Method for energizing mesenchymal stem cells

Abstract

The invention discloses a method for energizing mesenchymal stem cells, the method can control the cell size, the diameter of each cell is less than 15 [mu] m, the cells can easily pass through blood capillaries, and the risk of vascular embolism is avoided. The survival rate of each part of cells is greater than 98%, dead cells are few, and the energized MSCs have the ability to resist attack of different immune cells, such as macrophages, complements, blood coagulation factors, NK and T cells, high-expression chemotactic factors and blood vessel recognition molecules, good ability to interact with vascular endothelial cells, high-expression anti-apoptosis molecules, and high-expression anti-apoptosis molecules. The influence of adverse factors such as hypoxia, hyperinflammation and high free radicals in a severe microenvironment can be resisted. MSCs subgroups with high expression of stemness genes and high proliferation and cloning capacity are separated and selected, other polluted cells and senescent cells are removed, the MSCs group for amplification is more uniform in form and consistent in function, most of MSCs are controlled, a cell cycle with the consistent length is obtained, and heterogeneity can be reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and in particular, relates to a method for empowering mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) are multipotent adult stem cells with self-renewal and multidirectional differentiation functions. They play biological roles in immunoregulation, inflammation control, repair activation and regeneration and remodeling through paracrine active substances such as cytokines, chemical factors and regulatory molecules. They weakly express class II major histocompatibility complex (MHC-II) and co-stimulatory molecules, which means that allogeneic or autologous MSCs transplantation will not cause adverse reactions at the medical level, and also have strong potential for beauty, anti-aging, treatment of chronic diseases and prevention of lesions. MSCs from different sources have some different biological characteristics, namely heterogeneity, which will produce certain differences in therapeutic effects; for example, umbilical cord-derived MSCs have strong immunosuppressive function and inflammation control ability. MSCs that have been domesticated and educated can acquire certain specific functions and better therapeutic effects.

[0003] In the existing stem cell culture process, it is difficult to remove other contaminating cells and aging cells, and to separate and select MSCs subpopulations with high expression of stemness genes and high proliferation and cloning ability. Summary of the invention

[0004] In view of the problem that it is difficult to remove contaminated cells and senescent cells in the existing stem cell culture process, the present invention provides a method for empowering mesenchymal stem cells.

[0005] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for empowering mesenchymal stem cells, comprising the steps of:

[0007] S1. Place medical PBS containing 100 U / mL penicillin and 100 mg / L streptomycin in a sterile dish, obtain the umbilical cord fragments by aseptic processing, peel off the outer amniotic membrane and epithelial layer, and remove blood vessels and blood clots, leaving clean gelatinous tissue;

[0008] S2. In another culture dish, wash the separated gelatinous tissue with medical PBS, and then cut it into pieces about 2 mm in size. 3 Size of tissue blocks;

[0009] S3. Place the tissue block in a culture dish, invert it and store it at 4°C for 1-2 hours, then add 2 ml of low-glucose DMEM / F12 culture medium and place it in a 37°C, 5% CO2 incubator for culture;

[0010] S4. After 24 hours, add an equal amount of culture medium. After 5-7 days, change half of the medium. Thereafter, change half of the medium every 3 days. Observe the growth of cells around the adherent tissue. When the cells reach a confluence of about 85%, they can be digested and passaged.

[0011] S5. Digest the cells using TrypLEExpress pancreatic enzyme substitute and place them in a 37°C incubator or at room temperature (25°C) to digest the cells out of suspension.

[0012] S6. Add the same amount of medical PBS, use a pipette to pipette to form a single cell suspension, and add it to a centrifuge tube;

[0013] S7, the pre-seeded single cell suspension was placed in a 15 cm culture dish for 30 minutes, large cells larger than the target cells, miscellaneous cells and cells with strong adsorption were removed, all non-adherent cell suspensions were collected, and 0.5 ml was aspirated from them for trypan blue staining and counting;

[0014] S8. Count with a counting plate or cell counter at 5000 / cm 2 Density inoculated into new culture bottles, cultured in a 5% CO2, 37℃ incubator for 72h;

[0015] S9. When the cell density reaches 85-90%, it can be digested and subcultured.

[0016] Furthermore, the aseptic treatment method in step S1 specifically includes: using sterile surgical scissors to cut the umbilical cord into pieces of about 2.0 cm, using sterile pointed forceps to peel off the outer amniotic membrane and epithelial layer, and using a scalpel to longitudinally cut the umbilical cord to remove blood vessels and blood clots.

[0017] Furthermore, in step S5, the specific method of making the cells out of suspension is as follows: after digestion for 2 to 5 minutes, the culture bottle is placed under an inverted microscope for observation. When the cytoplasm is found to shrink and the intercellular space is increased, the bottom of the culture dish is gently tapped to make the cells out of suspension.

[0018] Furthermore, in step S6, the environment in the centrifuge tube is as follows: the single cell suspension is centrifuged at 1200 rpm for 5 min, the supernatant is removed by aspiration, and 10 ml of low-glucose DMEM / F12 culture medium is added.

[0019] Furthermore, the method further comprises step S10, repeating step S8 and step S9 for 3 times, culturing in a 5% CO2, 37OC incubator for 60 hours for the 4th time, adding a ZNB-enabling factor, culturing in a low oxygen environment for 12 hours, and then terminating the digestion with the supernatant, collecting the discrete suspended cells, centrifuging at 1200 r.min for 5 minutes, suspending the cells with physiological saline, counting and quality testing, and releasing them for use after passing the test.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention solves the problem of isolating and selecting MSCs subpopulations with high expression of stemness genes and high proliferation and cloning ability, removing other contaminating cells and senescent cells, making the MSCs population used for expansion more uniform in morphology and consistent in function, and controlling the vast majority of MSCs to obtain a cell cycle of consistent length to reduce heterogeneity. The present culture method has better anti-apoptosis ability, migration ability, regulation ability, inflammation control ability, activation ability and repair ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is an overall flow chart of a method for empowering mesenchymal stem cells in an embodiment of the present invention. DETAILED DESCRIPTION

[0023] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments and drawings. The contents mentioned in the implementation modes are not intended to limit the present invention.

[0024] like Figure 1 As shown, this embodiment provides a method for empowering mesenchymal stem cells, comprising the steps of:

[0025] S1. In a sterile dish, place medical PBS containing 100U / mL penicillin and 100mg / L streptomycin, and use a sterile treatment method to obtain umbilical cord fragments, peel off the outer amnion and epithelial layer, and remove blood vessels and blood clots, leaving clean gel-like tissue; the sterile treatment method in step S1 specifically includes: using sterile surgical scissors to cut the umbilical cord into fragments of about 2.0 cm, using sterile pointed tweezers to peel off the outer amnion and epithelial layer, and using a scalpel to longitudinally cut the umbilical cord to remove blood vessels and blood clots.

[0026] S2. In another culture dish, wash the separated gelatinous tissue with medical PBS, and then cut it into pieces about 2 mm in size. 3 Size of tissue blocks;

[0027] S3. Place the tissue block in a culture dish, invert it and store it at 4°C for 1-2 hours, then add 2 ml of low-glucose DMEM / F12 culture medium and place it in a 37°C, 5% CO2 incubator for culture;

[0028] S4. After 24 hours, add an equal amount of culture medium, and replace half of the medium after 5-7 days. Thereafter, replace half of the medium every 3 days. Observe the growth of cells around the adherent tissue. When the cells reach a confluence of about 85%, they can be digested and passaged.

[0029] S5. Digest the cells using TrypLEExpress pancreatic enzyme substitute, place them in a 37°C incubator or at room temperature (25°C) for digestion, and make the cells out of suspension. The specific method of making the cells out of suspension in step S5 is as follows: after digestion for 2 to 5 minutes, place the culture bottle under an inverted microscope for observation. When the cytoplasm shrinks and the cell gap increases, tap the bottom of the culture dish to make the cells out of suspension.

[0030] S6. Add the same amount of medical PBS, use a pipette to pipette to form a single cell suspension, and add it to a centrifuge tube; in step S6, the environment inside the centrifuge tube: centrifuge the single cell suspension at 1200 rpm for 5 min, remove the supernatant, and add 10 ml of low-glucose DMEM / F12 culture medium.

[0031] S7, the pre-seeded single cell suspension was placed in a 15 cm culture dish for 30 minutes, large cells larger than the target cells, miscellaneous cells and cells with strong adsorption were removed, all non-adherent cell suspensions were collected, and 0.5 ml was aspirated from them for trypan blue staining and counting;

[0032] S8. Count with a counting plate or cell counter at 5000 / cm 2 Density inoculated into new culture bottles, cultured in a 5% CO2, 37℃ incubator for 72h;

[0033] S9. When the cell density reaches 85-90%, it can be digested and subcultured.

[0034] The method also includes step S10, repeating step S8 and step S9 for 3 times, culturing in a 5% CO2, 37OC incubator for 60 hours for the 4th time, adding a ZNB-enabling factor, culturing in a low-oxygen environment for 12 hours, and then terminating the digestion with the supernatant, collecting discrete suspended cells, centrifuging at 1200 r.min for 5 minutes, suspending the cells with physiological saline, counting and quality testing, and releasing them for use after passing the test.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention can control the cell size, and the diameter of each cell is less than 15um, which is easy to pass through capillaries without the risk of vascular embolism. The survival rate of each cell is greater than 98%, and there are very few dead cells. The MSCs are empowered to resist the attack of different immune cells, such as macrophages, complement, coagulation factors, NK and T cells, highly express chemokines and vascular recognition molecules, have a good ability to interact with vascular endothelial cells, highly express anti-apoptosis molecules, and can resist the adverse factors of the harsh microenvironment such as hypoxia, high inflammation, high free radicals, etc.

[0037] Isolate and select MSCs subpopulations with high expression of stemness genes and high proliferation and cloning ability, remove other contaminating cells and aging cells, make the MSCs population used for expansion more uniform in morphology and consistent in function, control the cell cycle of most MSCs to obtain a consistent length, and reduce heterogeneity. This culture method has better anti-apoptosis ability, migration ability, regulation ability, inflammation control ability, activation ability and repair ability.

[0038] The above is a detailed introduction to a method for empowering mesenchymal stem cells provided by the present application. The description of the specific embodiments is only used to help understand the method and its core idea of ​​the present application. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A method for empowering mesenchymal stem cells, characterized in that: Includes steps: S1. Place medical PBS containing 100 U / mL penicillin and 100 mg / L streptomycin in a sterile dish, obtain the umbilical cord fragments by aseptic processing, peel off the outer amniotic membrane and epithelial layer, and remove blood vessels and blood clots, leaving clean gelatinous tissue; S2. In another culture dish, wash the separated gelatinous tissue with medical PBS and cut it into 2 mm pieces. 3 Size of tissue blocks; S3. Place the tissue block in a culture dish, invert it and store it at 4°C for 1-2 hours, then add 2 ml of low-glucose DMEM / F12 culture medium and place it in a 37°C, 5% CO2 incubator for culture; S4. After 24 hours, add an equal amount of culture medium. After 5-7 days, change half of the medium. Thereafter, change half of the medium every 3 days. Observe the growth of cells around the adherent tissue. When the cells reach 85% confluence, they can be digested and passaged. S5. Digest the cells using TrypLEExpress pancreatic enzyme substitute and place them in a 37°C incubator or at room temperature to remove the cells from suspension. S6. Add the same amount of medical PBS, use a pipette to pipette to form a single cell suspension, and add it to a centrifuge tube; S7, the pre-seeded single cell suspension was placed in a 15 cm culture dish for 30 minutes, large cells larger than the target cells, miscellaneous cells and cells with strong adsorption were removed, all non-adherent cell suspensions were collected, and 0.5 ml was aspirated from them for trypan blue staining and counting; S8. Count with a counting plate or cell counter at 5000 / cm 2 Density inoculated into new culture bottles, cultured in a 5% CO2, 37℃ incubator for 72h; S9. When the cell density reaches 85-90%, it can be digested and subcultured.

2. A method for empowering mesenchymal stem cells according to claim 1, characterized in that: The aseptic treatment method in step S1 specifically includes: using sterile surgical scissors to cut the umbilical cord into pieces of about 2.0 cm, using sterile pointed forceps to peel off the outer amniotic membrane and epithelial layer, and using a scalpel to longitudinally cut the umbilical cord to remove blood vessels and blood clots.

3. A method for empowering mesenchymal stem cells according to claim 2, characterized in that: The specific method of making the cells out of suspension in step S5 is as follows: after digestion for 2 to 5 minutes, the culture bottle is placed under an inverted microscope for observation. When the cytoplasm shrinks and the intercellular space increases, the bottom of the culture dish is gently tapped to make the cells out of suspension.

4. A method for empowering mesenchymal stem cells according to claim 3, characterized in that: In step S6, the environment in the centrifuge tube is as follows: the single cell suspension is centrifuged at 1200 rpm for 5 min, the supernatant is removed, and 10 ml of low-glucose DMEM / F12 medium is added.

5. A method for empowering mesenchymal stem cells according to claim 4, characterized in that: The method also includes step S10, repeating step S8 and step S9 for 3 times, culturing in a 5% CO2, 37OC incubator for 60 hours for the 4th time, adding a ZNB-enabling factor, culturing in a low-oxygen environment for 12 hours, and then terminating the digestion with the supernatant, collecting discrete suspended cells, centrifuging for 5 minutes at a centrifugal speed of 1200 r.min, suspending the cells with physiological saline, counting and quality testing, and releasing them for use after passing the test.

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