Method for separating human umbilical cord mesenchymal stem cells

Abstract

The invention provides a method for separating human umbilical cord mesenchymal stem cells. The umbilical cord mesenchymal stem cells are obtained by sequentially carrying out umbilical cord tissue treatment, umbilical cord sheet treatment and umbilical cord block culture and separation. The method disclosed by the invention is simple to operate, the separation efficiency of the umbilical cord tissue is improved, the purity of the obtained cells is higher and is obviously superior to the requirement of the ISCT lowest identification standard, the production cost can be effectively reduced, and industrial application is facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and more particularly, to a method for efficiently separating and culturing human umbilical cord mesenchymal stem cells. BACKGROUND

[0002] Mesenchymal stem cells (MSCs, also known as mesenchymal stromal cells) have unique immune regulation function and the characteristics of migrating to damaged tissues, and exhibit irreplaceable clinical application value in the fields of tissue repair, immunotherapy, and inflammation intervention. Compared with adult tissue sources such as bone marrow and fat, umbilical cord mesenchymal stem cells (UC-MSCs) are obtained from discarded umbilical cords after delivery, and the acquisition process does not cause damage to the mother and child, avoiding ethical controversy. At the same time, UC-MSCs have the industrialization advantages of lower immunogenicity, stronger proliferation activity, and stronger expansion capacity, and gradually become the ideal seed cells in the fields of cell therapy and regenerative medicine.

[0003] In recent years, the development of UC-MSCs-based cell therapy products has made significant progress. In January 2025, the National Medical Products Administration (NMPA) of China conditionally approved the first human umbilical cord mesenchymal stem cell injection "Amy Mesate Injection" (trade name: Rui Pisheng) for marketing, which is used for the treatment of acute graft-versus-host disease. This fully proves the clinical treatment value and industrialization prospect of UC-MSCs.

[0004] Efficient separation is a prerequisite for the clinical transformation of UC-MSCs. Existing separation technologies mainly develop around two core strategies of enzyme digestion method and tissue block adherence method. The enzyme digestion method uses biological enzymes such as collagenase and trypsin to degrade the extracellular matrix, and quickly releases UC-MSCs in the umbilical cord tissue. However, this method can damage cell viability and surface proteins, and small differences in enzyme digestion conditions can affect the purity of P0 generation MSCs cells (reference DOI: 10.1016 / j.jcyt.2020.06.002). The tissue block adherence method is a relatively direct method. The umbilical cord tissue is cut into 1-5mm³ small pieces and directly cultured, relying on the cell's own migration ability to crawl out of the tissue and adhere to the culture plate for proliferation. This method is simple and low in cost, and does not require complex enzyme reagents, and can maximize the preservation of cell interconnection structure, but has the defects of low cell harvest rate and long culture time, which is difficult to meet the needs of large-scale preparation. SUMMARY

[0005] In order to solve the problem of low separation efficiency of MSCs in the cell industry, the purpose of the present application is to establish a new method for efficiently separating MSCs.

[0006] To achieve the above object, the present application provides the following technical solutions.

[0007] In one aspect, the present application provides a method for isolating human umbilical cord mesenchymal stem cells, comprising the following steps:

[0008] (1) repeatedly washing the whole umbilical cord with DPBS or normal saline to remove blood clots, and cutting the umbilical cord into umbilical cord segments with a thickness of 3-5 cm;

[0009] (2) cutting the umbilical cord into umbilical cord pieces with a thickness of 0.5-1 cm, and repeatedly washing 3-5 times;

[0010] (3) cutting the umbilical cord pieces obtained in step (2) into uniform umbilical cord pieces with a thickness of 1-2 mm; 3

[0011] (4) evenly spreading the umbilical cord pieces cut in step (3) on the bottom of a T75 bottle, covering the bottle cap, and placing horizontally;

[0012] (5) placing the attached umbilical cord pieces in an incubator and incubating at 37°C for 1-16 h;

[0013] (6) after incubation, slowly adding the separation culture solution along the non-attached piece surface and continuing to culture in the incubator.

[0014] Preferably, after cutting the umbilical cord into umbilical cord segments in step (1), physical extrusion is performed, and each extrusion should compress the cross-sectional thickness of the umbilical cord segment to 40% to 60% of its initial thickness, and finally to the point where all the liquid is completely discharged.

[0015] The purpose of the physical extrusion in step (1) is to apply mechanical stimulation to the mesenchymal stem cells in the tissue to enhance their migration ability. To achieve this purpose and ensure the repeatability of the operation, each extrusion should compress the cross-sectional thickness of the umbilical cord segment to 40% to 60% of its initial thickness, and single or multiple extrusions can be used, with the point of complete discharge of the liquid in the umbilical cord segment as the end point. The initial thickness refers to the thickness of the umbilical cord segment in a natural relaxed state after washing and before extrusion. Sterile flat forceps or similar instruments can be used for extrusion, and by using instruments with scales, the thickness of the extruded tissue can be controlled within the above range.

[0016] Preferably, in step (3), the umbilical cord pieces are cut into umbilical cord pieces without removing any structural components, including arterial blood vessels and venous blood vessels.

[0017] Preferably, the incubation time in step (5) is 4-6 h.

[0018] ​Preferably, the separation culture solution in step (6) is DMF12 culture medium containing 10%-20% FBS, and 12 ml of the separation culture solution is added to each bottle.

[0019] Preferably, the separation culture solution in step (6) is DMF12 culture medium containing 20% FBS, and 12 ml of the separation culture solution is added to each bottle.

[0020] Preferably, the method further comprises: after the step (6) of placing in the incubator for continuous culture, semi-volume replacement is performed on the 5th to 7th day, and semi-volume or full-volume replacement is performed every 2-4 days thereafter; and the P0 generation of human umbilical cord mesenchymal stem cells is obtained by digestion when the cell fusion degree around the tissue is 80%-90%.

[0021] In another aspect, the present application provides the umbilical cord mesenchymal stem cells obtained by the above method.

[0022] In still another aspect, the present application provides the use of the umbilical cord mesenchymal stem cells obtained by the above method in the preparation of a medicament for treating immune and nervous system related diseases.

[0023] Preferably, the immune and nervous system related diseases are selected from Alzheimer's disease, atopic dermatitis, allergic rhinitis, rheumatoid arthritis, and graft-versus-host disease.

[0024] The present application can enhance the migration ability of MSCs, avoid the destruction of cell surface proteins, maintain cell viability, greatly improve the cell separation efficiency under the premise of ensuring the quality of cell separation, and obtain cells with high purity, a positive rate close to 100%, and a negative rate sum less than 0.2%, which is significantly better than the requirements of the ISCT minimum identification standard, is simple to operate, can effectively reduce production costs, solves the industrialization pain point problem, and can be used for industrial transformation of MSCs. The MSCs obtained by the present application show strong proliferation ability and biological efficacy, and can be used for the treatment of various indications. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Graph for average P0 generation MSCs harvested per bottle for comparison of different separation methods in Example 1

[0026] Figure 2 Graph for average P0 generation MSCs harvested per bottle for comparison of different separation solutions in Example 2

[0027] Figure 3 Graph for comparison of MSCs cell outgrowth for comparison of whether to remove umbilical cord blood vessels in Example 3

[0028] Figure 4Figure 5: Graph showing the effective outgrowth rate of day 5 umbilical cord tissue for Example 3 comparison of umbilical cord vessel removal

[0029] Figure 5 Figure 4: Graph showing the average harvest per flask of P0 MSCs for Example 4 comparison of different incubation times

[0030] Figure 6 Figure 5: Graph showing the effective outgrowth rate of day 5 umbilical cord tissue for Example 5 comparison of different incubation times

[0031] Figure 7 Figure 5: Graph showing the effective outgrowth rate of day 5 umbilical cord tissue for Example 5 comparison of different incubation times

[0032] Figure 8 Figure 5: Graph showing the P1 -unit cell HGF expression for Example 5 comparison of different incubation times

[0033] Figure 9 Figure 5: Graph showing the P1 -unit cell BDNF expression for Example 5 comparison of different incubation times

[0034] Figure 10 Figure 7: Graph showing the effect of MSC administration on mouse skin inflammation score in Example 7

[0035] Figure 11 Figure 7: Graph showing the effect of MSC administration on mouse serum inflammatory factor IL-4 in Example 7

[0036] Figure 12 Figure 7: Graph showing the effect of MSC administration on mouse serum inflammatory factor IgE in Example 7

[0037] Figure 13 Figure 7: Graph showing the effect of MSC administration on mouse sensitized site skin in Example 7

[0038] Figure 14 Figure 7: Graph showing the effect of MSC administration on mouse sensitized site skin mast cell number in Example 7 DETAILED DESCRIPTION

[0039] The present application will be further described with reference to the following specific examples and comparative examples, and the advantages and features of the present application will become apparent from the description. These examples and comparative examples are merely exemplary and do not limit the scope of the present application in any way.

[0040] Example 1 Comparative study of umbilical cord separation UC-MSCs method

[0041] Materials and Methods:

[0042] Reagents: DMEM / F12 Basic medium: Gibco; TrypLE™ enzyme: Gibco; FBS: Excell, DPBS: Tianjin Haoyang Biotechnology; 75% alcohol disinfectant: Tianjin Minsheng.

[0043] Instruments: AC2-4S8-CN biosafety cabinet: ESCO company; CCL-170B-8 carbon dioxide incubator: ESCO company; CKX53SF inverted phase contrast microscope: OLYMPUS company; Cellometer K2 automatic fluorescence analyzer: Nexcelom company; SorVall ST 16R table low-speed refrigerated centrifuge: Thermo company; 905GP ultra-low temperature refrigerator: Thermo company.

[0044] Separation method:

[0045] The whole umbilical cord was repeatedly washed with DPBS or normal saline to remove blood clots, and the umbilical cord was cut into 3-5 cm thick umbilical cord segments;

[0046] Method A: The umbilical cord segment was squeezed with a sterile hemostat, each time the cross-sectional thickness of the umbilical cord segment was compressed to 40% to 60% of its original thickness, and finally to all liquid was discharged, first cut the umbilical cord into 0.5-1 cm thick umbilical cord pieces, wash, then cut the umbilical cord pieces into 1-2 mm 3 uniform umbilical cord pieces;

[0047] Method B: The umbilical cord segment was torn open, flattened, and the arteries and veins were removed, washed, first cut the umbilical cord into 0.5-1 cm thick umbilical cord pieces, then cut the umbilical cord pieces into 1-2 mm 3 uniform umbilical cord pieces;

[0048] Next, the cut umbilical cord pieces were evenly spread on the bottom of the T75 bottle, and the culture bottles with umbilical cord pieces were placed in the incubator according to the group; After incubation, slowly add the separation culture solution (DMF12 medium containing 20% FBS) along the non-pasting block surface, add 12 ml of separation medium per bottle, and place it in the incubator for continuous culture. Then observe the state of the culture medium and perform half-volume replacement on the 5th-7th day, and perform half-volume or full-volume replacement every 2-4 days thereafter. On the 12th-14th day, carefully observe the cell crawling level, and when the cell fusion degree around the tissue is maximum and close to 80%-90%, digest to obtain P0 MSCs. Based on the cell separation rate of different groups, the number of P0 MSCs of the whole umbilical cord (length 15 cm) is calculated. Test results: see Table 1 and Figure 1 .

[0049] Table 1. Average number of P0 cells harvested per bottle from 3 batches of umbilical cord using different separation methods

[0051] Data shows that method A yielded significantly more P0 generation cells than method B, with method A yielding approximately 3.79 times more cells than method B.

[0052] Experimental summary: Separation method A can significantly improve the efficiency of primary cell separation.

[0053] Based on this separation efficiency, it is estimated that one umbilical cord can yield an average of 65.7 x 10^6 pieces using method A. 6 Cells can support over 2,500 clinical applications and can greatly solve the problem of cell industrialization.

[0054] Example 2: Comparative Study of Different Tissue Separation Fluids

[0055] Materials and Methods:

[0056] Reagents: DMEM / F12 Basic culture medium: Gibco; TrypLE™ enzyme: Gibco; FBS: Excell; DPBS: Tianjin Haoyang Biotechnology; 75% alcohol disinfectant: Tianjin Minsheng.

[0057] Instruments: AC2-4S8-CN biosafety cabinet: ESCO; CCL-170B-8 CO2 incubator: ESCO; CKX53SF inverted phase contrast microscope: OLYMPUS; Cellometer K2 fully automated fluorescence analyzer: Nexcelom; SorVall ST 16R benchtop low-speed refrigerated centrifuge: Thermo; 905GP ultra-low temperature freezer: Thermo.

[0058] Separation method:

[0059] Clean the entire umbilical cord repeatedly with DPBS or saline to remove blood clots. Cut the umbilical cord into 3-5cm thick segments. Use sterile hemostatic forceps to compress the umbilical cord segments, compressing the cross-sectional thickness of each segment to 40% to 60% of its initial thickness with each compression, until all fluid is drained. First, cut the umbilical cord into 0.5-1cm thick pieces, clean them, and then cut the pieces into 1-2mm thick strips. 3 Evenly spread the cut umbilical cord pieces evenly on the bottom of the T75 bottle, and place the culture bottles with the umbilical cord pieces attached into the incubator according to the group for incubation.

[0060] After incubation for the same amount of time, separation culture medium (Group A: 20% FBS-DMF12, Group B: 10% FBS-DMF12) was slowly added along the non-attached surface of the MSCs. 12 ml of separation culture medium was added to each MSC, and the MSCs were placed in an incubator for further culture. The culture medium status was observed on days 5-7, and half-volume medium was replaced. Thereafter, half-volume or full-volume medium replacements were performed every 2-4 days. On days 12-14, the cell migration level was carefully observed. When the confluence of cells around the tissue reached approximately 80%-90%, the MSCs were digested to obtain P0 generation MSCs. Grouping is shown in Table 2. Results: Results are shown in Table 3. Figure 2 .

[0061] Table 2. Grouping of the separated liquids

[0062] Group A Group B 20% FBS-DMF12 10% FBS-DMF12

[0063] Table 3. Average number of P0 cells harvested per bottle from two batches of different separation solutions for umbilical cord cord

[0064]

[0065] Data shows that the number of P0 generation cells harvested with 20% FBS-DMF12 is significantly higher than that harvested with 10% FBS-DMF12.

[0066] Experimental summary: Method A has higher cell separation efficiency.

[0067] Example 3: Comparative Study of Umbilical Cord Vessel Removal

[0068] Materials and Methods:

[0069] Reagents: DMEM / F12 Basic culture medium: Gibco; TrypLE™ enzyme: Gibco; FBS: Excell; DPBS: Tianjin Haoyang Biotechnology; 75% alcohol disinfectant: Tianjin Minsheng.

[0070] Instruments: AC2-4S8-CN biosafety cabinet: ESCO; CCL-170B-8 CO2 incubator: ESCO; CKX53SF inverted phase contrast microscope: OLYMPUS; Cellometer K2 fully automated fluorescence analyzer: Nexcelom; SorVall ST 16R benchtop low-speed refrigerated centrifuge: Thermo; 905GP ultra-low temperature freezer: Thermo.

[0071] Separation method:

[0072] Use DPBS or saline to repeatedly clean the entire umbilical cord, remove blood clots, cut the umbilical cord into 3-5cm thick segments, and squeeze the umbilical cord segments with sterile hemostatic forceps. Each squeeze should compress the cross-sectional thickness of the umbilical cord segment to 40% to 60% of its initial thickness, until all the fluid is drained.

[0073] Method A: Cut the umbilical cord segment into pieces 0.5-1cm thick, then cut the pieces into 1-2mm thick strips. 3 Uniform umbilical cord patches;

[0074] Method B: Tear the umbilical cord segment open, flatten it, remove the artery and vein, and cut the umbilical cord piece into 1-2mm pieces. 3 Uniform umbilical cord patches;

[0075] Next, the cut umbilical cord pieces were evenly spread across the bottom of the T75 flask, and the culture flasks with the umbilical cord pieces attached were placed in the incubator according to their groups for incubation. After incubation, separation culture medium (DMF12 medium containing 20% ​​FBS) was slowly added along the non-attached side, with 12 ml of separation culture medium added to each flask, and the flasks were placed in the incubator for continued incubation. The number of umbilical cord pieces with cells crawling out was observed and compared on day 5.

[0076] Experimental results: See [link to results] Figure 3 , Figure 4 .

[0077] The results showed that Method A had significantly more umbilical cord masses with cells emerging on day 5 than Method B.

[0078] Experimental summary: Method A has higher cell separation efficiency.

[0079] Example 4: Comparative Study of Different Incubation Times in Separation 1

[0080] Materials and Methods:

[0081] Reagents: DMEM / F12 Basic culture medium: Gibco; TrypLE™ enzyme: Gibco; FBS: Excell; DPBS: Tianjin Haoyang Biotechnology; 75% alcohol disinfectant: Tianjin Minsheng.

[0082] Instruments: AC2-4S8-CN biosafety cabinet: ESCO; CCL-170B-8 CO2 incubator: ESCO; CKX53SF inverted phase contrast microscope: OLYMPUS; Cellometer K2 fully automated fluorescence analyzer: Nexcelom; SorVall ST 16R benchtop low-speed refrigerated centrifuge: Thermo; 905GP ultra-low temperature freezer: Thermo.

[0083] Separation method:

[0084] Clean the entire umbilical cord repeatedly with DPBS or saline to remove blood clots. Cut the umbilical cord into 3-5cm thick segments. Use sterile hemostatic forceps to compress the umbilical cord segments, compressing each segment to 40% to 60% of its initial thickness with each compression, until all fluid is drained. Cut the umbilical cord segments into 0.5-1cm thick pieces, clean them, and then cut the pieces into 1-2mm pieces. 3 Evenly spread the cut umbilical cord pieces evenly on the bottom of the T75 bottle, and place the culture bottles with the umbilical cord pieces attached into the incubator according to the group for incubation. First, examine the effect of overnight incubation on umbilical cord separation.

[0085] After incubation according to the protocol, different groups were slowly added to the non-attached surface of the umbilical cord fragments using separation culture medium (DMF12 medium containing 20% ​​FBS), with 12 ml of separation culture medium added to each flask. The flasks were then placed in an incubator for further culture. On day 5 of primary culture, the culture flasks were photographed, and the effective attachment rate of the umbilical cord fragments and the proportion of cells that migrated from the umbilical cord fragments in each group were recorded. Half of the medium was then replaced.

[0086] On day 7, observe the culture medium status and perform a half-volume medium change. Thereafter, perform a half-volume or full-volume medium change every 2-4 days. On days 12-14, carefully observe the cell migration level. When the cell confluence around the tissue reaches its maximum of 80%-90%, digest the cells to obtain P0 generation MSCs.

[0087] Grouping is shown in Table 4. Experimental results: Results are shown in Table 5 and... Figure 5 .

[0088] Table 4. Incubation Time Grouping

[0089] Group A Group B Incubation X h (1-4 h) Incubation (X+12) h UC008 - Incubation 2 h UC008 - Incubation 14 h UC010 - Incubation 4 h UC010 - Incubation 16 h UC011 - Incubation 1 h UC011 - Incubation 13 h

[0090] Table 5. Average number of P0 cells harvested per bottle from three batches of umbilical cord cells at different incubation times

[0091]

[0092] The results showed that the number of P0 generation cells obtained by method A was significantly higher than that obtained by method B.

[0093] Experimental Summary: Method A has a higher cell separation efficiency. The number of P0 generation cells obtained from the same batch of umbilical cord cells separated without overnight incubation was more than twice that obtained from those separated with overnight incubation. This suggests that overnight incubation should not be performed, and the incubation time should not be too long, otherwise it will affect the separation efficiency.

[0094] Example 5: Comparative Study of Different Incubation Times in Separation 2

[0095] Materials and Methods:

[0096] Reagents: DMEM / F12 Basic culture medium: Gibco; TrypLE™ enzyme: Gibco; FBS: Excell; DPBS: Tianjin Haoyang Biotechnology; 75% alcohol disinfectant: Tianjin Minsheng.

[0097] Instruments: AC2-4S8-CN biosafety cabinet: ESCO; CCL-170B-8 CO2 incubator: ESCO; CKX53SF inverted phase contrast microscope: OLYMPUS; Cellometer K2 fully automated fluorescence analyzer: Nexcelom; SorVall ST 16R benchtop low-speed refrigerated centrifuge: Thermo; 905GP ultra-low temperature freezer: Thermo.

[0098] Separation method:

[0099] Clean the entire umbilical cord repeatedly with DPBS or saline to remove blood clots. Cut the umbilical cord into 3-5cm thick segments. Use sterile hemostatic forceps to compress the umbilical cord segments, compressing each segment to 40% to 60% of its initial thickness with each compression, until all fluid is drained. Cut the umbilical cord segments into 0.5-1cm thick pieces, clean them, and then cut the pieces into 1-2mm pieces. 3 Uniform umbilical cord pieces were prepared; the cut umbilical cord pieces were evenly spread on the bottom of the T75 bottle, and the culture bottles with the umbilical cord pieces were placed in the incubator according to the group to further investigate the effect of different incubation on umbilical cord separation.

[0100] After incubation according to the protocol, separation culture medium (DMF12 medium containing 20% ​​FBS) was slowly added along the non-attached surface of each group, with 12 ml of separation culture medium added to each bottle. The bottles were then placed in an incubator for further culture. On day 5 of primary culture, photographs were taken of the culture bottles to record the effective attachment rate of umbilical cord fragments and the proportion of cells that migrated from the umbilical cord fragments in each group. Half of the medium was replaced. Grouping is shown in Table 6.

[0101] Table 6. Grouping of incubation time

[0102] Group A Group B Group C Group D Incubation 1 h Incubation 2 h Incubation 4 h Incubation 6 h

[0103] On day 7, observe the condition of the culture medium and perform a half-volume medium change. Thereafter, perform a half-volume or full-volume medium change every 2-4 days.

[0104] On day 12, P0 generation cells from different groups were harvested. An appropriate amount of cells from each group were seeded and cultured at the same density to harvest P1 generation MSCs. The culture supernatant from the P1 generation cells before digestion was collected from each group, and the secretion levels of effector molecules HGF and BDNF were measured. Surface markers were then detected in the P1 generation cells.

[0105] Experimental results: See [link to results]Figure 6 , Figure 7 , Figure 8 , Figure 9 Table 10.

[0106] The results showed that the effective cell adhesion rate of groups C and D on day 5 was significantly higher than that of groups A and B. The proportion of cells that migrated out of the viviparous cell line in group C on day 5 was higher than that in other groups. However, the number of P1 passage cells harvested from each group was 1*10-1. 6 There was no significant difference in the amount of HGF and BDNF secreted by cells after 3 days, and there were no significant differences in the surface marker detection results of P1 generation cells from different groups. Furthermore, the surface marker detection results of P1 generation MSCs showed that the positive rate of MSCs obtained by this method was close to 100%, the total negative rate was less than 0.2%, and the purity was significantly higher than the minimum identification standard requirements of ISCT.

[0107] Study Summary: Considering the effective bottle adhesion rate, effective crawling rate, factor expression, and surface marker results, the optimal incubation method is 4-6 hours.

[0108] Table 10. Results of surface marker detection in P1 generation cells compared to different incubation times.

[0109]

[0110] Example 6: Representative Umbilical Cord Isolation Study (3 Batches)

[0111] Materials and Methods:

[0112] Reagents: DMEM / F12 Basic culture medium: Gibco; TrypLE™ enzyme: Gibco; FBS: Excell; DPBS: Tianjin Haoyang Biotechnology; 75% alcohol disinfectant: Tianjin Minsheng.

[0113] Instruments: AC2-4S8-CN biosafety cabinet: ESCO; CCL-170B-8 CO2 incubator: ESCO; CKX53SF inverted phase contrast microscope: OLYMPUS; Cellometer K2 fully automated fluorescence analyzer: Nexcelom; SorVall ST 16R benchtop low-speed refrigerated centrifuge: Thermo; 905GP ultra-low temperature freezer: Thermo.

[0114] Separation method:

[0115] Clean the entire umbilical cord repeatedly with DPBS or saline to remove blood clots. Cut the umbilical cord into 3-5cm thick segments. Use sterile hemostatic forceps to compress the umbilical cord segments, compressing each segment to 40% to 60% of its initial thickness with each compression, until all fluid is drained. Cut the umbilical cord segments into 0.5-1cm thick pieces, clean them, and then cut the pieces into 1-2mm pieces. 3 Obtain uniform small pieces of umbilical cord; evenly spread the cut umbilical cord pieces on the bottom of a T75 culture bottle, and place the culture bottles with the umbilical cord pieces attached into the incubator according to the group and incubate for 4-5 hours; after incubation, slowly add separation culture medium (DMF12 medium containing 20% ​​FBS) along the non-attached side, adding 12 ml of separation culture medium to each bottle, and place in the incubator to continue culturing. Then, observe the culture medium status on days 5-7 and perform a half-volume medium change, followed by a half-volume or full-volume medium change every 2-4 days thereafter. On days 12-14, carefully observe the cell migration level; when the cell confluence around the tissue reaches its maximum of approximately 80%-90%, digest and process to obtain P0 generation MSCs, and then grow them at 12000 cells / cm². 2 MSCs were cultured at high density, and the P1 generation of MSCs seed bank cells were harvested and their surface markers were detected.

[0116] The umbilical cord separation parameters and separation results are shown in Tables 7 and 8.

[0117] Table 7. Representative umbilical cord separation results from three batches

[0118]

[0119] Note: Clinical conversion counts are calculated as 1*10. 8 Calculation of injection dosage per cell / person.

[0120] Table 8. Detection results of surface markers in three representative batches of seed banks

[0121]

[0122] Results Summary: Three independent batches of umbilical cord isolation were carried out using the optimal method. The number of P0 cells harvested from each bottle met expectations. It is estimated that the clinical translational applications supported by these batches will exceed 2,500. The surface marker detection results were significantly better than the minimum identification standards required by ISCT.

[0123] Example 7: Pharmacological study of MSCs in the treatment of atopic dermatitis

[0124] Materials and Methods:

[0125] Reagents: DMEM / F12 Basic medium: Gibco; TrypLE™ enzyme: Gibco; FBS: Excell; DPBS: Tianjin Haoyang Biotechnology; Human serum albumin: Sichuan Yuanda Shuyang Pharmaceutical Co., Ltd. (Shuyang); Compound electrolyte injection: Shijiazhuang No.4 Pharmaceutical Co., Ltd.; DNFB (2,4-dinitrofluorobenzene): Shanghai Aladdin Biochemical Technology Co., Ltd.; Acetone: Tianjin Fengchuan Chemical Reagent Co., Ltd.; Olive oil: Olivoila.

[0126] Instruments: AC2-4S8-CN biosafety cabinet: ESCO; CCL-170B-8 CO2 incubator: ESCO; CKX53SF inverted phase contrast microscope: OLYMPUS; SorVall ST 16R benchtop low-speed refrigerated centrifuge: Thermo; ECA522 electronic balance: Nanjing Bernita Scientific Instruments Co., Ltd.; ECC2201 electronic balance: Nanjing Bernita Scientific Instruments Co., Ltd.; RT-6100 microplate reader: Redo Life Sciences Co., Ltd.

[0127] Atopic dermatitis efficacy study protocol: 30 7-8 week old BALB / c mice were divided into a normal control group, a model control group, and an MSC-treated group, with 10 mice in each group (see Table 9). After animal acclimatization observation, except for the normal control group, the other groups of mice underwent DNFB induction to establish a mouse atopic dermatitis model. On day 1, 150 μL of 0.5% DNFB solution was applied topically to the shaved area on the mouse's back for initial sensitization, for two consecutive days, with a four-day interval. Subsequently, on days 7, 9, 11, and 13, DNFB solution was applied to the shaved area on the mouse's back to challenge the model, for a total of 13 days. Successful modeling was indicated by the appearance of erythema, partial erosion, scabbing on the skin of the mouse's back, accompanied by increased scratching. The normal control group received an equal volume of solvent at the same frequency as the model group.

[0128] The subcutaneous administration dose of mesenchymal stem cells was set at 3 × 10⁻⁶. 5 Stem cells were administered subcutaneously around the wound on the back of each mouse (0.1 mL / mouse). The normal control group received an equal volume of solvent. The mice were administered the solution on days 7, 9, and 13 of modeling. On days 2, 4, 6, 8, 10, 12, 14, and 16 of modeling, back skin inflammation scores were assessed, and general observations were made, including behavioral activity, mental status, physical appearance, respiration, and secretions. On day 16, the mice were euthanized for further mechanistic studies, including detection of serum inflammatory factors, pathological examination of the back skin, and detection of mast cell infiltration in the back skin.

[0129] Table 9. Animal grouping for efficacy studies

[0130]

[0131] Results of a pharmacodynamic study on atopic dermatitis:

[0132] Quantitative data are expressed as mean ± standard error (Mean ± SEM) and statistically analyzed using SPSS 25.0 software. For data with homogeneous variances, one-way ANOVA was performed, and pairwise comparisons were conducted using the LSD test when significant differences were found. For data with unequal variances, the Kruskal-Wallis nonparametric test was performed, followed by pairwise comparisons. For categorical data, the Kruskal-Wallis nonparametric test was performed using SPSS 25.0 software, and pairwise comparisons were conducted. The significance level was set at α = 0.05, and p ≤ 0.05 was considered statistically significant.

[0133] The effect of human umbilical cord mesenchymal stem cell injection on skin inflammation scores in atopic dermatitis mice showed that the skin on the backs of mice in the normal control group was normal and smooth, with a skin inflammation score of 0. Compared with the normal control group, the model control group mice showed varying degrees of redness, swelling, skin thickening, crusting, and dryness with increasing modeling cycles, and the skin inflammation score was significantly increased from day 4 to day 16 of modeling. Compared with the model control group, the skin inflammation score of mice in the MSC subcutaneous administration group was significantly reduced from day 12 to day 16 of modeling. This suggests that subcutaneous administration of stem cell injection can improve skin inflammation in mice (see results below). Figure 10 ELISA results in mouse serum showed that, compared with the normal control group, the serum IL-4 and IgE levels in the model group mice were significantly increased; compared with the model control group, the IL-4 and IgE levels in the MSC subcutaneous administration group mice were significantly decreased (see results). Figure 11 , 12 HE results showed that the epidermis, dermis, and subcutaneous tissue of the back skin of the normal control group animals were intact, with clear hair follicles and sweat glands, and no obvious pathological changes in the stroma. Compared with the normal control group, the model control group animals showed epidermal necrosis; the epidermis in the non-necrotic areas was thickened, with extended epithelial pegs and visible keratotic cysts; crusts, hyperkeratosis, and / or parakeratosis were visible on the epidermis; the dermis showed degeneration / necrosis, inflammatory cell infiltration, fibrous tissue hyperplasia, and a reduction or disappearance of appendages; occasionally, inflammatory cell infiltration and edema were observed in the subcutaneous tissue. Compared with the model control group, the MSC subcutaneous administration group showed varying degrees of relief (see results). Figure 13 Toluidine blue staining results showed that, compared with the normal control group, the number of mast cells in the dorsal skin of the model control group was significantly increased; compared with the model control group, the number of mast cells in the sensitized areas of mice in the MSC subcutaneous drug administration group was significantly decreased (see results). Figure 14 The above results indicate that MSCs have a good preventive and therapeutic effect on DNFB-induced atopic dermatitis.

Claims

1. A method for isolating human umbilical cord mesenchymal stem cells, characterized in that, Includes the following steps: (1) Use DPBS or saline to repeatedly clean the entire umbilical cord, remove blood clots, and cut the umbilical cord into 3-5cm thick segments; (2) Cut the umbilical cord into pieces 0.5-1cm thick and clean it repeatedly 3-5 times; (3) Cut the umbilical cord piece obtained in step (2) into 1-2mm pieces. 3 Uniform umbilical cord patches; (4) Spread the small pieces of umbilical cord cut in step (3) evenly over the bottom of the T75 bottle, put on the bottle cap, and place it horizontally; (5) Place the attached umbilical cord patch in an incubator and incubate at 37°C for 1-16 hours; (6) After incubation, slowly add the separation culture medium along the non-attached surface and place it in an incubator for continued incubation.

2. The method according to claim 1, characterized in that: In step (1), after cutting the umbilical cord into segments, physical compression is performed. Each compression should compress the cross-sectional thickness of the umbilical cord segment to 40% to 60% of its initial thickness, and finally, the compression should continue until all the liquid is discharged.

3. The method according to claim 1, characterized in that: Before the umbilical cord piece is cut into small pieces in step (3), no structural components are removed, including arteries and veins.

4. The method according to claim 1, characterized in that: The incubation time in step (5) is 4-6 hours.

5. The method according to claim 1, characterized in that: The separation culture medium mentioned in step (6) is DMF12 medium containing 10%-20% FBS, with 12ml of separation culture medium added to each bottle.

6. The method according to claim 1, characterized in that: The separation culture medium mentioned in step (6) is DMF12 medium containing 20% ​​FBS, with 12ml of separation culture medium added to each bottle.

7. The method according to claim 1, characterized in that: After placing the culture in the incubator in step (6), half of the medium should be changed on the 5th to 7th day, and then half or full medium should be changed every 2 to 4 days thereafter; when the cell fusion around the tissue is 80%-90%, digestion treatment is performed to obtain P0 generation human umbilical cord mesenchymal stem cells.

8. Umbilical cord mesenchymal stem cells obtained by the method according to any one of claims 1-7.

9. Use of umbilical cord mesenchymal stem cells obtained by the method of any one of claims 1-7 in the preparation of medicaments for treating immune and nervous system-related diseases.

10. The use according to claim 9, characterized in that, The immune and nervous system-related diseases are selected from Alzheimer's disease, atopic dermatitis, allergic rhinitis, rheumatoid arthritis, and graft-versus-host disease.

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