CN112292447B

CN112292447B - Umbilical cord mesenchymal stem cell and preparation method of cell membrane thereof

Info

Publication number: CN112292447B
Application number: CN202080000064.5A
Authority: CN
Inventors: 刘东华; 常德华; 刘洋; 赵玉菲; 刘帅; 王淑玲; 王晓惠; 王芳
Original assignee: BOE Technology Group Co Ltd; BOE Regenerative Medicine Technology Co Ltd
Current assignee: BOE Technology Group Co Ltd; BOE Regenerative Medicine Technology Co Ltd
Priority date: 2019-02-28
Filing date: 2020-01-22
Publication date: 2023-03-31
Anticipated expiration: 2040-01-22
Also published as: JP2022521867A; KR102565022B1; WO2020173280A1; CN112292447A; KR20210018345A

Abstract

A preparation method of umbilical cord mesenchymal stem cells can obviously improve the cell yield. An umbilical cord mesenchymal stem cell membrane and a preparation method thereof. The umbilical cord mesenchymal stem cell membrane contains umbilical cord mesenchymal stem cells and all extracellular matrixes and growth factors secreted by the umbilical cord mesenchymal stem cells in the proliferation process, and the preparation method completely reserves the umbilical cord mesenchymal stem cells and the extracellular matrixes and growth factors secreted by the umbilical cord mesenchymal stem cells in the proliferation process and separates the umbilical cord mesenchymal stem cells from the surface of a culture dish under the conditions of not using enzyme and analogue digestion and not physically stripping, so that the lamellar cell membrane is obtained.

Description

Umbilical cord mesenchymal stem cell and preparation method of cell membrane thereof

The application is filed in 2019, month 02 and day 28, and the application number is CN 201910149006.8, which is the priority of the chinese patent application entitled "preparation method of umbilical cord mesenchymal stem cells and cell membrane thereof", and the disclosure of the chinese patent application is incorporated herein by reference in its entirety as a part of the present application.

Technical Field

The disclosure relates to the field of regenerative medicine and cell biology, in particular to a method for generating umbilical cord mesenchymal stem cells, an umbilical cord mesenchymal stem cell membrane and a preparation method thereof.

Background

Umbilical cord Mesenchymal Stem Cells (MSCs) are multifunctional Stem Cells existing in umbilical cord tissues of newborns, can be differentiated into a plurality of tissue Cells, and have wide clinical application prospects. Two methods for separating umbilical cord tissues are commonly used at present, namely enzyme digestion and tissue block adherence; the enzyme digestion has high cost, complex operation and difficult control, is easy to damage cells or has high risk of clinical application of cell mutation, and the tissue block mutation method has simple operation, low cost and small damage to cells and is suitable for clinical application. However, umbilical cord mesenchymal stem cells obtained by conventional tissue block adherence have long period, so that the number of primary cells is small, the purity is low, and the number of cells for clinical application cannot meet the international standard, so that the clinical application is difficult. Therefore, there is a need in the art for a method for preparing umbilical cord mesenchymal stem cells with high yield.

In addition, in the basic research and clinical application of the current umbilical cord mesenchymal stem cells in self tissue repair, a scheme of directly injecting cell suspension or transplanting the cell suspension after combining with a tissue engineering scaffold material is mainly adopted, and both have certain limitations. The direct injection of stem cell suspension can cause the loss of a large amount of stem cells, the utilization rate of the cells is low, and the function of the stem cells for tissue repair is limited; the problem of cell loss is solved by transplanting after the cells are combined with the tissue engineering scaffold, but the scaffold material may cause inflammatory reactions of different degrees in a living body, and degradation products of the material may change the microenvironment of local tissues to cause more serious pathological changes.

The cell sheet (cell sheet) technology is a new stem cell transplantation and application technology. The cell membrane forms an endogenous scaffold through extracellular matrix secreted by cells, and is favorable for interaction between cells and the extracellular matrix and transmission of genetic information. In a certain sense, the cell membrane engineering can simulate the process of embryonic development tissue formation to the maximum extent, and the effect and the value of the cell membrane engineering far exceed all exogenous biological scaffold materials.

Disclosure of Invention

In a first aspect, the present disclosure provides a method of producing umbilical cord mesenchymal stem cells, which significantly improves cell yield (i.e., expansion fold of cell generation) by adding culture medium in batches. Specifically, the method for generating umbilical cord mesenchymal stem cells of the present disclosure comprises the steps of: a. paving the umbilical cord tissue blocks in a culture container; b. adding complete culture medium in a fed-batch mode for culturing; c. isolating the cells attached to the culture vessel, thereby obtaining umbilical cord mesenchymal stem cells.

In certain embodiments, the complete medium is added to the culture vessel in step b in 2-5 batches, wherein the complete medium is added in an amount to keep the umbilical cord tissue mass moist but not covering the umbilical cord tissue mass except for the last addition; and adding the complete medium in an amount to cover the umbilical cord tissue pieces at the time of the last addition.

In certain embodiments, the 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 portions at intervals of 12-36 hours, for example at intervals of about 24 hours, in step b.

In certain embodiments, step a comprises:

a1. isolating Wharton's jelly from umbilical cord tissue;

a2. cutting up the Wharton's jelly to obtain a tissue mass; and

a3. the tissue pieces were plated in culture vessels.

In certain embodiments, step c comprises:

c1. when cells attached to a culture container appear around the tissue block, removing the tissue block, and adding a proper amount of complete culture medium into the culture container to continue culturing;

c2. isolating the cells attached to the culture vessel, thereby obtaining umbilical cord mesenchymal stem cells.

In certain embodiments, the method comprises the steps of:

(1) Isolating Wharton's jelly from umbilical cord tissue;

(2) Cutting up the Wharton's jelly to obtain a tissue mass;

(3) Spreading the tissue mass in a culture container;

(4) Dropwise adding a proper amount of complete culture medium into the tissue block in the step (3), and culturing;

(5) Dropwise adding a proper amount of complete culture medium into the tissue block in the step (4), and continuously culturing;

(6) Adding a proper amount of complete medium to the culture container to cover the tissue mass, and continuing culturing;

(7) When cells attached to a culture container appear around the tissue block, removing the tissue block, and adding a proper amount of complete culture medium into the culture container to continue culturing;

(8) Isolating the cells attached to the culture vessel, thereby obtaining umbilical cord mesenchymal stem cells.

In certain embodiments, the cells attached to the culture vessel in step (7) are umbilical cord mesenchymal stem cells of generation P0. In certain embodiments, when the degree of confluence of the cells attached to the culture vessel as described in step (7) is not less than about 80% (e.g., not less than about 85%, not less than about 90%, or not less than about 95%), the cells may be separated from the culture vessel, thereby obtaining the umbilical cord mesenchymal stem cells of the P0 generation.

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

In certain embodiments, the complete medium is a serum-free medium comprising a serum replacement.

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

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

In certain embodiments, in step (1), the Wharton's gum is isolated by: removing umbilical cord adventitia and blood vessel of umbilical cord tissue, and stripping Wharton's jelly.

In certain embodiments, in step (2), the gordonia gel is sheared into tissue pieces using sterile scissors.

In certain embodiments, the tissue mass has a volume of about 1-2mm 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 pieces are uniformly spread in the culture vessel at a spacing of about 2-30 mm.

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

In certain embodiments, in steps (4) - (7), 5% CO at 37 ℃% ₂ The incubator of (2) for cultivation.

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

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

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

In certain embodiments, in step (5), the amount of complete medium is about 20-200. Mu.l.

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

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

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

In certain embodiments, after step (7), the method further comprises the steps of: the cells are passaged when the cells have a confluence of greater than or equal to about 85% (e.g., greater than or equal to about 90%, greater than or equal to about 95%, or greater than or equal to about 100%).

In certain embodiments, at about 1 × 10 ⁶ Cell density at/ml was passaged.

Methods for passaging cells are well known to those skilled in the art. For example, the method may include: the cells are separated from the culture vessel and uniformly dispersed in a medium, and then inoculated in the culture vessel. Adding proper amount of culture medium, replacing proper amount of fresh culture medium every 1-5 days according to the cell growth state, and repeating the passage operation when the cells grow to 70-100% confluence. The passage number of cells increases by 1 each time the cells are passaged. The umbilical cord mesenchymal stem cells grow adherently, are in a fibroid shape and are uniform in shape.

In certain embodiments, methods of separating the cells from the culture vessel include, but are not limited to, pancreatin and similar digestion, use of cell scraping, and the like.

In certain embodiments, the cells are uniformly dispersed in the medium by stirring, vortexing, or the like, and then seeded.

Optionally, after obtaining umbilical cord mesenchymal stem cells by culture, a cell growth curve may be measured by MTT method, WST method, DNA content detection method, ATP detection method, or the like to evaluate the growth activity of umbilical cord mesenchymal stem cells. In addition, the isolated and cultured umbilical cord mesenchymal stem cells can be identified by detecting cell surface markers through flow cytometry, detecting cell expression genes through three-way differentiation assay and detecting cell expression genes through a PCR method.

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

In certain embodiments, at about 2X 10 ⁶ The cells were frozen at a cell density of ml.

In a second aspect, the present disclosure provides a method of preparing an umbilical cord mesenchymal stem cell patch, characterized in that: umbilical cord mesenchymal stem cells and extracellular matrix and growth factors secreted by the umbilical cord mesenchymal stem cells in the proliferation process are completely retained and separated from the surface of a culture dish without enzyme and analog digestion to form an umbilical cord mesenchymal stem cell membrane.

Specifically, the method for preparing the umbilical cord mesenchymal stem cell membrane comprises the following steps:

adding a coating solution into a temperature-sensitive culture dish for incubation, wherein the coating solution contains serum;

adding umbilical cord mesenchymal stem cells into the temperature-sensitive culture dish for culture;

and adding the precooled buffer solution into the temperature-sensitive culture dish, and separating the umbilical cord mesenchymal stem cells and the extracellular matrix secreted by the umbilical cord mesenchymal stem cells into sheets to obtain the umbilical cord mesenchymal stem cell sheets.

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

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

In certain embodiments, the cell suspension of umbilical cord mesenchymal stem cells is added to a temperature sensitive culture dish for culturing.

In certain embodiments, the umbilical cord mesenchymal stem cell is a passage P0-P20 umbilical cord mesenchymal stem cell. In certain embodiments, the umbilical cord mesenchymal stem cell is a P2-P10 generation umbilical cord mesenchymal stem cell.

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

The "temperature-sensitive culture dish" refers to a culture dish coated with a temperature-sensitive high molecular substance on the surface, wherein the high molecular substance has different molecular chain segment stretching conditions at different temperatures, so that the high molecular substance shows hydrophilicity or hydrophobicity, and the hydrophilicity and the hydrophobicity of the high molecular substance can be changed along with the change of external temperature. When the surface of the temperature-sensitive culture dish is hydrophilic, the adhesion with cells and extracellular matrix secreted by the cells is poor, and the cells are exfoliated in layers. In one embodiment, when the temperature is lowered below the Lower Critical Solution Temperature (LCST) of the polymer, the surface of the temperature sensitive petri dish is hydrophilic, so that cells will be exfoliated.

The method successfully realizes that the mesenchymal stem cells forming the sheet layer are separated from the bottom of the temperature-sensitive culture dish under the condition of not using enzyme and analogue digestion nor physical stripping to form the cell sheet with complete connection of extracellular 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, i.e., is serum isolated from autologous peripheral blood. As used herein, "autologous" means that the serum isolated from human peripheral blood is obtained and isolated from a subject and administered to the same subject using the umbilical cord mesenchymal stem cell patch obtained from the serum, i.e., the donor and recipient are the same. In such embodiments, 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 fluid is 100% serum. The amount of the adhesion factors contained in the coating solution and the coating time directly affect the formation of the cell patch, for example, if the adhesion factors are too small, the adhesion factors cannot adhere well to the cells, and if the adhesion factors are too large, the growth of the cells is hindered, so that the control of the amount of the adhesion factors and the action time thereof are important for the formation of the cell patch. The inventors of the present application have unexpectedly found that when coating for 12-24h with 100% serum, the content of adhesion factors in the coating system is suitable for both cell attachment and cell growth, thereby facilitating patch formation.

In certain embodiments, the coating is a basal medium comprising 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%) serum. In some embodiments, the basal medium is selected from DMEM/F12, alpha MEM, or DMEM. In such embodiments, the serum is FBS.

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

In certain embodiments, the amount of coating solution is about 0.05 to 0.3ml/cm ² (area of the bottom of the culture dish), for example, about 0.09ml/cm ² About 0.14ml/cm ² Or about 0.25ml/cm ² . In certain embodiments, the amount of coating is about 5ml when the temperature sensitive petri dish has a diameter of 100 mm. In certain embodiments, when the temperature sensitive petri dish has a diameter of 60mm, the amount of coating liquid is about 3ml. In certain embodiments, when the temperature sensitive petri dish has a diameter of 35mm, the amount of coating liquid is about 2ml.

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

In certain embodiments, the coating is added to a temperature sensitive petri dish; will be described inTemperature sensitive Petri dish was incubated at 37 ℃ and 5% CO ₂ Incubating for about 12-24 hours in the incubator of (1); optionally, any remaining coating solution in the temperature sensitive petri dish is discarded.

In certain embodiments, the cell suspension of umbilical cord mesenchymal stem cells is administered at about 1x 10 ⁶ -1×10 ⁷ Individual cell/cm ² (e.g., about 2X 10) ⁶ -4×10 ⁶ Per cm ² About 2.5X 10 ⁶ -6.0×10 ⁷ Per cm ² About 5.5X 10 ⁶ -6.5×10 ⁶ Per cm ² ) Is added into the temperature-sensitive culture dish. In certain embodiments, when the temperature sensitive culture dish is 100mm in diameter, the seeded cell concentration is: about 6X 10 ⁷ -7×10 ⁷ Seed/ml, inoculum volume was about 5ml. In certain embodiments, when the temperature sensitive culture dish has a diameter of 60mm, the seeded cell concentration is: about 2X 10 ⁷ -4×10 ⁷ Pieces/ml, inoculation volume about 3ml. In certain embodiments, when the temperature sensitive culture dish has a diameter of 35mm, the seeded cell concentration is: about 8X 10 ⁶ -1.5×10 ⁷ Pieces/ml, inoculation volume about 2ml.

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

In certain embodiments, the culture conditions are 12-36h. In certain embodiments, the culture conditions are 37 ℃ and 5% CO ₂ 。

In certain embodiments, the buffer is selected from HBSS, PBS or physiological saline. In certain embodiments, a 4 ℃ pre-chilled buffer (e.g., HBSS, PBS, or saline) is added. After the precooled buffer solution is added, the lamellar umbilical cord mesenchymal stem cells are gradually separated from the bottom surface of the temperature-sensitive culture dish to form a cell membrane which keeps complete connection of extracellular matrix.

In certain embodimentsThe amount of said pre-cooled buffer is about 0.05-0.3ml/cm ² (area of the bottom of the culture dish), for example, about 0.09ml/cm ² About 0.14ml/cm ² Or about 0.25ml/cm ² . In certain embodiments, the amount of buffer is about 5ml when the temperature sensitive culture dish is 100mm in diameter. In certain embodiments, when the temperature sensitive petri dish is 60mm in diameter, the amount of buffer is about 3ml. In certain embodiments, when the temperature sensitive petri dish is 35mm in diameter, the amount of buffer is about 2ml.

In certain embodiments, the method further comprises the step of transferring the umbilical cord mesenchymal stem cell patch into a storage container. In certain embodiments, the storage container is a cell culture dish.

In some embodiments, the umbilical cord mesenchymal stem cell patch may be transferred to a storage container by: using scissors (e.g., sterile) to cut off about 1/3 of the tip of a pipette tip (e.g., a 1ml tip); the clipped tip was used and the membrane was sucked up by a pipette and transferred to a storage vessel.

In other embodiments, the umbilical cord mesenchymal stem cell patch may be transferred to a storage container by: and pouring the liquid in the temperature-sensitive culture dish together with the cell membrane into a storage container. When the temperature-sensitive culture dish is poured, the cell membrane separated from the bottom of the dish flows into the storage container along with the flow of the liquid. In the transfer process, the cell membrane floats on the liquid, so that the cell membrane is ensured not to be directly adhered to the edge of a temperature-sensitive culture dish or a storage container, and the cell membrane is prevented from being torn or damaged.

In certain embodiments, the amount of liquid (i.e., the amount of buffer) in the temperature sensitive culture dish in which the cell patch to be transferred is placed is maintained at about 0.05-0.4ml/cm ² (area of the bottom of the culture dish), for example, about 0.09 to 0.18ml/cm ² About 0.14-0.24ml/cm ² Or about 0.25 to 0.38ml/cm ² . In certain embodiments, when the temperature sensitive culture dish is 100mm in diameter, the amount of liquid is about 5-10ml. In some embodiments, when the temperature sensitive culture dish has a diameter of 60mm, the temperature sensitive culture dish is used for culturing a culture dishThe amount of liquid is about 3-5ml. In certain embodiments, when the temperature sensitive petri dish has a diameter of 35mm, the amount of liquid is about 2-3ml.

In other embodiments, the umbilical cord mesenchymal stem cell membrane may be scooped up and transferred to a storage container using a membrane scoop. The membrane spatula is any spatula-like article that can be used with cells, such as a specialized membrane spatula or cell staining spatula.

In a third aspect, the present disclosure provides an umbilical cord mesenchymal stem cell patch prepared by the method of the second aspect. The umbilical cord mesenchymal stem cell membrane prepared by the method disclosed by the invention contains umbilical cord mesenchymal stem cells and all extracellular matrix and growth factors secreted by the umbilical cord mesenchymal stem cells in the proliferation process. In addition, because enzymes and analogues are not used for digestion, and a physical method is not used for stripping, the umbilical cord mesenchymal stem cells in the umbilical cord mesenchymal stem cell diaphragm disclosed by the invention have high density, uniform thickness and regular edges. The umbilical cord mesenchymal stem cell membrane disclosed by the invention can secrete various cytokines including angiogenesis and immunoregulation, and participates in the repair of tissues and organs.

Optionally, after the umbilical cord mesenchymal stem cell membrane is prepared, the surface structure of the cell membrane can be observed by a scanning electron microscope. In addition, the amount of cytokines secreted by the cell membrane, proteins contained in the extracellular matrix in the cell membrane, and the like can be detected.

In certain embodiments, the sheet of umbilical cord mesenchymal stem cell membranes has a surface that does not contact the culture dish during the preparation process and a basal surface that contacts the culture dish, the surface being smooth and the basal surface being rough.

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

In certain embodiments, the umbilical cord mesenchymal stem cell patch has an extracellular matrix (e.g., a substantially continuous layer of extracellular matrix) distributed on at least a basal surface thereof. In certain embodiments, the extracellular matrix comprises at least one of a polyamino acid, a collagen, a polysaccharide, a fibronectin, a vitronectin, a laminin, and may be, for example, a mixture of fibronectin and laminin. In certain embodiments, the umbilical cord mesenchymal stem cell patch comprises umbilical cord mesenchymal stem cells comprising a junction location comprising the foregoing.

In certain embodiments, the umbilical cord mesenchymal stem cell membrane is off-white, compact in structure, and smooth and flat in surface.

In certain embodiments, the umbilical cord mesenchymal stem cell patch is enriched in fibronectin and integrin beta 1.

In certain embodiments, the retinal pigment epithelial cells in the umbilical cord mesenchymal stem cell patch are capable of secreting a plurality of angiogenic and immunomodulatory factors. For example, the angiogenic and immunoregulatory 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).

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, the method comprising the step of topically applying the umbilical cord mesenchymal stem cell patch of the second aspect of the present disclosure to the site of damage of the subject.

In certain embodiments, the disease is heart failure. In certain embodiments, the heart failure is ischemic heart failure, e.g., acute ischemic heart failure.

In a fifth aspect, the present disclosure relates to the use of the umbilical cord mesenchymal stem cell sheet of the second aspect of the present disclosure in the treatment of a disease 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 cord mesenchymal stem cell membrane tablet of the second aspect of the present disclosure for the preparation 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, e.g., acute ischemic heart failure.

The method has the advantage that the cell yield of the mesenchymal stem cells is remarkably improved by adding the culture medium in batches. The higher the cell yield is, the more cells are obtained, and the dosage of clinical treatment is more easily met.

According to the method, by utilizing the temperature-sensitive culture dish and controlling the serum dosage and the coating time in the process of coating the temperature-sensitive culture dish, under the conditions of not using enzymes and analogues for digestion and not physically stripping, umbilical cord mesenchymal stem cells and extracellular matrix and growth factors secreted in the proliferation process are completely reserved and separated from the surface of the culture dish, and the lamellar cell membrane is obtained. The cell patch obtained by the method has high cell density, uniform thickness and complete structure. The umbilical cord mesenchymal stem cell membrane prepared by the method disclosed by the invention has abundant natural extracellular matrix, most of fibronectin and laminin can be kept, suturing is not needed during in vivo transplantation, and adhesion molecules and extracellular matrix in the membrane can be directly adhered to a diseased tissue, so that the cells can act on the damaged part of an organism, thereby improving the regeneration and repair effect of the tissue by cell transplantation and better keeping the activity of the transplanted cells.

Drawings

Fig. 1 shows representative photographs of primary day 0, primary day 5, and umbilical cord mesenchymal stem cell P0, respectively. Wherein, primary refers to umbilical cord tissue block, and P0 refers to umbilical cord mesenchymal stem cells which have been crawled out of the tissue block but are not passaged.

Fig. 2 shows representative photographs under x 4-fold objective lens and x 10-fold objective lens at day 5 of umbilical cord mesenchymal stem cell P2, respectively.

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

FIG. 4 shows the results of the measurement of the adipogenic and osteogenic differentiation functions of umbilical cord mesenchymal stem cells.

Figure 5 shows representative photographs of umbilical cord mesenchymal stem cell patches.

Figure 6 shows the scanning electron microscope image photograph of the umbilical cord mesenchymal stem cell membrane. FIG. 6A: surface of the cell patch (upper surface). FIG. 6B: basal surface of cell patch.

Figure 7 shows an immunofluorescence imaging photograph of umbilical cord mesenchymal stem cell patch. FIG. 7A: fibronectin. FIG. 7B: integrin beta 1.

Fig. 8 shows the results of detecting cytokine expression in the umbilical cord mesenchymal stem cell patch culture supernatant using the ELISA method.

Fig. 9 shows the characterization of the constructed heart failure mouse disease model. FIG. 9A: photographs of hearts of disease model mice; FIG. 9B: electrocardiographic results of disease model mice.

Figure 10 shows echocardiographic results of mice at different time points. FIG. 10A: before modeling; FIG. 10B: 1 week after modeling; FIG. 10C: 4 weeks after modeling. Left side: control group animals; right side: cell patch transplant group animals.

Figure 11 shows the left ventricular ejection fraction of mice as a function of time before and after modeling.

Figure 12 shows the mouse left ventricular short axis shortening index as a function of time before and after modeling.

FIG. 13 shows the curves of the mouse left ventricle inner diameter as a function of time before and after modeling.

Fig. 14 shows the curves of the left ventricular volume of the mice before and after modeling as a function of time.

Figure 15 shows a graph of Masson staining results of mouse heart tissue sections at the end of the experiment (day 28 post-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 only these examples.

Example 1 isolation and culture of umbilical cord mesenchymal Stem cells

The fresh umbilical cord was repeatedly washed with 1xPBS buffer solution without the double cyan antibody to remove blood stains. Removing umbilical cord adventitia and blood vessel to obtain Wharton's jelly-like group in umbilical cord tissueAnd (5) weaving. Cutting into pieces of 1-2mm with sterile scissors ³ The tissue blocks of (1) are spread in a 100mm petri dish, 20-100ul of complete medium is added dropwise to each tissue block, the mixture is placed at 37 ℃ and 5% CO ₂ An incubator. After 24 hours, 20-200ul of complete medium was added dropwise. The 48 hour plate was filled with 3ml of complete medium. After 3-5 days, the cells were allowed to creep out of the medium, the tissue blocks were removed, and 5ml of medium was added to each dish. When the cells grow to 85 percent and are merged, passage is carried out, and the cell passage density is 1 multiplied by 10 ⁶ . The passage number of cells increases by 1 each time the cells are passaged. The umbilical cord mesenchymal stem cells grow adherently, are in a fibroid shape and are uniform in shape. Representative pictures of the umbilical cord mesenchymal stem cells at the P0 and P2 generations are shown in FIGS. 1-2. The detection shows that the cell yield of the 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 umbilical cord mesenchymal Stem cell surface markers

The umbilical cord mesenchymal stem cells are dispersed in a culture medium and then centrifuged, and the cells are stained with cell surface marker proteins, including but not limited to CD73, CD90, CD105, CD34, CD11B, CD19, CD45 and HLA-DR, according to the purchased reagent instructions in serum or isotonic physiological solution with the serum protein content of 1-20%. Wherein the phenotype of CD73, CD90 and CD105 is positive, the ratio is not less than 95%, the phenotype of CD34, CD11B, CD19, CD45 and HLA-DR is negative, and the ratio is not more than 2%. The results are shown in FIG. 3, in which CD105/CD34 99.64%/0.02%, CD105/CD 31.04%/0.00%, and CD105/CD117 95.53%/0.51%.

2.2 three-dimensional induced differentiation of umbilical cord mesenchymal Stem cells

The umbilical cord mesenchymal stem cells prepared in the example 1 are inoculated into a proper culture vessel according to the proportion of the three-dimensional induced differentiation reagent specification, and when the cells detected by osteogenesis are grown to 50-90% confluence and the cells detected by adipogenesis are grown to more than 90% confluence, osteogenesis and adipogenesis induction culture media are respectively added. When in chondrogenesis induction, a certain amount of cells are centrifuged to the bottom of a centrifuge tube, then chondrogenesis induction culture medium is added, and after the cells are agglomerated into small balls, the small balls of the cells are separated from the tube bottom to ensure that the cells are completely contacted with the induction culture medium.

The cells were tested after induction culture for more than 7 days. Osteogenic induction can be stained with, but not limited to alizarin red, anti-Osteocalcin, adipogenic induction can be stained with, but not limited to, oil Red O, anti-mFABP4, chondrogenic induction can be stained with, but not limited to, alnew blue, safranin O, anti-Aggrecan.

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 Membrane

1. Coating with serum: coating temperature-sensitive culture dishes with 100% serum, wherein the addition amount in different culture dishes is as follows: 35mm/2ml, 60mm/3ml, 100mm/5ml. Coating time and temperature: 12-24h,37 ℃ and 5% CO ₂ An incubator.

2. Cell culture: after the coating was completed, the liquid in the culture dish was discarded, and the umbilical cord mesenchymal stem cells obtained in example 1 were seeded at a cell seeding concentration: the 35mm dish seeded cell concentration was: 8X 10 ⁶ --1.5×10 ⁷ Cell/ml, 60mm dish seeded cell concentration: 2X 10 ⁷ --4×10 ⁷ Cell/ml, 100mm dish seeded cell concentration: 6X 10 ⁷ --7×10 ⁷ Each/ml. 37 ℃ and 5% of CO ₂ Culturing in an incubator for 12-36h.

3. Stripping the membrane: taking out the incubator from the incubator, and sucking and discarding the culture medium; add 4 ℃ pre-chilled HBSS: 35mm/2ml, 60mm/3ml, 100mm/5ml; after 10-30 minutes, the umbilical cord mesenchymal stem cells which are laminated are separated from the edge of the dish to form a cell membrane which is completely connected with the retained extracellular matrix, the macroscopic morphology of the cell membrane is shown in figure 5, and the umbilical cord mesenchymal stem cell membrane is grey white, compact in structure and smooth and flat in surface.

4. Transferring the membrane: transferring the completely stripped cell membrane to a common culture dish, adding HBSS liquid to wash the membrane for 2-3 times: 35mm/2ml, 60mm/3ml, 100mm/5ml.

5. Temporarily storing the prepared membrane at 4 ℃.

Example 4 structural characterization of umbilical cord mesenchymal stem cell patch

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

First, umbilical cord mesenchymal stem cell sheets were prepared by the method as described in example 3. The cell membrane is separated from the bottom of the temperature-sensitive intelligent culture dish, and the formed membrane is completely connected with extracellular matrix. The cell membrane is fixed by 2.5 percent glutaraldehyde, subjected to alcohol gradient dehydration, air drying and other steps to prepare a sample, and then is shot by a scanning electron microscope. As shown in fig. 6, the cell patch has a surface (upper surface, fig. 6A) not in contact with the culture dish and a basal surface (lower surface, fig. 6B) in contact with the culture dish, and there is a difference in structure: the surface formed by natural sedimentation of cells is smooth; the basal surface is in contact with the material of the cuvette and is relatively rough. Due to the structural characteristics, the basal surface can provide larger friction force, and is favorable for better attachment to an application part when the cell membrane is applied.

The expression of fibronectin and integrin beta 1 in the umbilical cord mesenchymal stem cell membrane is further detected by an immunofluorescence method. The patches were frozen sections after fixation with fixative, stained with fluorescein-labeled fibronectin and integrin beta 1 antibody, and analyzed by immunofluorescence imaging. As a result, as shown in fig. 7, the cell patch prepared by the method of the present disclosure contains a large amount of fibronectin (fig. 7A) and integrin β 1 (fig. 7B).

Fibronectin is widely present in animal tissues and interstitial fluid and has the function of promoting the adhesion growth of cells, which is a necessary condition for maintaining and repairing the tissue structure of the body. Integrin β 1 is an important member of the integrin family, which plays an important role in mediating cell-to-cell, cell-to-extracellular matrix (ECM) adhesion, and bidirectional signaling between cells and ECM, and is closely associated with tissue repair and fibrosis formation. The above results indicate that the umbilical cord mesenchymal stem cell membrane of the present disclosure is not simply formed by stacking cells, but formed by connecting extracellular matrix, and has compact organization and biological activity. Also, high levels of fibronectin and integrin β 1 expression in the cell membrane sheet indicate that it has a tissue repair function, and can be used in diseases associated with damage to tissues such as heart, liver, pancreas, and uterus to achieve tissue repair.

Example 5 structural characterization of umbilical cord mesenchymal stem cell patch

To further characterize the function of the umbilical cord mesenchymal stem cell membrane of the present disclosure, the following cytokines secreted therefrom 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 epithelial-mesenchymal transition (EMT) process, has a regulatory effect on various tissues and cells, and can promote cell movement and division; IL-6 and IL-8 are involved in regulating immune responses and various physiological processes of immune cells; VEGF has the functions of promoting endothelial cell proliferation and inducing angiogenesis. The above cytokines have the functions of promoting cell growth and differentiation and promoting angiogenesis process, and have important effects on tissue repair.

The culture supernatant was taken during the preparation of the cell patch, and the cytokine in the supernatant was detected by ELISA, and the detection results are shown in fig. 8. The results show that the four cytokines are expressed in the supernatant, and the expression levels of HGF and IL-8 are higher. The result shows that the umbilical cord mesenchymal stem cell membrane disclosed by the invention can secrete various cell factors including angiogenesis factors and immunoregulatory factors, and the umbilical cord mesenchymal stem cell membrane is proved to have high biological activity and function and can promote local angiogenesis and tissue repair processes. In addition, the high IL-8 expression level shows that the cell membrane has the functions of promoting immune response and inhibiting bacteria in the using process, and is favorable for the cell membrane to better exert the biological function.

Example 6 use of umbilical cord mesenchymal stem cell patch in the treatment of heart failure

In this example, a heart failure mouse disease model was constructed and the repair function of the umbilical cord mesenchymal stem cell membrane sheet of the present disclosure on heart tissue was evaluated in this model. Firstly, an acute ischemia type heart failure animal model is constructed in male C57BL/6 mice (about 12 weeks old) by a coronary artery ligation method, and the specific steps comprise:

(1) Anesthetizing the mice with isoflurane mixed with oxygen (isoflurane concentration is about 3.5-5%) to remove hair;

(2) Performing tracheal intubation by direct vision through neck transillumination, pumping anesthetic gas by using a respirator to maintain anesthesia, and measuring electrocardiosignals of a mouse;

(3) Open chest and expose heart, mice left anterior descending (approximately 1.5mm below the left auricle) were ligated using 7-0 surgical suture for modeling;

(4) Chest sutures and post-operative procedures.

After modeling in step (3), whitening of the left ventricular wall of the mice was observed (fig. 9A); the electrocardiogram result shows that the ST segment is elevated and shows a myocardial infarction state (figure 9B), which indicates that the modeling of the heart failure animal model is successful. For the umbilical cord mesenchymal stem cell diaphragm treatment group mice, after the step (3), the umbilical cord mesenchymal stem cell diaphragm which is cut into a circle with the diameter of about 2-5mm or an appropriate shape with an approximate area is attached to the surface of the left ventricle of the model animal. After standing for 3 to 5 minutes, the above step (4) is carried out. Animals without attached cell patches served as controls. Cell patch treated and control groups were 10 mice each.

Echocardiography was performed on mice before modeling (fig. 10A), 1 week after modeling (fig. 10B), and 4 weeks after modeling (fig. 10C), and echocardiography was observed with the parasternal short-axis section marked with a section at the level of the left ventricular papillary muscle. As can be seen from the results in fig. 10B and 10C, the heart of the heart failure model animal after modeling had a significant phenomenon of motion reduction. Also, the heart movement was stronger in the cell patch-transplanted animals (right panels) than in the control animals (left panels).

The left ventricular ejection fraction of the mice before and after the operation was calculated from the echocardiogram and plotted against time (fig. 11) and the left ventricular short axis shortening index (fig. 12). Left ventricular ejection fraction is an important index for evaluating left ventricular function. As shown in the results in fig. 11, the left ventricular ejection fraction values of the heart failure model animals after modeling were significantly decreased, but the ejection fractions of the cell patch-transplanted group animals were significantly higher than those of the control group animals. The left ventricle short axis shortening index refers to the ratio of the short axis of the left ventricle in contraction and relaxation, and a larger ratio indicates a stronger systolic function. As shown in the results in fig. 12, the left ventricular short axis shortening index values of the post-modeling heart failure model animals were significantly decreased, but the left ventricular short axis shortening index values of the cell patch-transplanted animals were significantly higher than those of the control animals.

A plot of left ventricular inside diameter over time (fig. 13) and a plot of left ventricular volume over time (fig. 14), both of which can be used to describe left ventricular volume, are also calculated and plotted from echocardiograms. After the animal model is prepared, compensatory remodeling of the left ventricle occurs and the ventricle volume becomes large due to ischemic heart failure. As shown in the results in fig. 13 and fig. 14, after modeling, the inner diameter and volume (systolic phase and diastolic phase) of the left ventricle of the cell patch transplanted animal are significantly lower than those of the control animal, which indicates that the use of the cell patch has a significant effect on inhibiting the left ventricle remodeling caused by ischemic heart failure, and can significantly improve the cardiac function.

At the end of the experiment (day 28 post-modeling), mice were sacrificed and heart tissue was taken for fixation, sectioning and staining. The results of sectioning (fig. 15) show that the left ventricular wall of the mice in the mesenchymal stem cell patch-transplanted group was thicker, ventricular remodeling was less, and fibrosis degree was less (Masson staining, collagen fibers appeared blue) compared to the control group animals. The results show that the degree of fibrosis of the left ventricle of the mouse transplanted with the cell sheet is obviously lower compared with the control group of animals.

While specific embodiments of the invention have been described above, it will be understood by those skilled in the art that these are by way of example only, and that the scope of the invention is defined by the appended claims. Various changes and modifications to these embodiments may be made by those skilled in the art without departing from the spirit and scope of the invention, and these changes and modifications are within the scope of the invention.

Claims

1. A method for preparing an umbilical cord mesenchymal stem cell sheet, comprising the steps of:

generating umbilical cord mesenchymal stem cells by:

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

b. adding complete culture medium in a fed-batch mode for culturing;

c. isolating the cells attached to the culture vessel, thereby obtaining umbilical cord mesenchymal stem cells;

wherein the complete medium is added to the culture vessel in 2-5 portions at intervals of 12-36 hours in step b, wherein the complete medium is added in an amount to keep the umbilical cord tissue pieces wet but not covering the umbilical cord tissue pieces except for the last addition; and adding the complete medium in an amount to cover the umbilical cord tissue pieces at the time of the last addition;

adding a coating solution into a temperature-sensitive culture dish for incubation, wherein the coating solution is 100% serum and the coating time is 12-24h, and discarding the coating solution in the temperature-sensitive culture dish after coating;

adding the cell suspension of the umbilical cord mesenchymal stem cells into the temperature-sensitive culture dish for culture;

2. The method of claim 1, wherein the complete medium is added to the culture vessel in 3 or 4 batches in step b.

3. The method of claim 1 or 2, wherein the complete medium is added in portions at intervals of 24 hours in step b.

4. The method of any one of claims 1-3, wherein step a comprises:

a1. isolating Wharton's jelly from umbilical cord tissue;

a2. cutting up the Wharton's jelly to obtain a tissue mass; and

a3. the tissue pieces were plated in culture vessels.

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

c1. when cells attached to the culture container appear around the tissue block, removing the tissue block, and adding a proper amount of complete culture medium into the culture container to continue culturing;

6. The method of claim 1, wherein the umbilical cord mesenchymal stem cells are generated by:

(1) Isolating Wharton's jelly from umbilical cord tissue;

(2) Cutting up the Wharton's jelly to obtain a tissue mass;

(3) Spreading the tissue mass in a culture container;

7. The method of any one of claims 1-6, wherein the method further comprises the step of passaging the umbilical cord mesenchymal stem cells after step c.

8. The method of claim 7, wherein the umbilical cord mesenchymal stem cells are passaged when they are greater than or equal to 85% confluent.

9. The method of any one of claims 1-8, wherein the complete medium is selected from DMEM/F12, α MEM, or DMEM comprising 10% fetal bovine serum.

10. The method of any one of claims 1-8, wherein the complete medium comprises serum-free medium Lonza (12-725 f) and serum replacement Pall (15950-017).

11. The method of any one of claims 6-10, wherein, in step (4) and/or step (5), the culturing time is 24h; and/or the amount of the complete medium is 20 to 100. Mu.l.

12. The method of any one of claims 6 to 11, wherein, in step (6), the culturing time is 3 to 5 days; and/or the amount of complete medium is 3ml.

13. The method of any one of claims 1-12, wherein the serum is selected from fetal bovine serum or serum isolated from human peripheral blood.

14. The method of any one of claims 1-13, which is characterized by one or more of the following features:

(1) The amount of the coating liquid is 0.05-0.3ml/cm ² (area of culture dish bottom);

(2) Coating conditions 37 ℃ and 5% CO ₂ 。

15. The method of claim 14, wherein the amount of coating solution is 0.09ml/cm ² 、0.14ml/cm ² Or 0.25ml/cm ² 。

16. The method of any one of claims 1-15, which is characterized by one or more of the following features:

(1) the cell suspension of the umbilical cord mesenchymal stem cells is 1x 10 ⁶ -1×10 ⁷ Individual cell/cm ² Is added to the temperature sensitive petri dish;

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

(3) The culture condition of the umbilical cord mesenchymal stem cells in the temperature-sensitive culture dish is 12-36h;

(4) The culture conditions of the umbilical cord mesenchymal stem cells in the temperature-sensitive culture dish are 37 ℃, 5% CO ₂ 。

17. The method of claim 16, wherein the complete medium is selected from DMEM/F12, α MEM, or DMEM comprising 10% fetal bovine serum; alternatively, the complete medium was serum-free medium Lonza (12-725 f) containing serum replacement Pall (15950-017).

18. The method of any one of claims 1-17, wherein the buffer is selected from HBSS, PBS or physiological saline.

19. The method of any of claims 1-18, wherein the method further comprises the step of transferring the umbilical cord mesenchymal stem cell patch into a storage container.

20. The method of claim 19, wherein 1/3 of the pipette tip end is sheared off using scissors; using the shortened suction head, sucking the umbilical cord mesenchymal stem cell membrane by a liquid transfer machine, and transferring the umbilical cord mesenchymal stem cell membrane into a storage container.

21. The method of claim 19, wherein the coating solution in the temperature-sensitive culture dish is poured into a storage container together with the umbilical cord mesenchymal stem cell sheet.

22. The method of claim 19, wherein the umbilical cord mesenchymal stem cell membrane is scooped up and transferred to a storage container using a membrane scoop.

23. An umbilical cord mesenchymal stem cell patch prepared by the method of any one of claims 1-22.

24. The umbilical cord mesenchymal stem cell patch of claim 23, wherein the cell patch has a surface that does not contact a culture dish during preparation and a basal surface that contacts a culture dish, the surface being smooth and the basal surface being rough.

25. The umbilical cord mesenchymal stem cell patch of claim 23 or 24, comprising a monolayer or multilayer interconnected cellular structure that exhibits substantially uniform cellular directionality and substantially retains extracellular matrix secreted by umbilical cord mesenchymal stem cells.

26. The umbilical cord mesenchymal stem cell membrane of claim 25, wherein the umbilical cord mesenchymal stem cell membrane has an extracellular matrix distributed on at least a basal surface thereof.

27. The umbilical cord mesenchymal stem cell patch of any one of claims 23-26, wherein the cell patch is enriched in fibronectin and integrin beta 1.

28. The umbilical cord mesenchymal stem cell sheet of any of claims 23-27, wherein umbilical cord mesenchymal stem cells in the cell sheet are capable of secreting a plurality of angiogenic factors and immunomodulatory factors.

29. The umbilical cord mesenchymal stem cell sheet of claim 28, wherein the angiogenic and immunomodulatory factors comprise one or more of Hepatocyte Growth Factor (HGF), interleukin-6 (IL-6), interleukin-8 (IL-8) and Vascular Endothelial Growth Factor (VEGF).

30. Use of umbilical cord mesenchymal stem cell membrane tablet of any one of claims 23-29 in the preparation of a composition for the treatment of cardiac tissue damage or a cardiac insufficiency related disease in a subject.

31. The use according to claim 30, wherein the disease is heart failure.

32. The use of claim 31, wherein the heart failure is ischemic heart failure.

33. The use of claim 32, wherein the ischemic heart failure is acute ischemic heart failure.