adhesion factor for the formation of the cell sheet. The inventors of the present application unexpectedly found that when using 100% serum and coating for 12-24 hours, the content of adhesion factor in the coating system is suitable for both cell adhesion and cell growth, which is advantageous for the formation of sheets.

In certain embodiments, the coating solution is at least 10% (v/v) (e.g. at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) is a basal medium containing serum. In some embodiments, the basal medium is selected from DMEM/F12, αMEM or DMEM. In this embodiment, the serum is FBS.

In another embodiment, the basal medium is serum-free medium (SFM), such as Lonza (12-725f). In this embodiment, the serum is serum from human peripheral blood.

In certain embodiments, the amount of coating solution is about 0.05-0.3 ml/cm 2 (the bottom area of the Petri dish), such as about 0.09 ml/cm 2, about 0.14 ml/cm 2, or about 0.25 ml/cm 2. In certain embodiments, when the diameter of the heat-sensitive Petri dish is 100 mm, the amount of coating solution is about 5 ml. In certain embodiments, when the diameter of the heat-sensitive Petri dish is 60 mm, the amount of coating solution is about 3 ml. In certain embodiments, when the diameter of the heat-sensitive Petri dish is 35 mm, the amount of coating solution is about 2 ml.

In certain embodiments, the coating time is about 12-24 hours. In certain embodiments, the coating conditions are 37° C., 5% CO ₂ .

In certain embodiments, the coating solution is added to a heat-sensitive Petri dish; The heat-sensitive Petri dish was placed in an incubator at 37° C., 5% CO ₂ and incubated for about 12-24 hours; Optionally, the coating solution remaining in the heat-sensitive Petri dish is discarded.

In certain embodiments, the cell suspension of umbilical mesenchymal stem cells is about 1×10 ⁶ -1×10 ⁷ cells/cm 2 (e.g., about 2×10 ⁶ -4×10 ⁶ cells/cm 2, about 2.5× 10 ⁶ -6.0×10 ⁷ pieces/cm 2, about 5.5×10 ⁶ -6.5×10 ⁶ pieces/cm 2 ), added to the heat-sensitive Petri dish. In certain embodiments, when the diameter of the heat-sensitive Petri dish is 100 mm, the concentration of seeded cells is about 6×10 ⁷ -7×10 ⁷ cells/ml and the seeded volume is about 5 ml. In certain embodiments, when the diameter of the heat-sensitive Petri dish is 60 mm, the concentration of seeded cells is about 2×10 ⁷ -4×10 ⁷ cells/ml and the seeded volume is about 3 ml. In certain embodiments, when the diameter of the heat-sensitive Petri dish is 35 mm, the concentration of seeded cells is about 8×10 ⁶ -1.5×10 ⁷ cells/ml and the seeded volume is about 2 ml.

In certain embodiments, the cell suspension is a complete medium containing umbilical mesenchymal stem cells. In some embodiments, the complete medium is selected from DMEM/F12, αMEM or DMEM containing 10% fetal bovine serum. In some other embodiments, the complete medium is a serum-free medium containing a serum substitute, such as a serum-free medium Lonza (12-725f) containing a serum substitute Paul (15950-017).

In certain embodiments, the culture conditions are about 12-36 h. In certain embodiments, the culture conditions are 37° C., 5% CO ₂ .

In certain embodiments, the buffer is selected from HBSS, PBS or saline. In certain embodiments, a 4° C. pre-cooled buffer (eg, HBSS, PBS or saline) is added. When the pre-cooled buffer is added, the layered umbilical umbilical mesenchymal stem cells gradually separate from the bottom surface of the heat-sensitive Petri dish and become a cell sheet that maintains the complete connectivity of the extracellular matrix.

In certain embodiments, the amount of pre-cooled buffer is about 0.05-0.3 ml/cm 2 (bottom area of the Petri dish), such as about 0.09 ml/cm 2, about 0.14 ml/cm 2, or about 0.25 ml/cm 2. In certain embodiments, when the diameter of the heat-sensitive Petri dish is 100 mm, the amount of buffer is about 5 ml. In certain embodiments, when the diameter of the heat-sensitive Petri dish is 60 mm, the amount of buffer is about 3 ml. In certain embodiments, when the diameter of the heat-sensitive Petri dish is 35 mm, the amount of buffer is about 2 ml.

In certain embodiments, the method further comprises transferring the umbilical cord mesenchymal stem cell sheet to a storage container. In certain embodiments, the storage container is a cell culture dish.

In some embodiments, the umbilical mesenchymal stem cell sheet can be transferred to a storage container by the following steps: about a third of the tip of a pipette tip (such as a 1 ml pipette tip) using scissors (e.g., sterile) Being cut; The sheet is drawn by a pipette using a shortened tip and the sheet is transferred to a storage container.

In some other embodiments, the umbilical mesenchymal stem cell sheet can be transferred to a storage container by the following steps: The liquid in the heat-sensitive Petri dish is poured into the reservoir along with the cell sheet. When pouring the heat-sensitive Petri dish, the cell sheet separated from the bottom of the dish flows into the storage container with a flow of liquid. During the transfer process, since the cell sheet floats on the liquid, it is ensured that the cell sheet does not adhere directly to the edge of the heat-sensitive Petri dish or storage container, thereby preventing the cell sheet from being torn or damaged.

In certain embodiments, the amount of liquid in the heat-sensitive Petri dish in which the cell sheet to be transferred is maintained (i.e., the amount of buffer) is about 0.05-0.4 ml/cm 2 (bottom area of the Petri dish), such as about 0.09-0.18 ml /Cm2, about 0.14-0.24 ml/cm2, or about 0.25-0.38 ml/cm2. In certain embodiments, when the diameter of the heat-sensitive Petri dish is 100 mm, the amount of liquid is about 5-10 ml. In certain embodiments, when the diameter of the heat-sensitive Petri dish is 60 mm, the amount of liquid is about 3-5 ml. In certain embodiments, when the diameter of the heat-sensitive Petri dish is 35 mm, the amount of liquid is about 2-3 ml.

In some other embodiments, the umbilical mesenchymal stem cell sheet can be scooped using a sheet spatula and transferred to a storage container. Sheet spatula is any spatula-like product that can be used for cells, such as special sheet spatula or cell stain spatula.

In a third aspect, the present disclosure provides an umbilical mesenchymal stem cell sheet prepared by the method described in the second aspect. The umbilical mesenchymal stem cell sheet prepared by the method of the present disclosure contains umbilical mesenchymal stem cells and all extracellular matrix and growth factors secreted by them during the proliferation process. In addition, since no enzymes or analogs are used for digestion and no physical method is used for exfoliation, the umbilical mesenchymal stem cells in the umbilical mesenchymal stem cell sheet of the present disclosure have a high density, uniform sheet thickness, and neat edges. Has. The umbilical mesenchymal stem cell sheet of the present disclosure is capable of secreting a variety of cytokines, including angiogenic factors and immunomodulatory factors, which are involved in the repair of tissues and organs.

Optionally, after preparation of the umbilical mesenchymal stem cell sheet, the surface structure of the cell sheet can be observed with a scanning electron microscope. In addition, the amount of cytokines secreted by the cell sheet and the protein contained in the extracellular matrix in the cell sheet can be tested.

In certain embodiments, the umbilical mesenchymal stem cell sheet has a surface that does not contact a Petri dish during the manufacturing process and a basal surface that contacts the Petri dish, wherein the surface is smooth and the basal surface is rough.

In certain embodiments, the umbilical mesenchymal stem cell sheet comprises a monolayer or multilayer interconnected cellular structure that exhibits a substantially uniform cell orientation and substantially retains the extracellular matrix secreted by the umbilical mesenchymal stem cells.

In certain embodiments, the umbilical mesenchymal stem cell sheet has an extracellular matrix (eg, a substantially continuous layer of extracellular matrix) distributed on at least its basal surface. In certain embodiments, the extracellular matrix comprises at least one of a polyamino acid, collagen, polysaccharide, fibronectin, vitronectin, laminin, such as a mixture of fibronectin and laminin. In certain embodiments, the site of connection between the umbilical mesenchymal stem cells contained in the umbilical mesenchymal stem cell sheet comprises said material.

In certain embodiments, the umbilical mesenchymal stem cell sheet is off-white and has a compact structure with a smooth, flat surface.

In certain embodiments, the umbilical mesenchymal stem cell sheet is rich in fibronectin and integrin β1.

In certain embodiments, the umbilical mesenchymal stem cells within the umbilical mesenchymal stem cell sheet are capable of secreting a variety of angiogenic and immunomodulatory factors. For example, angiogenic factors and immunomodulatory factors may include one or more of hepatocyte growth factor (HGF), interleukin-6 (IL-6), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF). I can.

In a fourth aspect, the present disclosure relates to a method of treating a disease associated with cardiac tissue damage or cardiac insufficiency in a subject, wherein the method comprises applying the umbilical umbilical mesenchymal stem cell sheet of the second aspect of the present disclosure to injury to a subject. Topically applying to the site.

In certain embodiments, the disease is heart failure. In certain embodiments, the heart failure is ischemic heart failure, such as acute ischemic heart failure.

In a fifth aspect, the present disclosure relates to the use of the umbilical mesenchymal stem cell sheet of the second aspect of the present disclosure in the treatment of diseases associated with cardiac tissue damage or cardiac insufficiency in a subject.

In a sixth aspect, the present disclosure relates to the use of the umbilical mesenchymal stem cell sheet of the second aspect of the present disclosure in the manufacture of a composition for treating a disease associated with cardiac tissue damage or cardiac insufficiency in a subject.

In certain embodiments of the fifth and sixth aspects, the disease is heart failure. In certain embodiments, the heart failure is ischemic heart failure, such as acute ischemic heart failure.

The present disclosure employs the addition of culture medium in a batch that significantly improves the yield of mesenchymal stem cells. The higher the cell yield, the more cells are obtained, and the easier it is to meet the dosage requirements in clinical treatment.

By using a heat-sensitive Petri dish and controlling the amount of serum and coating time in the process of coating the heat-sensitive Petri dish, the present disclosure provides the umbilical mesenchymal stem cells as well as the extracellular matrix secreted by them during the proliferation process. And the growth factor is completely maintained, after which it is separated from the surface of the Petri dish to obtain a layered cell sheet. The cell sheet obtained by this method has a high cell density, a uniform thickness, and an intact structure. Umbilical umbilical mesenchymal stem cell sheets prepared by the methods of the present disclosure have an abundant natural extracellular matrix and are capable of retaining most of fibronectin and laminin. There is no need to suture during in vivo implantation. The adhesion molecule and extracellular matrix can directly adhere to the diseased tissue, so the cells can act perfectly on the damaged site of the body, thereby improving the regeneration and repair effect of cell transplantation into the tissue and the activity of the transplanted cells. I can.

1 shows representative photographs of umbilical mesenchymal stem cells of primary, and P0 on days 0 and 5, respectively. The primary of these refers to the umbilical tissue block, and P0 refers to the umbilical mesenchymal stem cells that have migrated from the tissue block but have not been passaged.
2 shows a representative picture of umbilical mesenchymal stem cells of P2 on day 5 under a ×4 objective lens and a ×10 objective lens.
3 shows the results of flow cytometry for umbilical mesenchymal stem cell surface markers.
4 shows the results of adipogenesis and osteogenic differentiation functions of umbilical mesenchymal stem cells.
5 shows a representative photograph of an umbilical mesenchymal stem cell sheet.
6 shows a scanning electron microscope imaging photograph of an umbilical mesenchymal stem cell sheet. 6A: The surface of the cell sheet (top surface). 6B: Basal surface of the cell sheet.
7 shows an immunofluorescence imaging photograph of an umbilical mesenchymal stem cell sheet. Figure 7A: Fibronectin. 7B: Integrin β1.
8 shows the results of cytokine expression in the culture supernatant of the umbilical cord mesenchymal stem cell sheet by ELISA method.
9 shows the characterization of the established heart failure mouse disease model. 9A: Picture of the heart of a disease model mouse; 9B: ECG results of disease model mice.
10 shows the results of echocardiography of mice at different time points. 10A: Before modeling; 10B: 1 week after modeling; 10C: 4 weeks after modeling. Left: control animals; Right: Animals in the cell sheet transplant group.
11 shows a curve of a left ventricular ejection fraction of a mouse over time before and after modeling.
12 shows a curve of the left ventricular fraction shortening index of a mouse over time before and after modeling.
13 shows a curve of the left ventricle diameter of a mouse over time before and after modeling.
14 shows a curve of the volume of the left ventricle of the mouse over time before and after modeling.
Fig. 15 shows the results of Masson staining of mouse heart tissue sections at the end of the experiment (day 28 after modeling). Left: control animals; Right: Animals in the cell sheet transplant group.

<Detailed explanation>

Although the present invention is described below based on examples, the present invention is not limited to these examples.

Example 1. Isolation and culture of umbilical cord mesenchymal stem cells

Fresh umbilical cords were rinsed repeatedly with 1xPBS buffer without penicillin or streptomycin to remove bloodstains. The outer membrane of the umbilical cord and blood vessels were removed, and the Wharton jelly-like tissue was maintained in the umbilical cord tissue. The tissue was cut into 1-2 mm 3 tissue blocks with sterile scissors, and plated on a 100 mm Petri dish. 20-100 μl of complete medium was added dropwise to each tissue block and placed in a 37° C., 5% CO ₂ incubator. After 24 hours, 20-200 μl of complete medium was added dropwise. 3 ml of complete medium was added to the Petri dish at 48 hours. After 3-5 days, the culture medium can be changed after the cells have migrated, the tissue block is removed, and 5 ml of the culture medium is added to each Petri dish. When the cells grew to 85% confluence, the cells were passaged at a density of 1×10 ⁶ . Each time a cell was passaged, the number of passages increased by 1. Umbilical mesenchymal stem cells were grown adhesively, fibrous and uniform in shape. Representative photographs of umbilical mesenchymal stem cells of passages P0 and P2 are shown in Figs. 1-2. After determination, the cell yield of this method is 8.3 times, while the cell yield of the conventional method is 3-5 times.

Example 2. Identification of umbilical cord mesenchymal stem cells

2.1 Identification of surface markers for umbilical mesenchymal stem cells

The umbilical mesenchymal stem cells were dispersed in a medium and then centrifuged. Cell surface marker proteins including, but not limited to, CD73, CD90, CD105, CD34, CD11B, CD19, CD45, HLA-DR are purchased in isotonic physiological solutions containing 1-20% serum or serum protein. Staining according to the instructions for one reagent. Among the markers, the expression of CD73, CD90 and CD105 is positive and the proportion should be at least 95%; The expression of CD34, CD11B, CD19, CD45 and HLA-DR is negative and the ratio should not be more than 2%. The results are shown in Figure 3, where CD105/CD34 is 99.64%/0.02%, CD105/CD31 is 99.04%/0.00%, and CD105/CD117 is 95.53%/0.51%.

2.2 3-way induced differentiation of umbilical mesenchymal stem cells

The umbilical cord mesenchymal stem cells prepared in Example 1 were seeded in a suitable culture vessel at a ratio according to the instructions of the 3-way induced differentiation reagent. Bone formation and adipogenesis induction medium were added, respectively, when cells tested for induction of osteogenesis grew with 50-90% confluent growth and cells tested for induction of adipogenesis grew with more than 90% confluent growth. During chondrogenesis induction, a certain number of cells were centrifuged to the bottom of the centrifuge tube, and then chondrogenesis induction medium was added. After the cells formed a pellet, the cell pellet was allowed to leave the bottom of the tube to ensure complete contact with the induction medium.

Cells were tested when cultured under induction for more than 7 days. Induction of bone formation can be stained with reagents including, but not limited to, alizarin red and anti-osteocalcin; Adipogenesis induction can be stained with reagents including, but not limited to, Oil Red O and anti-mFABP4; Chondrogenesis induction can be stained with reagents including, but not limited to, Alcian Blue, Saffron O and anti-agrecan.

The results of osteogenic differentiation (alizarin red staining) and adipogenic differentiation (oil red O staining) are shown in FIG. 4.

Example 3. Preparation of umbilical cord mesenchymal stem cell sheet

1. Serum coating: Heat-sensitive Petri dishes were coated with 100% serum. The amounts added to the different Petri dishes were as follows: 35 mm/2 ml, 60 mm/3 ml, 100 mm/5 ml. Coating time and temperature: 12-24 h, 37° C., in 5% CO ₂ incubator.

2. Cell culture: After the coating was completed, the liquid in the Petri dish was discarded, and the umbilical mesenchymal stem cells obtained in Example 1 were seeded. Cell concentration for seeding: In the case of a 35 mm dish, the concentration of the seeded cells is 8×10 ⁶ -1.5×10 ⁷ cells/ml; In the case of a 60 mm dish, the concentration of seeded cells is 2×10 ⁷ -4×10 ⁷ cells/ml; In the case of a 100 mm dish, the concentration of the seeded cells was 6×10 ⁷ -7×10 ⁷ cells/ml. Petri dishes were incubated in a 37° C., 5% CO ₂ incubator for 12-36 h.

3. Sheet peeling: remove the heat-sensitive dish from the incubator and discard the culture medium; 4° C. pre-cooled HBSS solution was added: 35 mm/2 ml, 60 mm/3 ml, 100 mm/5 ml; After 10-30 minutes, the layered umbilical mesenchymal stem cells were separated from the edge of the dish, and a cell sheet was formed that maintained the complete connection of the extracellular matrix. The macroscopic appearance of the cell sheet is shown in FIG. 5. The umbilical mesenchymal stem cell sheet was off-white and had a compact structure with a smooth and flat surface.

4. Sheet transfer: The completely exfoliated cell sheet was transferred to a normal Petri dish, and the sheet was washed 2-3 times by adding HBSS solution: 35 mm/2 ml, 60 mm/3 ml, 100 mm/5 ml.

5. The prepared sheet was temporarily stored at 4°C.

Example 4. Structural characterization of umbilical mesenchymal stem cell sheets

In this Example, the structure of the prepared umbilical mesenchymal stem cell sheet was characterized using scanning electron microscopy and immunofluorescence imaging.

First, an umbilical cord mesenchymal stem cell sheet was prepared by the method described in Example 3. The cell sheet was separated from the bottom of the heat-sensitive smart Petri dish, and the obtained sheet maintained the complete connection of the extracellular matrix. After preparation through steps such as 2.5% glutaraldehyde fixation, alcohol gradient dehydration, and air drying, cell sheets were photographed by scanning electron microscopy. As shown in Fig. 6, the cell sheet has a surface that does not contact the Petri dish (upper surface, Fig. 6A) and a basal surface (lower surface, Fig. 6B) in contact with the Petri dish, and the structure is different: the surface formed is Relatively smooth due to the natural sedimentation of cells; The base surface is in contact with the material of the heat dish and is relatively rough. Due to its structural features, the base surface can provide greater friction, which is advantageous for cell sheets that adhere better to the application site.

In addition, the expression of fibronectin and integrin β1 in the umbilical mesenchymal stem cell sheet was tested by immunofluorescence. Sheets were fixed by fixation solution prior to freezing and sectioning, stained with fluorescein-labeled fibronectin and integrin β1 antibodies, and analyzed by immunofluorescence imaging. As shown in Figure 7, the cell sheet prepared by the method of the present disclosure contained large amounts of fibronectin (Figure 7A) and integrin β1 (Figure 7B).

Fibronectin, which is widely present in animal tissues and tissue fluids, has a function of promoting cell adhesion and growth, and cell adhesion and growth are essential for maintenance and repair of body tissue structure. Integrin β1 is an important member of the integrin family, which is important for mediating cell-cell adhesion, cell-cell extracellular matrix (ECM) adhesion, and bidirectional signaling between cells and ECM, and is closely related to tissue repair and fibrosis formation. The above results indicate that the umbilical mesenchymal stem cell sheet of the present disclosure is not a simple accumulation of cells, but a dense tissue and biologically active sheet formed by extracellular matrix linkage. In addition, high levels of fibronectin and integrin β1 in the cell sheet indicate that it has the function of tissue repair and can be used to achieve tissue repair in diseases associated with damage to tissues such as the heart, liver, pancreas and uterus.

Example 5. Functional characterization of umbilical mesenchymal stem cell sheets

To further characterize the function of the umbilical mesenchymal stem cell sheets of the present disclosure, the following cytokines secreted were tested: hepatocyte growth factor (HGF); Interleukin-6 (IL-6), interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF). HGF is produced by mesenchymal stem cells, has a regulatory effect on various tissues and cells, and is involved in the epithelial-mesenchymal transition (EMT) process, which can promote cell migration and division; IL-6 and IL-8 are involved in regulating the body's immune response and various physiological processes of immune cells; VEGF has the function of promoting endothelial cell proliferation and inducing angiogenesis. The cytokine has a function of promoting cell growth and differentiation, promoting angiogenesis process, and plays an important role in tissue repair.

During the preparation of the cell sheet, the culture supernatant was sampled and the cytokines in the supernatant were tested by ELISA method. The results are shown in Figure 8. The results indicate that all of the four cytokines are expressed in the supernatant, and the expression levels of HGF and IL-8 are relatively high. The above results show that the umbilical mesenchymal stem cell sheet of the present disclosure secretes various cytokines including angiogenic factors and immunomodulatory factors, thereby exhibiting high biological activity and function, and promoting local angiogenesis and tissue repair processes. Indicates that you can. Moreover, the high expression level of IL-8 indicates that the cell sheet has the function of promoting an immune response and inhibiting bacteria during use, indicating that the cell sheet is advantageous in exerting its biological function better.

Example 6. Application of Umbilical Cord Mesenchymal Stem Cell Sheet in Heart Failure Treatment

In this example, a mouse model of heart failure was established, and the function of the umbilical mesenchymal stem cell sheet of the present disclosure on repair of heart tissue was evaluated in the model. First, an animal model of acute ischemic heart failure was established by coronary artery ligation in male C57BL/6 mice (approximately 12 weeks old). Specific steps include:

(1) anesthetizing the mouse with isoflurane mixed with oxygen (the concentration of isoflurane is about 3.5-5%) and removing hair;

(2) performing transluminal intubation through cervical spine permeation illumination, pumping anesthetic gas using a ventilator to maintain anesthesia, and measuring ECG signals of mice;

(3) opening the chest, exposing the heart, and ligating the left anterior descending limb (about 1.5 mm from the lower edge of the left atrium) of the mouse using a 7-0 surgical suture to establish a model;

(4) Chest suture and postoperative treatment.

After modeling in step (3), whitening of the left ventricular wall of the mouse was observed (Fig. 9A); ECG results indicate ST segment elevation and myocardial infarction (FIG. 9B ), indicating that an animal model of heart failure was successfully built. In the case of mice in the umbilical mesenchymal stem cell sheet treatment group, the umbilical mesenchymal stem cell sheet cut into a round shape having a diameter of about 2-5 mm or an appropriate shape having a similar area was prepared in the left ventricle of the model animal after step (3). Attached to the surface. After allowing to stand for 3-5 minutes, step (4) was carried out. An animal to which the cell sheet was not attached was used as a control. There were 10 mice in each of the cell sheet treatment group and the control group.

Echocardiography was performed in mice before modeling (FIG. 10A), 1 week after modeling (FIG. 10B) and 4 weeks after modeling (FIG. 10C). The short axis of the sternum with the horizontal cross section of the left ventricular papillary muscle as a mark point was observed by echocardiography. From the results of FIGS. 10B and 10C, it can be seen that the heart of the heart failure model animal had apparent impaired movement after modeling. In addition, compared to the control group (left panel), the cell sheet transplant group (right panel) had stronger cardiac motion.

The curve of the left ventricular ejection fraction of the mouse over time before and after surgery (FIG. 11) and the curve of the left ventricular fraction shortening index of the mouse over time (FIG. 12) were calculated and plotted according to echocardiography. The left ventricular ejection fraction is an important indicator for evaluating left ventricular function. As shown in the results of FIG. 11, the left ventricular ejection fraction of the heart failure model animals significantly decreased after modeling, but the animals of the cell sheet transplant group had significantly higher ejection fractions than the control group. The left ventricular fractional shortening index refers to the ratio of the shortening of the left ventricle during the systolic versus diastolic phase. The larger the ratio, the stronger the contractile function of the heart. As shown in the results of FIG. 12, the value of the left ventricular fractional shortening index of the heart failure model animals was significantly decreased after modeling, but the value of the left ventricular fractional shortening index of the animals of the cell sheet transplant group was significantly higher than that of the control group.

The curve of the left ventricle diameter over time (Fig. 13) and the curve of the left ventricle volume over time (Fig. 14) were calculated and plotted according to echocardiography, all of which can be used to describe the left ventricular volume. After preparing the animal model, compensatory remodeling took place in the left ventricle, and the volume of the ventricle became larger due to ischemic heart failure. As shown in the results of FIGS. 13 and 14, after modeling, the left ventricle diameter and volume (systolic phase and diastolic phase) of the animals in the cell sheet transplant group were significantly lower than that of the control group, and this indicates that the use of cell sheets was caused by ischemic heart failure. It has a significant effect on inhibition of left ventricular remodeling and indicates that it can significantly improve cardiac function.

At the end of the experiment (day 28 after modeling), mice were sacrificed, and heart tissue was collected for fixation, sectioning and staining. The results of sectioning (FIG. 15) show that the left ventricular wall of the mice of the mesenchymal stem cell sheet transplant group is thicker, the ventricular remodeling is less severe and the degree of fibrosis is lower than that of the control animals (wear staining, collagen fibers appear blue) Represents. The above results indicate that the degree of fibrosis of the left ventricle of the mouse implanted with the cell sheet was significantly lower than that of the control animals.

While certain embodiments of the present invention have been described above, those skilled in the art should understand that these are merely examples, and the scope of protection of the present invention is defined by the appended claims. A person skilled in the art can make various changes or modifications to these embodiments without departing from the principle and essence of the present invention, but all these changes and modifications fall within the protection scope of the present invention.

Claims (41)

A method of producing umbilical mesenchymal stem cells, comprising the following steps:
a. Plating the umbilical cord tissue block in the culture vessel;
b. Adding complete medium as a batch feed for cultivation;
c. Isolating the cells attached to the culture vessel to obtain umbilical mesenchymal stem cells. The method of claim 1, wherein complete medium is added to the culture vessel in 2-5 batches in step b, wherein the complete medium is in an amount that keeps the umbilical tissue block wet except for the last addition, but does not cover the umbilical tissue block. Added; The method wherein the complete medium is added in an amount covering the umbilical cord tissue block at the last addition. The method of claim 2, wherein the complete medium is added to the culture vessel in 3 or 4 batches in step b. The method according to any one of the preceding claims, wherein the complete medium is added in batches at a time interval of 12-36 hours in step b, such as at a time interval of about 24 hours. The method according to any one of claims 1 to 4, wherein step a comprises the following steps:
a1. Separating Wharton jelly from the umbilical cord tissue;
a2. Slicing the Wharton jelly to obtain a tissue block; And
a3. Plating the tissue block in the culture vessel. The method according to any one of claims 1 to 5, wherein step c comprises the following steps:
c1. When cells attached to the culture vessel are observed around the tissue block, removing the tissue block and adding an appropriate amount of complete medium to the culture vessel to continue culturing;
c2. Isolating the cells attached to the culture vessel to obtain umbilical mesenchymal stem cells. The method of claim 1 comprising the steps of:
(1) separating Wharton jelly from umbilical cord tissue;
(2) slicing the Wharton jelly to obtain a tissue block;
(3) plating the tissue block in the culture vessel;
(4) adding an appropriate amount of complete medium dropwise to the tissue block in step (3) and performing culture;
(5) adding an appropriate amount of complete medium dropwise to the tissue block in step (4) and continuing the culture;
(6) adding an appropriate amount of complete medium to the culture vessel to cover the tissue block and continue the culture;
(7) when cells adhering to the culture vessel are observed around the tissue block, removing the tissue block and adding an appropriate amount of complete medium to the culture vessel to continue culturing;
(8) Isolating the cells attached to the culture vessel to obtain umbilical cord mesenchymal stem cells. 8. The method of any one of claims 1-7, further comprising passage of umbilical mesenchymal stem cells after step c. 9. The method of claim 8, comprising passage of the umbilical mesenchymal stem cells when confluence is at least about 85%. The method according to any one of claims 1 to 9, wherein the complete medium is selected from DMEM/F12, αMEM or DMEM containing 10% fetal bovine serum. 10. The method of any one of claims 1-9, wherein the complete medium comprises serum-free medium Lonza (12-725f) and serum substitute Pall (15950-017). 12. The method of any one of claims 7 to 11, wherein the incubation time in step (4) and/or step (5) is about 24 h; The method in which the amount of complete medium in step (4) and/or step (5) is about 20-100 μl. 13. The method of any one of claims 7-12, wherein the incubation time in step (6) is about 3-5 days; In step (6), the amount of complete medium is about 3 ml. A method of preparing an umbilical mesenchymal stem cell sheet comprising the following steps:
Adding a coating solution to a heat-sensitive Petri dish for incubation, wherein the coating solution contains serum;
Adding umbilical mesenchymal stem cells to a heat-sensitive Petri dish for culture;
Adding a pre-cooled buffer solution to a heat-sensitive Petri dish, and separating the umbilical mesenchymal stem cells and the extracellular matrix secreted by them into layers to obtain an umbilical mesenchymal stem cell sheet. The method of claim 14, wherein the umbilical mesenchymal stem cells are produced by the method of any one of claims 1 to 13. The method according to claim 14 or 15, wherein the serum is selected from fetal bovine serum or serum isolated from human peripheral blood. 17. The method of any one of claims 14-16, wherein the coating solution is 100% serum. 17. The method according to any one of claims 14 to 16, wherein the coating solution is a basal medium containing at least 10% (v/v) serum. The method of claim 18, wherein the basal medium is selected from DMEM/F12, αMEM or DMEM, and the serum is FBS. 19. The method of claim 18, wherein the basal medium is Lonza (12-725f) and the serum is serum isolated from human peripheral blood. The method of any one of claims 14-20, having one or more of the following characteristics:
(1) the amount of the coating solution is about 0.05-0.3 ml/cm 2 (the bottom area of the Petri dish), such as about 0.09 ml/cm 2, about 0.14 ml/cm 2, or about 0.25 ml/cm 2;
(2) the coating time is about 12-24h;
(3) The coating condition is 37℃, 5% CO ₂ . The method according to any one of claims 14 to 21, having one or more of the following characteristics:
(1) A cell suspension of umbilical mesenchymal stem cells was added to a heat-sensitive Petri dish at a density of about 1×10 ⁶ -1×10 ⁷ cells/cm 2;
(2) the cell suspension is a complete medium containing umbilical mesenchymal stem cells;
(3) the culture condition is about 12-36h;
(4) The culture conditions are 37℃, 5% CO ₂ . 23. The method of claim 22, wherein the complete medium is selected from DMEM/F12, αMEM or DMEM containing 10% fetal bovine serum; The method in which the complete medium is serum-free medium Lonza (12-725f) containing serum substitute Paul (15950-017). 24. The method of any one of claims 14-23, wherein the buffer is selected from HBSS, PBS or saline. 25. The method of any one of claims 14 to 24, further comprising transferring the umbilical mesenchymal stem cell sheet to a storage container. 26. The method of claim 25, wherein about a third of the tip of the pipette is cut with scissors; A method comprising allowing the umbilical mesenchymal stem cell sheet to be stretched by a pipette using a shortened tip and transferring the umbilical mesenchymal stem cell sheet to a storage container. The method of claim 25, wherein the coating solution in the heat-sensitive Petri dish is poured into the storage container along with the umbilical mesenchymal stem cell sheet. The method of claim 25, wherein the umbilical mesenchymal stem cell sheet is lifted using a sheet spatula and transferred to a storage container. The umbilical cord mesenchymal stem cell sheet prepared by the method of any one of claims 14 to 28. 30. The umbilical mesenchymal stem cell sheet of claim 29, wherein the cell sheet has a surface not in contact with the Petri dish during the manufacturing process and a basal surface in contact with the Petri dish, wherein the surface is smooth and the basal surface is rough. The umbilical cord mesenchymal stem cell of claim 29 or 30 comprising a monolayer or multilayer interconnected cellular structure that exhibits a substantially uniform cellular orientation and substantially retains the extracellular matrix secreted by the umbilical mesenchymal stem cells. Sheet. 32. The umbilical mesenchymal stem cell sheet according to claim 31, having an extracellular matrix distributed on at least its basal surface. The retinal pigment epithelial cell sheet according to any one of claims 29 to 32, wherein the cell sheet is rich in fibronectin and integrin β1. The method according to any one of claims 29 to 33, wherein the retinal pigment epithelial cells in the cell sheet are capable of secreting various angiogenic factors and immunomodulatory factors, for example, angiogenic factors and immunomodulatory factors are hepatocyte growth. Retinal pigment epithelial cell sheet comprising one or more of factor (HGF), interleukin-6 (IL-6), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF). A method of treating a disease associated with cardiac tissue damage or cardiac dysfunction in a subject, comprising the step of topically applying the umbilical mesenchymal stem cell sheet of any one of claims 29 to 34 to an injured site of the subject. 36. The method of claim 35, wherein the disease is heart failure. 36. The method of claim 35, wherein the heart failure is ischemic heart failure, such as acute ischemic heart failure. The use of the umbilical cord mesenchymal stem cell sheet according to any one of claims 29 to 34 in the treatment of diseases associated with cardiac tissue damage or cardiac insufficiency in a subject. Use of the umbilical cord mesenchymal stem cell sheet according to any one of claims 29 to 34 in the manufacture of a composition for treating diseases associated with cardiac tissue damage or cardiac insufficiency in a subject. 40. Use according to claim 38 or 39, wherein the disease is heart failure. 41. Use according to claim 40, wherein the heart failure is ischemic heart failure, such as acute ischemic heart failure.

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