WO2020173280A1

WO2020173280A1 - Preparation method for umbilical cord mesenchymal stem cells and cell sheets thereof

Info

Publication number: WO2020173280A1
Application number: PCT/CN2020/073812
Authority: WO
Inventors: 刘东华; 常德华; 刘洋; 赵玉菲; 刘帅; 王淑玲; 王晓惠; 王芳
Original assignee: 京东方科技集团股份有限公司
Priority date: 2019-02-28
Filing date: 2020-01-22
Publication date: 2020-09-03
Also published as: JP7538048B2; JP2022521867A; KR102565022B1; CN112292447B; KR20210018345A; CN112292447A

Abstract

Disclosed is a preparation method for umbilical cord mesenchymal stem cells, which can significantly improve the cell yield. According to the umbilical cord mesenchymal stem cell sheets and the preparation method therefor, the umbilical cord mesenchymal stem cells contain umbilical cord mesenchymal stem cells and all extracellular matrix and growth factors secreted during breeding process thereof; the preparation method can, without using enzyme and analogues for digestion or carrying out physical stripping, completely reserve the umbilical cord mesenchymal stem cells and extracellular matrix and growth factors secreted during breeding process thereof and separate same from the surface of the culture dish, so as to obtain layer-shaped cell sheets.

Description

The preparation method of umbilical cord mesenchymal stem cells and cell membranes. This application requires that the application number submitted on February 28, 2019 is CN 201910149006.8, and the title of the invention is ``method for preparing umbilical cord mesenchymal stem cells and cell membranes'' in China For the priority of the patent application, the content disclosed in the above-mentioned Chinese patent application is quoted here in full as a part of this application. Technical field

The present disclosure relates to the fields of regenerative medicine and cell biology, and in particular to a method for producing umbilical cord mesenchymal stem cells, as well as a umbilical cord mesenchymal stem cell membrane sheet and a preparation method thereof. Background technique

Umbilical cord mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells that exist in neonatal umbilical cord tissue. They can differentiate into many kinds of tissue cells and have broad clinical application prospects. Currently, there are two commonly used methods for separating umbilical cord tissue: enzyme digestion and tissue block attachment; enzyme digestion is costly and the operation is complicated and difficult to control, and it is easy to cause cell damage or cell mutation. The clinical application risk is high. The tissue block mutation method is simple to operate. Low cost, less damage to cells, suitable for clinical applications. However, the umbilical cord mesenchymal stem cells obtained by conventional tissue block adherence have a long cycle, resulting in a small number of primary cells and low purity. The number of cells used in clinical applications cannot meet international standards, resulting in difficulties in clinical applications. Therefore, the art needs a method for preparing umbilical cord mesenchymal stem cells with high yield.

In addition, in the current basic research and clinical application of umbilical cord mesenchymal stem cells in self-tissue repair, the direct injection of cell suspension or the combination of tissue engineering scaffold materials and transplantation are mainly used, both of which have certain limitations. Direct injection of stem cell suspension will result in the loss of a large number of stem cells, low cell utilization, and limited function of stem cells to perform tissue repair; while transplantation after the combination of cells and tissue engineering scaffolds solves the problem of cell loss, the scaffold material may be harmful in vivo Causes different degrees of inflammation, and the degradation products of the material may change the microenvironment of local tissues and cause more serious lesions.

Cell sheet technology is a new technology for stem cell transplantation and application. The cell membrane forms an endogenous scaffold through the extracellular matrix secreted by the cell itself, which is beneficial to the cell and Interaction between cells, between cells and extracellular matrix, and transmission of genetic information. In a certain sense, cell membrane engineering can simulate the process of embryonic developmental tissue formation to the greatest extent, and its function and value far exceed all exogenous biological scaffold materials. Summary of the invention

In the first aspect, the present disclosure provides a method for generating umbilical cord mesenchymal stem cells, which significantly improves the cell yield (ie, the expansion multiple of one generation of cells) by adding medium in batches. Specifically, the method for producing umbilical cord mesenchymal stem cells of the present disclosure includes the following steps: a. spreading umbilical cord tissue blocks in a culture container; b. adding complete medium in a batch fed manner for culturing; c. separating and attaching to Culture the cells in the container to obtain umbilical cord mesenchymal stem cells.

In some embodiments, in step b, the complete medium is added to the culture vessel in 2-5 batches, where except for the last addition, to keep the umbilical cord tissue block moist but not covered The complete medium is added to the amount of umbilical cord tissue mass; and the complete medium is added in an amount that covers the umbilical cord tissue mass at the last addition.

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

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

In some embodiments, step a includes:

al. Separate Wharton's glue from umbilical cord tissue;

a2. Cut the Wharton's glue into pieces to obtain tissue pieces; and

a3. Spread the tissue block in a culture container.

In some embodiments, step c includes:

cl. When cells attached to the culture container appear around the tissue block, remove the tissue block, and add an appropriate amount of complete medium to the culture container to continue the culture;

c2. Separate the cells attached to the culture container to obtain umbilical cord mesenchymal stem cells.

In some embodiments, the method includes the following steps:

(1) Separate Wharton's glue from umbilical cord tissue;

(2) Chopping the Wharton's glue to obtain tissue pieces;

(3) Spread the tissue block in a culture container; (4) Drop an appropriate amount of complete medium to the tissue block described in step (3), and culture;

(5) Add an appropriate amount of complete medium to the tissue block described in step (4), and continue to culture;

(6) Add an appropriate amount of complete medium to the culture container to cover the tissue mass, and continue the culture;

(7) When cells attached to the culture container appear around the tissue block, remove the tissue block and add an appropriate amount of complete medium to the culture container to continue the culture;

(8) Separate the cells attached to the culture container to obtain umbilical cord mesenchymal stem cells.

In some embodiments, the cells attached to the culture vessel described in step (7) are umbilical cord mesenchymal stem cells of generation P0. In some embodiments, when the confluence of the cells attached to the culture container described in step (7) is not less than about 80% (for example, not less than about 85%, not less than about 90%, or not less At about 95%), the cells can be separated from the culture container to obtain P0 generation umbilical cord mesenchymal stem cells.

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

In some embodiments, the complete medium is a serum-free medium containing serum replacement.

In some embodiments, the complete medium comprises serum-free medium

Lonza (l 2-725f) and serum replacement Pall (l 5950-017).

In some embodiments, before step (1), the method further includes the following steps: (i) providing fresh umbilical cord tissue; (ii) washing the umbilical cord tissue to remove blood stains. In some embodiments, the umbilical cord tissue is washed with PBS buffer or saline. In some embodiments, the PBS buffer or saline does not contain streptomycin and penicillin.

In some embodiments, in step (1), the Wharton's glue is separated by the following steps: removing the umbilical cord adventitia and blood vessels of the umbilical cord tissue, and peeling off the Wharton's glue.

In some embodiments, in step (2), sterile scissors are used to cut the Warton's strands into tissue pieces.

In some embodiments, the volume of the tissue mass is about 1-2 mm ³ .

In some embodiments, in step (3), the culture container is cell culture. In some embodiments, the culture container is a cell culture with a diameter of 100 mm. In some embodiments, the tissue blocks are evenly spread on the culture volume at an interval of about 2-30 mm. 器中。

In some embodiments, in steps (4)-(7), the culture condition is 37 ^° C, 5% CO ₂ . In some embodiments, in steps (4)-(7), culture is performed in an incubator at 37 ^° C. and 5% CO ₂ .

In some embodiments, in step (4), the incubation time is about 24 hours.

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

In some embodiments, in step (5), the incubation time is about 24 hours.

In certain embodiments, in step (5), the amount of complete medium is about 20-200fil _o

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

In some embodiments, in step (6), the amount of the complete medium is about 3 ml. In some embodiments, in step (7), the amount of the complete medium is about 5 ml. In some embodiments, after step (7), the method further includes the following step: when the cell confluence is greater than or equal to about 85% (eg, greater than or equal to about 90%, greater than or equal to about 95%, or When it is greater than or equal to about 100%), the cells are passaged.

In some embodiments, passage is performed at a cell density of about 1 x 10 ⁶ /ml.

The method of passage of cells is well known to those skilled in the art. For example, the method may include: separating the cells from the culture container and uniformly dispersing the cells in the culture medium, and then inoculating the culture container. Add an appropriate amount of medium, and change an appropriate amount of fresh medium every 1 to 5 days according to the cell growth status. When the cells grow to 70-100% confluence, repeat the subculture operation. Each time the cells are subcultured, the number of generations increases by one. Umbilical cord mesenchymal stem cells grow adherently, are fibrous, and have a uniform shape.

In some embodiments, the method for separating the cells from the culture container includes but not limited to trypsin and similar substance digestion, cell scraping, and the like.

In some embodiments, the cells are evenly dispersed in the culture medium by stirring, vortexing, etc., and then seeded.

Optionally, after the umbilical cord mesenchymal stem cells are obtained by culturing, the cell growth curve can be determined by the MTT method, WST method, DNA content detection method, ATP detection method, etc., to evaluate the growth activity of the umbilical cord mesenchymal stem cells. In addition, flow cytometry can be used to detect cell surface markers, Three-way differentiation assay and PCR method to detect cell expression genes to identify the isolated and cultured umbilical cord mesenchymal stem cells.

In some embodiments, after step (8), the method further includes a step of freezing the umbilical cord mesenchymal stem cells.

In some embodiments, cryopreservation is performed at a cell density of about 2 x 10 ⁶ /ml.

In the second aspect, the present disclosure provides a method for preparing umbilical cord mesenchymal stem cell membranes, which is characterized in that: without using enzymes and the like for digestion, umbilical cord mesenchymal stem cells and their extracellular secreted during proliferation The matrix and growth factors are completely retained and separated from the culture solitary surface to form a membrane of umbilical cord mesenchymal stem cells.

Specifically, the method for preparing umbilical cord mesenchymal stem cell membranes of the present disclosure includes the following steps: adding a coating solution to a temperature-sensitive culture for incubation, the coating solution containing serum; adding umbilical cord mesenchymal stem cells to the temperature-sensitive culture Cultivation during cultivation;

The pre-cooled buffer is added to the above-mentioned temperature-sensitive culture sol, and the umbilical cord mesenchymal stem cells and their secreted extracellular matrix are separated in layers to obtain the umbilical cord mesenchymal stem cell membrane.

In some embodiments, the Shengzhuang mesenchymal stem cells are prepared by the method of the first aspect of the present disclosure.

In some embodiments, the umbilical cord mesenchymal stem cells are umbilical cord mesenchymal stem cells with passage numbers P0-P20, for example, umbilical cord mesenchymal stem cells with passage numbers P2-P10.

In some embodiments, a cell suspension of umbilical cord mesenchymal stem cells is added to a temperature-sensitive culture for culture.

In certain embodiments, the umbilical cord mesenchymal stem cells are umbilical cord mesenchymal stem cells of generation P 0 -P 20. In certain embodiments, the umbilical cord mesenchymal stem cells are umbilical cord mesenchymal stem cells of generation P2-P10.

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

The “temperature-sensitive culture isolation” mentioned herein refers to a culture with a temperature-sensitive polymer material coated on the surface. The molecular chain stretches of the polymer material at different temperatures are different, thus exhibiting hydrophilicity or Hydrophobicity enables the hydrophilicity and hydrophobicity of the polymer substance to change with changes in external temperature. When the surface of the temperature-sensitive culture solitary is hydrophilic, the adhesion to the cells and the extracellular matrix secreted by the cells becomes poor, and the cells will fall off in layers. In a specific embodiment, when the temperature is lowered When the temperature is below the low critical solution temperature (LCST) of the polymer substance, the temperature-sensitive culture sol surface is hydrophilic, so that the cells will fall off in layers.

The present disclosure utilizes temperature-sensitive culture cells to successfully achieve lamellar mesenchymal stem cells detached from the bottom of the temperature-sensitive culture without using enzymes and the like for digestion or peeling by physical methods, and become retained cells. Cell membranes intactly connected by the outer matrix.

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, that is, it is serum isolated from autologous peripheral blood. "Autologous" as used herein means that the serum isolated from human peripheral blood is obtained and separated from a subject, and the umbilical cord mesenchymal stem cell patch obtained using the serum is administered to the same subject, that is The body and the receptor are the same. In such embodiments, without being bound by theory, it is believed that the use of autologous serum will be expected to reduce or eliminate the immune response from the subject.

In some embodiments, the coating solution is 100% serum. The amount of adhesion factor contained in the coating solution and the coating time directly affect the formation of cell membranes. For example, if the adhesion factor is too small, the cells will not adhere well, and if the adhesion factor is too much, it will It hinders the growth of cells, so controlling the amount of adhesion factors and their action time is crucial for the formation of cell membranes. The inventor of the present application unexpectedly discovered that when 100% serum is used for coating for 12-24 hours, the content of adhesion factors in the coating system is suitable for cell adhesion and cell growth, which is beneficial to the membrane. Formation.

In some embodiments, the coating liquid contains at least 10% (v/v) (eg 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%) serum basal medium. In some embodiments, the basal medium is selected from DMEM/F12, aMEM or DMEM. In such embodiments, the serum is FBS.

In other embodiments, the basal medium is a serum-free medium (SFM), such as Lonza (12-725f). In such embodiments, the serum is serum derived from human peripheral blood.

In some embodiments, the amount of the coating solution is about 0.05-0.3 ml/cm ² (culture isolated bottom area), for example, about 0.09 ml/cm ² , about 0.14 ml/cm ² , or about 0.25 ml /cm ² . In some embodiments, when the diameter of the temperature-sensitive culture solitary is 100 mm, the amount of the coating solution is about 5 ml. In some embodiments, when the diameter of the temperature-sensitive culture solitary is 60 mm, the amount of the coating solution is about 3 ml. In some embodiments, when the diameter of the temperature-sensitive culture is 35 mm, the amount of the coating solution is about 2ml _o

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

In some embodiments, the coating solution is added to the temperature-sensitive culture; the temperature-sensitive culture is placed in an incubator at 37°C and 5% CO ₂ and incubated for about 12-24 hours; optionally , Discard the coating solution remaining in the temperature-sensitive culture.

In some embodiments, the cell suspension of umbilical cord mesenchymal stem cells is adjusted to about 1×10 ⁶ -1×10 ⁷ cells/cm ² (for example, about 2×10 ⁶ -4×10 ⁶ cells/cm ² , about 2.5 x 10 ⁶ -6.0 x 10 ⁷ th / cm ^2, from about 5.5 xl 0 ^⁶ -6.5 x l0 ⁶ th / cm ²⁾ was added a density in the Wen Minpei keep solitary. In certain embodiments, when Wen Minpei keep solitary diameter of 100mm, cells are seeded at a concentration of: from about 6x l0 ^⁷ -7 x l0 ⁷ / ml, and inoculated with a volume of about 5ml. In some embodiments, when the diameter of the temperature-sensitive culture is 60 mm, the inoculation cell concentration is: about 2 >< 10 ⁷ -4 >< 10 ⁷ cells/1111, and the inoculation volume is about 3 ml. In some embodiments, when the temperature-sensitive culture solitary diameter is 35 mm, the inoculation cell concentration is about ⁸ ×10 ⁶ -1.5×10 ⁷ cells/ml, and the inoculation volume is about 2 ml.

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

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

In some embodiments, the buffer is selected from HBSS, PBS or physiological saline. In some embodiments, a 4°C pre-cooled buffer (eg, HBSS, PBS, or saline) is added. When the pre-cooled buffer is added, the layered umbilical cord mesenchymal stem cells will gradually detach from the bottom surface of the temperature-sensitive culture and become a cell membrane that retains the complete connection of the extracellular matrix.

In some embodiments, the amount of the pre-cooled buffer is about 0.05-0.3 ml/cm ² (culture bottom area), for example, about 0.09 ml/cm ² , about 0.14 ml/cm ² , or about 0.25 ml/cm ² . In certain embodiments, when the temperature-sensitive culture solitary diameter is 100 mm, the amount of the buffer is about 5 ml. In some embodiments, when the temperature-sensitive culture solitary diameter is 60 mm, the amount of the buffer is about 3 ml. In some embodiments, when the temperature-sensitive culture solitary diameter is 35mm, the buffer solution The amount is about 2ml.

In some embodiments, the method further includes the step of transferring the umbilical cord mesenchymal stem cell membrane sheet to a storage container. In some embodiments, the storage container is cell culture.

In some embodiments, the umbilical cord mesenchymal stem cell membrane can be transferred to the storage container by the following steps: Use scissors (for example, sterile) to cut off the end of the pipette tip (for example, 1ml tip) About 1/3; Use the short tip and suck the membrane with a pipette, and transfer the membrane to the storage container.

In other embodiments, the umbilical cord mesenchymal stem cell membrane may be transferred to a storage container by the following steps: Pour the liquid in the temperature-sensitive culture together with the cell membrane into the storage container. When the temperature-sensitive culture is poured, the cell membrane that has been separated from the bottom will flow into the storage container along with the flow of liquid. During this transfer process, since the cell membrane is floating on the liquid, it is ensured that the cell membrane will not directly stick to the edge of the temperature-sensitive culture or storage container, thereby preventing the cell membrane from being torn or damaged.

In certain embodiments, the membrane sheet to be transferred Wenmin Pei Yang where the amount of liquid in the isolated (i.e., the amount of buffer) is maintained at about 0.05-0.41111 / ⁰¹¹¹² (culture lone bottom area), for example, from about 0.09 to 0.18ml/cm ² , about 0.14-0.24ml/cm ² , or about 0.25-0.38ml/cm ² . In some embodiments, when the temperature-sensitive culture solitary diameter is 100 mm, the liquid volume is about 5-10 ml. In some embodiments, when the temperature-sensitive culture solitary diameter is 60 mm, the liquid volume is about 3-5 ml. In some embodiments, when the temperature-sensitive culture solitary diameter is 35 mm, the liquid volume is about 2-3 ml.

In other embodiments, the umbilical cord mesenchymal stem cell membrane can be scooped up using a membrane scoop and transferred to a storage container. The membrane shovel is any shovel product that can be used for cells, such as a special membrane production or cell staining production.

In a third aspect, the present disclosure provides an umbilical cord mesenchymal stem cell membrane sheet, which is prepared by the method described in the second aspect. The umbilical cord mesenchymal stem cell membrane prepared by the method of the present disclosure contains the umbilical cord mesenchymal stem cells and all the extracellular matrix and growth factors secreted during the proliferation process. In addition, since no enzymes and the like are used for digestion and no physical method is used for stripping, the umbilical cord mesenchymal stem cell membrane of the present disclosure has a high density of umbilical cord mesenchymal stem cells, the membrane thickness is uniform, and the edges are neat. The umbilical cord mesenchymal stem cell membrane of the present disclosure can secrete various cytokines including angiogenesis and immune regulation, and participate in the repair of tissues and organs. Optionally, after the umbilical cord mesenchymal stem cell membrane sheet is prepared, the surface structure of the cell membrane sheet can be observed through a scanning electron microscope. In addition, the amount of cytokines secreted by the cell membrane and the protein contained in the extracellular matrix in the cell membrane can be detected.

In some embodiments, the umbilical cord mesenchymal stem cell membrane sheet has a surface that does not contact the culture during the preparation process and a base surface that contacts the culture, the surface is relatively smooth and the base surface is relatively rough.

In some embodiments, the umbilical cord mesenchymal stem cell membrane comprises a single-layer or multi-layer interconnected cell structure that substantially exhibits uniform cell directionality, and substantially retains extracellular secretion from umbilical cord mesenchymal stem cells. Matrix.

In some embodiments, the umbilical cord mesenchymal stem cell membrane sheet has an extracellular matrix (e.g., a substantially continuous layer of extracellular matrix) distributed on at least its base surface. In some embodiments, the extracellular matrix comprises at least one of polyamino acid, collagen, polysaccharide, fibronectin, vitrone, and laminin, for example, it may be fibronectin and laminin. A mixture of egg whites. In some embodiments, the junction between the umbilical cord mesenchymal stem cells contained in the umbilical cord mesenchymal stem cell membrane sheet contains the above-mentioned substances.

In some embodiments, the umbilical cord mesenchymal stem cell membrane is off-white, with a dense structure, and a smooth and flat surface.

In some embodiments, the umbilical cord mesenchymal stem cell patch is rich in fibronectin and integrin (31.

In some embodiments, the retinal pigment epithelial cells in the umbilical cord mesenchymal stem cell patch can secrete a variety of angiogenic factors and immunomodulatory factors. For example, the angiogenic factors and immunoregulatory factors may include hepatocyte growth factor (HGF), interleukin-6 (IL-6), interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) One or more of.

In a fourth aspect, the present disclosure relates to a method for treating a disease related to heart tissue damage or cardiac insufficiency in a subject, the method comprising locally applying the umbilical cord of the second aspect of the present disclosure to the injured site of the subject Mesenchymal stem cell membrane steps.

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 cord mesenchymal stem cell patch of the second aspect of the present disclosure in the treatment of diseases related to cardiac tissue damage or cardiac insufficiency in a subject. In the sixth aspect, the present disclosure relates to the use of the umbilical cord mesenchymal stem cell membrane sheet of the second aspect of the present disclosure in preparing a composition for treating cardiac tissue damage or diseases related to 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 uses the method of adding the medium in batches to significantly improve the cell yield of mesenchymal stem cells. The higher the cell yield, the more cells can be obtained, which is easier to meet the dosage of clinical treatment.

The present disclosure utilizes temperature-sensitive culturing and controlling the amount of serum and coating time during the process of coating temperature-sensitive culturing, without using enzymes and the like for digestion and without physical stripping, the umbilical cord mesenchymal stem cells and The extracellular matrix and growth factors secreted during the proliferation process are completely retained and separated from the culture solitary surface to obtain a sheet-like cell membrane. The cell patch obtained by this method has high cell density, uniform thickness and complete structure. The umbilical cord mesenchymal stem cell membrane prepared by the method of the present disclosure has abundant natural extracellular matrix, and can retain most of the fibronectin and laminin, and does not need to be sutured when transplanted in the body. Adhesive molecules and extracellular matrix can directly adhere to diseased tissues, so that 100% of the cells act on the damaged parts of the body, thereby improving the regeneration and repair effect of cell transplantation on tissues and the transplanted cells better retain their activity. Description of the drawings

Figure 1 shows representative photographs of primary day 0, primary day 5, and umbilical cord mesenchymal stem cells P0. Among them, the primary generation refers to the umbilical cord tissue block, and P0 refers to the umbilical cord mesenchymal stem cells that have crawled out of the tissue block but have not been passaged.

Figure 2 shows the representative photos of the umbilical cord mesenchymal stem cells P2 on the 5th day under the x4x objective lens and the x10x objective lens.

Figure 3 shows the results of flow cytometric detection of surface markers of umbilical cord mesenchymal stem cells.

Figure 4 shows the test results of the adipogenic and osteogenic differentiation of umbilical cord mesenchymal stem cells.

Figure 5 shows representative photographs of membranes of umbilical cord mesenchymal stem cells.

Figure 6 shows a scanning electron microscopic image of an umbilical cord mesenchymal stem cell patch. Figure 6A: Surface (upper surface) of cell membrane. Figure 6B: Basal surface of cell membrane. Figure 7 shows an immunofluorescence imaging photograph of a membrane of umbilical cord mesenchymal stem cells. Figure 7A: Fibronectin. Figure 7B: Integrin (31.

Figure 8 shows the results of using the ELISA method to detect cytokine expression in the culture supernatant of the optic cord mesenchymal stem cell patch.

Figure 9 shows the characterization of the constructed mouse disease model of heart failure. Figure 9A: Heart photos of disease model mice; Figure 9B: ECG results of disease model mice.

Figure 10 shows the mouse echocardiogram results at different time points. Figure 10A: Before modeling; Figure 10B: One week after modeling; Figure 10C: Four weeks after modeling. Left: control group animals; right: cell patch transplantation group animals.

Figure 11 shows the curve of the left ventricular ejection fraction of mice before and after modeling with time.

Figure 12 shows the curve of the short axis shortening index of the left ventricle of mice before and after modeling with time. Figure 13 shows the curve of the left ventricle diameter of the mouse before and after modeling with time.

Figure 14 shows the curve of the volume of the left ventricle of the mouse before and after modeling with time.

Figure 15 shows the results of Masson staining of mouse heart tissue sections at the end of the experiment (day 28 after modeling). Left side: control group animals; right side: cell patch transplantation group animals. detailed description

The present invention will be described below based on examples, but the present invention is not limited to these examples.

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

Rinse the fresh umbilical cord repeatedly with lxPBS buffer without blue chain double antibody to remove blood stains. The outer membrane and blood vessels of the umbilical cord are removed to obtain the Wharton's gel-like tissue in the umbilical cord tissue. Cut into 1-2 mm ³ tissue blocks with sterile scissors, spread them flat in a 100mm culture solitary, add 20-100ul of complete medium to each tissue block and place it in a 37°C, 5% CO ₂ incubator. After 24 hours, add 20-200ul complete medium dropwise. Add 3 ml of complete medium during the 48-hour culture. In 3-5 days, the cells crawl out and replace the medium, remove the tissue mass, and add 5ml medium to each culture sol. When the cells grow to 85% confluence, they are passaged, and the cell passage density is 1×10 ⁶ . Each time the cells are passaged, the number of passages increases by 1. Umbilical cord mesenchymal stem cells grow adherently, are fibrous, and have a uniform shape. Representative pictures of P0 and P2 umbilical cord mesenchymal stem cells are shown in Figure 1-2. After testing, the cell yield of this method is 8.3 times, and the cell yield of the general method is 3-5 times. Example 2. Identification of umbilical cord mesenchymal stem cells

2.1 Identification of surface markers of umbilical cord mesenchymal stem cells

Disperse the umbilical cord mesenchymal stem cells in the culture medium and centrifuge. Use serum or serum protein content of 1-20% and isotonic physiological solution to stain the cell surface marker protein according to the purchased reagent instructions, including but not Limited to CD73, CD90, CD105, CD34, CD11B, CD19, CD45, HLA-DR. Among them, the phenotypes of CD73, CD90, and CD105 are positive, and the ratio should be no less than 95%, and the phenotypes of CD34, CD11B, CD19, CD45, and HLA-DR are negative, and the ratio should be no more than 2%. The results are shown in Figure 3, where CD105/CD34 99.64%/0.02%, CD105/CD31 99.04%/0.00%, and CD105/CD117 95.53%/0.51%.

2.2 Three-way induced differentiation of umbilical cord mesenchymal stem cells

The umbilical cord mesenchymal stem cells prepared in Example 1 were inoculated into a suitable incubator according to the ratio of the three-way induction differentiation reagent specification, and the cells to be tested for osteogenic induction grew to 50~90% confluence, and became the cells for fat induction test. When it grows to more than 90% confluence, add osteogenic and adipogenic induction media respectively. During chondrogenesis induction, a certain number of cells are centrifuged to the bottom of the centrifuge tube, and then chondrogenic induction medium is added. After the cell clusters become pellets, the cell pellets are allowed to leave the bottom of the tube to ensure complete contact with the induction medium.

The cells were tested when they were induced and cultured for more than 7 days. Osteogenesis can be induced by staining including but not limited to Alizarin Red and anti-Osteocalcin, fat induction can include but not limited to Oil Red 0, anti-mFABP4 staining, and cartilage induction can be used including but not limited to Alcian Blue and Safranin. , Anti-Aggrecan dyeing.

Figure 4 shows the results of osteogenic differentiation (alizarin red staining) and adipogenic differentiation (oil red 0 staining). Example 3. Preparation of Umbilical Cord Mesenchymal Stem Cell Patch

1. Serum coating: Use 100% serum to coat the temperature-sensitive culture i, and add the following amounts in different cultures: 35mm/2ml, 60mm/3ml, 100mm/5ml. Coating time and temperature: 12-24h, n°c, 5%co ₂ incubator.

2. Cell culture: After the coating is completed, discard the liquid in the culture Jia and inoculate the umbilical cord mesenchymal stem cells obtained in Example 1. The cell inoculation concentration: 35mm The inoculation cell concentration is:

8>< 10 ⁶ --1 .5 X 10 ⁷ cells/ml, 60mmJ! 2 The inoculation cell concentration is: 2>< 10 ⁷ --4>< 10 ⁷ cells/ml, 100mm The concentration of orphan inoculation cells is: 6 x 10 ^{7 -7} x 10 ⁷ cells/ml. Incubate for 12-36h in a 37°C, 5%CO ₂ incubator.

3. Membrane stripping: Take out Wengu from the incubator, aspirate and discard the medium; add 4°C pre-cooled HBSS solution: 35mm/2ml, 60mm/3ml, 100mm/5ml; 10-30 minutes later, it becomes a sheet The mesenchymal stem cells of the singular cord will detach from the solitary edge and become a cell membrane with the complete connection of the extracellular matrix. The macroscopic appearance of the cell membrane is shown in Figure 5. The membrane of umbilical cord mesenchymal stem cells is off-white with dense structure. The surface is smooth and flat.

4. Membrane transfer: Move the completely stripped cell membrane to a normal culture sol, and add HBSS solution to wash the membrane 2-3 times: 35mm/2ml, 60mm/3ml, 100mm/5ml.

5. Temporarily store the prepared diaphragm at 4°C. Example 4. Structural characterization of umbilical cord mesenchymal stem cell membrane

In this example, scanning electron microscopy and immunofluorescence imaging were used to characterize the structure of the prepared umbilical cord mesenchymal stem cell membrane.

First, the umbilical cord mesenchymal stem cell membrane sheet was prepared by the method described in Example 3. Separate the cell membrane from the bottom of the temperature-sensitive intelligent culture, and the formed membrane retains the complete connection of the extracellular matrix. The cell membranes were fixed by 2.5% glutaraldehyde, alcohol gradient dehydration, and air-dried, and then sampled by scanning electron microscopy. As shown in Figure 6, the cell membrane has a surface that is not in contact with the culture (upper surface, Figure 6A) and a basal surface (lower surface, Figure 6B) that is in contact with the culture sol. There are differences in structure: the surface is due to the natural sedimentation of cells, The formed surface is relatively smooth; the base surface is relatively rough in contact with the warm material. Due to its structural characteristics, the base surface can provide greater friction, which is beneficial for the cell membrane to better adhere to the application site during application.

Furthermore, the expression of fibronectin and integrin (31) in the membrane of umbilical cord mesenchymal stem cells was detected by immunofluorescence. The membrane was fixed in fixative and then frozen sectioned, using fluorescein-labeled fibronectin And integrin (31 antibody was stained, and immunofluorescence imaging analysis was performed. The result is shown in Figure 7. The cell membrane prepared by the method of the present disclosure contains a large amount of fibronectin (Figure 7A) and integrin (31 ( Figure 7B).

Fibronectin is widely present in animal tissues and tissue fluids, and has the function of promoting the adhesion and growth of cells, and the adhesion and growth of cells is a necessary condition for maintaining and repairing the tissue structure of the body. Integrin (31 is an important member of the integrin family, which plays an important role in mediating the mutual adhesion between cells, cells and extracellular matrix (ECM), and bidirectional signal transduction between cells and ECM , And is closely related to tissue repair and fibrosis formation. The above results show that the umbilical cord of the present disclosure Mesenchymal stem cell membranes are not simply formed by the accumulation of cells, but are densely organized and biologically active membranes connected by extracellular matrix. In addition, the high levels of fibronectin and integrin (31 expression in the cell membrane) indicate that it has the function of tissue repair and can be used in diseases related to tissue damage such as the heart, liver, pancreas, and uterus to achieve tissue Repair. Example 5. Structural characterization of umbilical cord mesenchymal stem cell membrane

In order to further characterize the function of the umbilical cord mesenchymal stem cell membrane of the present disclosure, the following cytokines secreted by it 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 and participates in the epithelial-mesenchymal transition (EMT) process. It has a regulatory effect on a variety of tissues and cells, and can promote cell movement and division; IL-6 and IL-8 participate 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 above-mentioned cytokines have the function of promoting cell growth and differentiation and promoting the angiogenesis process, and play an important role in tissue repair.

During the preparation of the cell patch, the culture supernatant was taken, and the cytokine in the supernatant was detected by ELISA. The detection result is shown in Figure 8. The results showed that the above-mentioned four cytokines were all expressed in the supernatant, and the expression levels of HGF and IL-8 were high. The above results indicate that the umbilical cord mesenchymal stem cell patch of the present disclosure can secrete a variety of cytokines, including angiogenic factors and immunoregulatory factors, proving that it has high biological activity and functions, and can promote local angiogenesis and tissue repair processes. Moreover, the high level of IL-8 expression indicates that the cell membrane has the function of promoting immune response and inhibiting bacteria during use, which is beneficial to the cell membrane to better perform its biological functions. Example 6. Application of umbilical cord mesenchymal stem cell patch in the treatment of heart failure

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

(1) Use isoflurane mixed with oxygen (isoflurane concentration is about 3.5-5%) to anesthetize the mouse and perform hair removal treatment;

(2) Perform tracheal intubation through direct vision through the neck, use a ventilator to pump anesthetic gas to maintain anesthesia, and measure the mouse's ECG signal; (3) Open the chest and expose the heart. Use 7-0 sutures to ligate the left anterior descending branch of the mouse (about 1.5mm at the lower edge of the left atrial appendage) for modeling;

(4) Thoracic suturing and postoperative treatment.

After modeling in step (3), the left ventricular wall of the mouse was observed to be whitish (Figure 9A); the electrocardiogram results showed that the ST segment was elevated, showing a state of myocardial infarction (Figure 9B), indicating that the heart failure animal model was successfully modeled . For mice in the umbilical cord mesenchymal stem cell patch treatment group, after step (3), the umbilical cord mesenchymal stem cell patch cut into a circle with a diameter of about 2-5 mm or an appropriate shape with an approximate area is attached to the model The surface of the animal's left ventricle. After standing for 3-5 minutes, proceed to the above step (4). Animals without cell patch were used as controls. There were 10 mice in each of the cell patch treatment group and the control group.

Before modeling (Figure 10A), 1 week after modeling (Figure 10B) and 4 weeks after modeling (Figure 10C), mice were subjected to echocardiography. The parasternal short-axis view was taken at the level of the left ventricular papillary muscle. Mark points can be observed echocardiogram. From the results in Fig. 10B and Fig. 10C, it can be seen that the heart of the heart failure model animal has obvious weakening of motion after modeling. In addition, compared with the control animals (left panel), the cell patch transplantation group animals (right panel) had stronger heart movements.

According to the echocardiogram, the curve of the left ventricular ejection fraction with time before and after the operation (Figure 11) and the curve of the left ventricular short axis shortening index with time were calculated and drawn (Figure 12). Left ventricular ejection fraction is an important indicator for evaluating left ventricular function. As shown in the results in Figure 11, after modeling, the left ventricular ejection fraction of the heart failure model animals decreased significantly, but the ejection fraction of the cell patch transplantation group was significantly higher than that of the control group. The left ventricular short-axis shortening index refers to the ratio of the short-axis of left ventricle contraction and diastole. The larger the ratio, the stronger the systolic function of the heart. As shown in the results in Figure 12, the left ventricular short axis shortening index value of the heart failure model animals decreased significantly after modeling, but the left ventricular short axis shortening index value of the cell patch transplantation group was significantly higher than that of the control group.

The curve of left ventricular diameter with time (Figure 13) and the curve of left ventricular volume with time (Figure 14) were also calculated and drawn based on echocardiogram, both of which can be used to describe left ventricular volume. After preparing the animal model, due to ischemic heart failure, the left ventricle undergoes compensatory remodeling and the ventricular volume becomes larger. As shown in the results in Figure 13 and Figure 14, after modeling, the left ventricular diameter and volume (systolic and diastolic) of the cell patch transplantation group were significantly lower than those of the control group, indicating that the use of cell patch has a lack of inhibition. The left ventricular remodeling caused by bloody heart failure has a significant effect and can significantly improve heart function.

At the end of the experiment (day 28 after modeling), the mice were sacrificed and heart tissues were taken for fixation, sectioning and staining. The section results (Figure 15) showed that compared with the control animals, the mesenchymal stem cell membrane The left ventricular wall of the mice in the transplantation group was thicker, the ventricular remodeling was lighter, and the degree of fibrosis was less (Masson staining, collagen fibers were blue). The above results indicate that the degree of fibrosis of the left ventricle of the mice transplanted with the cell patch is significantly lower than that of the control animals. Although the specific embodiments of the present invention are described above, those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. Those 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 these changes and modifications all fall within the protection scope of the present invention.

Claims

1. A method for producing umbilical cord mesenchymal stem cells, which comprises the following steps:

a. Place the umbilical cord tissue block in the culture container;

b. Add complete medium for cultivation in batch feeding mode;

c. Separate the cells attached to the culture container to obtain umbilical cord mesenchymal stem cells.

2. The method of claim 1, wherein in step b, the complete medium is added to the culture vessel in 2-5 batches, wherein except for the last addition, the umbilical cord tissue mass is maintained The complete medium is added in an amount that is moist but does not cover the umbilical cord tissue mass; and the complete medium is added in an amount that covers the umbilical cord tissue mass at the last addition.

3. The method of claim 2, wherein in step b, the complete medium is added to the culture vessel in 3 or 4 batches.

4. The method of any one of claims 1-3, wherein in step b, the complete medium is added in batches at a time interval of 12-36 hours, for example, at a time interval of about 24 hours. Complete medium.

5. The method of any one of claims 1-4, wherein step a comprises:

al. Separate Wharton's glue from umbilical cord tissue;

a2. Cut the Wharton's glue into pieces to obtain tissue pieces; and

a3. Spread the tissue block in a culture container.

6. The method of any one of claims 1-5, wherein step c comprises:

7. The method of claim 1, wherein the method comprises the following steps: (1) Separate Wharton's glue from umbilical cord tissue;

(2) Chopping the Wharton's glue to obtain tissue pieces;

(3) Spread the tissue block in a culture container;

(4) Drop an appropriate amount of complete medium to the tissue block described in step (3), and culture;

8. The method according to any one of claims 1-7, wherein the method further comprises a step of passage of the umbilical cord mesenchymal stem cells after step c.

9. The method of claim 8, wherein the method passages the umbilical cord mesenchymal stem cells when their confluence is greater than or equal to about 85%.

10. The method of any one of claims 1-9, wherein the complete medium is selected from DMEM/F12, aMEM or DMEM containing 10% fetal bovine serum.

11. The method of any one of claims 1-9, wherein said medium comprises a serum-free complete medium Lonza (12_ ⁷ 2 ⁵ f) and serum replacement Pall (l ^⁵ 9 ⁵ 0-01 ⁷ ).

12. The method of any one of claims 7-11, in step (4) and/or step (5), the culture time is about 24h; and/or, the amount of the complete medium is About 20-100fxl.

13. The method of any one of claims 7-12, in step (6), the culture time is about 3-5 days; and/or, the amount of the complete medium is about 3 ml.

14. A method for preparing umbilical cord mesenchymal stem cell membrane sheet, which comprises the following steps: Adding a coating solution to a temperature-sensitive culture for incubation, where the coating solution contains serum; adding umbilical cord mesenchymal stem cells to the temperature-sensitive culture for culture;

15. The method of claim 14, wherein the umbilical cord mesenchymal stem cells are prepared by the method according to any one of claims 1-13.

16. 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.

18. The method of any one of claims 14-16, wherein the coating liquid is a basal medium containing at least 10% (v/v) serum.

19. The method of claim 18, wherein the basal medium is selected from DMEM/F12, aMEM or DMEM, and the serum is FBS.

20. The method of claim 18, wherein the basal medium is Lonza (12-725f), and the serum is serum isolated from human peripheral blood.

21. The method of any one of claims 14-20, which has one or more of the following characteristics:

(1) The amount of the coating solution is about 0.05-0.3ml/cm ² (culture bottom area), for example, about 0.09ml/cm ² , about 0.14ml/cm ² , or about 0.25ml/cm ² ;

(2) The coating time is about 12-24h;

(3) The coating conditions are 37°C, 5% CO ₂ .

22. The method of any one of claims 14-21, which has one or more of the following characteristics:

(1) The cell suspension of the umbilical cord mesenchymal stem cells is added to the temperature-sensitive culture at a density of about 1×10 ⁶ -1×10 ⁷ cells/cm ² ;

(2) The cell suspension is a complete medium containing umbilical cord mesenchymal stem cells;

(3) The culture condition is about 12-36h;

(4) The culture conditions are 37°C, 5% CO ₂ .

23. The method of claim 22, wherein the complete medium is selected from DMEM/F12, aMEM, or DMEM containing 10% fetal bovine serum; or, the complete medium is a serum substitute Pall (l ⁵ 9 ^{^50-017)} serum-free medium ^{^{Lonza (12_ 7 2 5 f)}} .

24. The method of any one of claims 14-23, wherein the buffer is selected from HBSS, PBS, or physiological saline.

25. The method according to any one of claims 14-24, wherein the method further comprises the step of transferring the umbilical cord mesenchymal stem cell membrane into a storage container.

26. The method of claim 25, wherein scissors are used to cut off about 1/3 of the end of the pipette tip; the cut pipette is used to suck the umbilical cord mesenchymal stem cell membrane through the pipette, And transfer the umbilical cord mesenchymal stem cell patch to the storage container.

27. The method of claim 25, wherein the coating solution in the temperature-sensitive culture is poured into a storage container together with the umbilical cord mesenchymal stem cell membrane.

28. The method of claim 25, wherein the umbilical cord mesenchymal stem cell membrane is scooped up using a membrane scoop and transferred to a storage container.

29. An umbilical cord mesenchymal stem cell membrane prepared by the method of any one of claims 14-28.

30. The umbilical cord mesenchymal stem cell membrane sheet according to claim 29, wherein the cell membrane sheet has a surface that does not contact the culture JI2 and a base surface that contacts the culture JI2 during the preparation process, the surface is relatively smooth and the substrate The surface is rough.

31. The umbilical cord mesenchymal stem cell membrane according to claim 29 or 30, which comprises a monolayer or multi-layer interconnected cell structure substantially exhibiting uniform cell directionality, and substantially retains umbilical cord mesenchymal stem cells Secreted extracellular matrix.

32. The umbilical cord mesenchymal stem cell membrane sheet of claim 31, wherein the umbilical cord mesenchymal stem cell membrane sheet has an extracellular matrix distributed at least on its base surface.

33. The retinal pigment epithelial cell patch of any one of claims 29-32, wherein the cell patch is rich in fibronectin and integrin (31.

34. The retinal pigment epithelial cell patch of any one of claims 29-33, wherein the retinal pigment epithelial cells in the cell patch can secrete a variety of angiogenic factors and immunoregulatory factors, such as the angiogenic factors and The immunoregulatory factors include one or more of hepatocyte growth factor (HGF), interleukin-6 (IL-6), interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF).

35. A method for treating a disease related to cardiac tissue damage or cardiac insufficiency in a subject, the method comprising locally applying the method of any one of claims 29-34 to the injured site of the subject Steps of umbilical cord mesenchymal stem cell patch.

36. The method of claim 35, wherein the disease is heart failure.

37. The method of claim 35, wherein the heart failure is ischemic heart failure, such as acute ischemic heart failure.

38. Use of the umbilical cord mesenchymal stem cell patch according to any one of claims 29 to 34 in the treatment of cardiac tissue damage or diseases related to cardiac insufficiency in a subject.

39. Use of the umbilical cord mesenchymal stem cell membrane sheet according to any one of claims 29 to 34 in the preparation of a composition for treating cardiac tissue damage or diseases related to cardiac insufficiency in a subject.

40. The use according to claim 38 or 39, wherein the disease is heart failure.

41. The use according to claim 40, wherein the heart failure is ischemic heart failure, such as acute ischemic heart failure.