Culture medium of umbilical cord mesenchymal stem cells and isolated culture method of umbilical cord mesenchymal stem cells

Abstract

The invention discloses a culture medium of umbilical cord mesenchymal stem cells and an isolated culture method of the umbilical cord mesenchymal stem cells, and belongs to the technical field of biological medicines.The culture medium is high in proliferation speed, large in quantity and stable in phenotype due to mutual cooperation of all components, and on the premise that animal origin, antibiotics and serum are not added in the whole preparation process, the yield of the umbilical cord mesenchymal stem cells is increased. The umbilical cord mesenchymal stem cells with high purity, high motility rate and high yield are separated from Wharton's jelly. By improving the purity and motility rate of the umbilical cord mesenchymal stem cells, the effectiveness and timeliness of clinical treatment of diseases by the umbilical cord mesenchymal stem cells are improved. Meanwhile, the cost of clinical use of stem cell treatment is reduced by increasing the yield of the umbilical cord mesenchymal stem cells. The umbilical cord mesenchymal stem cells obtained through separation culture have good multiplication capacity and multidirectional differentiation potential, and the umbilical cord mesenchymal stem cells obtained through separation culture can be rapidly induced and differentiated into osteoblasts, adipoblasts and chondroblasts in an induced differentiation culture medium.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a culture medium for umbilical cord mesenchymal stem cells and a method for separating and culturing umbilical cord mesenchymal stem cells. Background Art

[0002] Umbilical cord mesenchymal stem cells (UC-MSCs) are stem cells derived from the mesenchymal tissue of the umbilical cord of newborns, with functions such as multidirectional differentiation, self-renewal and immunoregulation. At the same time, they are non-tumorigenic, highly safe, and rich in sources and easy to obtain. The collection process avoids secondary trauma to the human body and ethical and legal issues. They also have low immunogenicity and do not produce rejection reactions when used allogeneically. They are a suitable source of materials for clinical research and application, and are widely used in stem cell therapy for tissue repair and regeneration, with broad prospects for clinical application.

[0003] Stem cell transplantation therapy is to transplant healthy stem cells into the patient's body to repair or replace damaged cells or tissues, thereby achieving the purpose of healing. Stem cells have the potential for self-renewal and multidirectional differentiation, and can treat nervous system diseases, immune system diseases, metabolic diseases and some aging-related damage diseases.

[0004] At present, 21 stem cell therapy products have been approved for marketing worldwide, of which 10 are mesenchymal stem cell products. By the end of 2023, China has 129 stem cell clinical studies that have been filed, of which 74 are mesenchymal stem cell products; 68 stem cell preparations have obtained IND implied approval, of which 43 are mesenchymal stem cell products.

[0005] Stem cells are considered to be an ideal cell source for replacement therapy. However, the earliest developed embryonic stem cells are subject to ethical and legal restrictions, and there are risks such as tumorigenicity and unexpected differentiation, making it difficult to develop into cell preparations. Induced pluripotent stem cells can simulate the pluripotency of embryonic stem cells and are not ethically controversial, but there are still risks such as tumorigenicity and unexpected differentiation. Adult mesenchymal stem cells are not ethically controversial and can be autologously transplanted, making them a very attractive and promising option. The earliest mesenchymal stem cells used were bone marrow mesenchymal stem cells, but due to the difficulty in obtaining bone marrow tissue, researchers began to look for alternative sources in other tissues and found mesenchymal stem cells in tissues such as umbilical cord, fat, and peripheral blood. At present, umbilical cord mesenchymal stem cells are widely used in cell therapy to treat diseases because they are easy to obtain, safe and reliable to use, easy to obtain, sufficient in number, and have strong proliferation ability.

[0006] The human umbilical cord is formed in the fifteenth week of the embryonic period. By the normal delivery stage, the average length of the umbilical cord of a full-term baby is 50cm to 60cm, the weight varies from 30g to 60g, and the diameter varies from 0.6cm to 2cm. Umbilical cord mesenchymal stem cells can be isolated and obtained from different parts of the umbilical cord, including umbilical cord blood, umbilical vein subendothelial layer, umbilical cord epithelial layer, and Wharton's jelly layer. Among them, Wharton's jelly refers to the gelatinous tissue that surrounds the umbilical artery and umbilical vein and is rich in water. It is rich in a large number of high-quality umbilical cord mesenchymal stem cells.

[0007] However, under the current technical background, in the process of culturing umbilical cord mesenchymal stem cells, antibiotics are added to ensure that they are not contaminated, and non-clinical grade culture reagents containing human or animal serum, allogeneic human serum or plasma components are added to improve the proliferation efficiency. As a result, the yield, viability and purity of the harvested umbilical cord mesenchymal stem cells are low, and do not meet the quality standard requirements for clinical-grade umbilical cord mesenchymal stem cell injection, which may have a negative impact on the user's body. Summary of the invention

[0008] The purpose of the present invention is to provide a culture medium for umbilical cord mesenchymal stem cells and a method for separating and culturing umbilical cord mesenchymal stem cells in view of the existing problems.

[0009] The present invention is achieved through the following technical solutions:

[0010] A method for isolating and culturing umbilical cord mesenchymal stem cells comprises the following steps:

[0011] (1) Cleaning of umbilical cord tissue:

[0012] Place the umbilical cord in a biosafety cabinet, remove the umbilical cord with sterile hook-tooth forceps, place it in a 10 cm petri dish containing saline solution prepared in advance, wash it 2-3 times, clean the blood stains on the surface of the tissue, cut the umbilical cord into small pieces with surgical scissors, and place it in a 10 cm petri dish containing saline solution and continue washing it 2-3 times;

[0013] (2) Umbilical cord tissue stripping:

[0014] Use sterile scissors to cut the umbilical cord parallel to the vein, then use hook-toothed forceps to tear off the venous membrane along the cut side, then spread the umbilical cord flat and find the two arteries;

[0015] Use hook-toothed forceps to remove the two arteries in the umbilical cord. The remaining part is Wharton's jelly and the amniotic membrane on the surface of the umbilical cord. Use hook-toothed forceps to gently peel off the Wharton's jelly, put it in a 25ml glass beaker, and cut it into 1cm pieces with scissors. 2 Small tissue masses;

[0016] (3) Primary cell inoculation:

[0017] Suspend the tissue block with the prepared culture medium and inoculate it in a 10 cm culture dish, about 3 to 4 ml per dish;

[0018] Shake it evenly to make it spread evenly on the ground, and put it into the incubator for cultivation, which is recorded as P0;

[0019] (4) Primary cell collection:

[0020] On the 10th to 12th day of cell inoculation, observe the growth of MSCs in the dish, and perform P0-P1 subculture operations according to the growth of cell colonies;

[0021] The upper culture medium was discarded, and the cells were washed twice with physiological saline, digested with Accutase enzyme solution, and the cell suspension was collected into a 50 ml centrifuge tube, centrifuged at 200g for 5 min, and the cells were collected and washed once with physiological saline at 200g for 5 min;

[0022] Add pre-warmed culture medium to the 50 ml centrifuge tube containing cells, resuspend the cells, and calculate the cell number according to 1.0*10 6 The cells were inoculated in a 15 cm culture dish, and each dish was filled with pre-warmed culture medium to 20 ml, and placed in a carbon dioxide incubator for culture, which was recorded as P1;

[0023] (5) Cell passaging:

[0024] Observe the growth of MSCs every day, and carry out subculture expansion when the cell density is ≥85%. Perform P1 to P2 subculture expansion. According to the cell count, the number of cells is 1.0*10 6 cells were seeded in 15 cm culture dishes at a density of 10;

[0025] (6) Cell cryopreservation:

[0026] Observe the growth status of the cells to be collected. When the cell density reaches more than 85%, add physiological saline to the culture dish, gently shake the culture bottle, and discard the physiological saline;

[0027] Add Accutase to the culture flask or dish, place at 37°C, and observe the cell digestion under a microscope;

[0028] When most of the cells are detached from the wall, collect the cell suspension into a 50ml centrifuge tube and centrifuge at 200g for 5min to collect the cells. After centrifugation, discard the supernatant, resuspend the cells with 50ml of normal saline, take 50ul of sample for counting, and wash once at 200g for 5min.

[0029] According to the number of cells, add an appropriate amount of pre-warmed freezing solution to adjust the cell suspension density to 2×10 6 / ml, 1ml per tube, and marked P2. The cells were placed in a -80℃ refrigerator overnight and transferred to -196℃ liquid nitrogen for long-term storage the next day.

[0030] (7) Cell counting:

[0031] The P1 cell collection count can reach 1.5-2.5*10 8 The cell density was 2×10 6 / ml for freezing, 70 to 100 tubes can be frozen;

[0032] The P2 generation cell collection count can reach 7.0~8.0*10 8 The cell density was 2×10 6 / ml for freezing, 300 to 400 tubes can be frozen;

[0033] (8) Cell detection:

[0034] Cell surface marker detection and stem cell tri-lineage differentiation function detection were performed before cell freezing.

[0035] Sterility testing, activity testing, purity testing.

[0036] The culture medium comprises the following components: basic culture medium, vitamin C, vitamin E, Centella asiatica extract, protein fiber, L-glycine, L-arginine, tyrosine, lanthanum nitrate and cerium nitrate.

[0037] Furthermore, the umbilical cord is cut into small segments of 3 to 5 cm using surgical scissors as described in step (1).

[0038] Furthermore, during the subculturing operation described in step (4), the cell density is between 85% and 90%.

[0039] Furthermore, the basic culture medium is MSCBM culture medium.

[0040] Furthermore, the final concentrations of the vitamin E and vitamin C are the same, both of which are 8.2-10.4 μg / L.

[0041] Furthermore, the final concentration of the Centella asiatica extract is 3.6-5.2 μg / L.

[0042] Furthermore, the final concentration of the protein fiber is 10.3-11.1 μg / L.

[0043] Furthermore, the final concentrations of L-glycine, L-arginine and tyrosine are 20.3-21.3 ng / L, 12.5-13.7 ng / L and 8.2-8.8 ng / L, respectively.

[0044] Furthermore, the final concentrations of the lanthanum nitrate and cerium nitrate are 6.3-8.3 μg / L and 5.1-7.1 μg / L, respectively.

[0045] Compared with the prior art, the present invention has the following advantages:

[0046] 1. The present invention provides a culture medium for umbilical cord mesenchymal stem cells. Vitamin C, vitamin E, Centella asiatica extract, protein fiber, L-glycine, L-arginine, tyrosine, lanthanum nitrate, and cerium nitrate in the culture medium are used in combination to improve the adhesion of cells and the stability of cell growth. Rare earth elements are combined with nutrients such as amino acids to shorten the delay phase and logarithmic phase of cell growth, intensify the logarithmic phase growth of cells, eliminate the stable phase, increase the cell growth rate and proliferation amount, promote cell growth, and improve cell activity.

[0047] 2. The present invention provides a method for isolating and culturing umbilical cord mesenchymal stem cells and their application, which aims to isolate and obtain high-purity, high-viability, and high-yield umbilical cord mesenchymal stem cells from Wharton's jelly without adding animal-derived substances, antibiotics, and serum throughout the preparation process. By improving the purity and viability of umbilical cord mesenchymal stem cells, the effectiveness and timeliness of clinical treatment of diseases with umbilical cord mesenchymal stem cells are improved. At the same time, by increasing the yield of umbilical cord mesenchymal stem cells, the cost of clinical use of stem cell therapy is reduced. The umbilical cord mesenchymal stem cells obtained by separation and culture have good proliferation ability and multidirectional differentiation potential. The umbilical cord mesenchymal stem cells obtained by the above separation and culture can be rapidly induced to differentiate into osteoblasts, adipocytes, and chondrocytes under the induction differentiation medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the flow cytometry image of HUC-MSCs (CD34);

[0049] Figure 2 This is the flow cytometry image of HUC-MSCs (CD44);

[0050] Figure 3 This is the flow cytometry image of HUC-MSCs (CD45);

[0051] Figure 4 This is the flow cytometry image of HUC-MSCs (CD73);

[0052] Figure 5 This is the flow cytometry image of HUC-MSCs (CD90);

[0053] Figure 6 This is the flow cytometry image of HUC-MSCs (CD105);

[0054] Figure 7This is the flow cytometry image of HUC-MSCs (HLA-DR);

[0055] Figure 8 This is the staining diagram for adipogenic differentiation;

[0056] Fig. 9 Staining diagram for osteogenic differentiation;

[0057] Fig.10 This is a staining image for chondrogenic differentiation. DETAILED DESCRIPTION

[0058] In order to further explain the present invention, it is described below in conjunction with the following specific embodiments.

[0059] Example 1

[0060] A method for isolating and culturing umbilical cord mesenchymal stem cells comprises the following steps:

[0061] (1) Cleaning of umbilical cord tissue:

[0062] Place the umbilical cord in a biosafety cabinet, remove it with sterile hook-tooth forceps, place it in a 10 cm petri dish containing saline solution prepared in advance, wash it 2-3 times, clean the blood stains on the surface of the tissue, cut the umbilical cord into 3-5 cm small pieces with surgical scissors, and place it in a 10 cm petri dish containing saline solution and continue washing it 2-3 times;

[0063] (2) Umbilical cord tissue stripping:

[0064] Use sterile scissors to cut the umbilical cord parallel to the vein, then use hook-toothed forceps to tear off the venous membrane along the cut side, then spread the umbilical cord flat and find the two arteries;

[0065] Use hook-toothed forceps to remove the two arteries in the umbilical cord. The remaining part is Wharton's jelly and the amniotic membrane on the surface of the umbilical cord. Use hook-toothed forceps to gently peel off the Wharton's jelly, put it in a 25ml glass beaker, and cut it into 1cm pieces with scissors. 2 Small tissue masses;

[0066] (3) Primary cell inoculation:

[0067] Suspend the tissue block with the prepared culture medium and inoculate it in a 10 cm culture dish, about 3 to 4 ml per dish;

[0068] Shake it evenly to make it spread evenly on the ground, and put it into the incubator for cultivation, which is recorded as P0;

[0069] (4) Primary cell collection:

[0070] On the 10th to 12th day after cell inoculation, observe the growth of MSCs in the dish, and perform P0-P1 subculture operations (density at 85-90%) according to the growth of cell colonies;

[0071] The upper culture medium was discarded, and the cells were washed twice with physiological saline, digested with Accutase enzyme solution, and the cell suspension was collected into a 50 ml centrifuge tube, centrifuged at 200g for 5 min, and the cells were collected and washed once with physiological saline at 200g for 5 min;

[0072] Add pre-warmed culture medium to the 50 ml centrifuge tube containing cells, resuspend the cells, and calculate the cell number according to 1.0*10 6 The cells were inoculated in a 15 cm culture dish, and each dish was filled with pre-warmed culture medium to 20 ml, and placed in a carbon dioxide incubator for culture, which was recorded as P1;

[0073] (5) Cell passaging:

[0074] Observe the growth of MSCs every day, and carry out subculture expansion when the cell density is ≥85%. Perform P1 to P2 subculture expansion. According to the cell count, the number of cells is 1.0*10 6 cells were seeded in 15 cm culture dishes at a density of 10;

[0075] (6) Cell cryopreservation:

[0076] Observe the growth status of the cells to be collected. When the cell density reaches more than 80%, add physiological saline to the culture dish, gently shake the culture bottle, and discard the physiological saline;

[0077] Add Accutase to the culture flask or dish, place at 37°C, and observe the cell digestion under a microscope;

[0078] When most of the cells are detached from the wall, collect the cell suspension into a 50ml centrifuge tube and centrifuge at 200g for 5min to collect the cells. After centrifugation, discard the supernatant, resuspend the cells with 50ml of normal saline, take 50ul of sample for counting, and wash once at 200g for 5min.

[0079] According to the number of cells, add an appropriate amount of pre-warmed freezing solution to adjust the cell suspension density to 2×10 6 / ml, 1ml per tube, and marked P2. The cells were placed in a -80℃ refrigerator overnight and transferred to -196℃ liquid nitrogen for long-term storage the next day.

[0080] (7) Cell counting:

[0081] The P1 cell collection count can reach 1.5-2.5*10 8 The cell density was 2×10 6 / ml for freezing, 70 to 100 tubes can be frozen;

[0082] The P2 generation cell collection count can reach 7.0~8.0*10 8 The cell density was 2×10 6 / ml for freezing, 300 to 400 tubes can be frozen;

[0083] (8) Cell detection:

[0084] Cell surface marker detection and stem cell tri-lineage differentiation function detection were performed before cell cryopreservation;

[0085] Preparation of culture medium: Add the following components (in final concentration) to the basal culture medium MSCBM: including the following components: vitamin C 8.2 μg / L, vitamin E 8.2 μg / L, Centella asiatica extract 3.6 μg / L, protein fiber 10.3 μg / L, L-glycine 20.3 ng / L, L-arginine 12.5 ng / L, tyrosine 8.2 ng / L, lanthanum nitrate 6.3 μg / L, and cerium nitrate 5.1 μg / L.

[0086] Example 2 provides a method for isolating and culturing umbilical cord mesenchymal stem cells, which differs from Example 1 in that the components of the culture medium are different, and the rest are the same as Example 1. Example 2 Preparation of the culture medium: The following components (in terms of final concentration) are added to the basal culture medium MSCBM: including the following components: vitamin C 9.3 μg / L, vitamin E 9.3 μg / L, Centella asiatica extract 4.4 μg / L, protein fiber 10.7 μg / L, L-glycine 20.8 ng / L, L-arginine 13.1 ng / L, tyrosine 8.5 ng / L, lanthanum nitrate 7.3 μg / L, cerium nitrate 6.1 μg / L.

[0087] Example 3 provides a method for isolating and culturing umbilical cord mesenchymal stem cells, which differs from Example 1 in that the components of the culture medium are different, and the rest are the same as Example 1. Example 2 Preparation of culture medium: The following components (in final concentration) are added to the basal culture medium MSCBM: including the following components: vitamin C 10.4 μg / L, vitamin E 10.4 μg / L, Centella asiatica extract 5.2 μg / L, protein fiber 11.1 μg / L, L-glycine 21.3 ng / L, L-arginine 13.7 ng / L, tyrosine 8.8 ng / L, lanthanum nitrate 8.3 μg / L, cerium nitrate 7.1 μg / L.

[0088] Example 4 provides a method for isolating and culturing umbilical cord mesenchymal stem cells, which differs from Example 1 in that the composition of the culture medium is different, and the rest is the same as Example 1. Example 4 Preparation of culture medium: 10% fetal bovine serum is added to the basic culture medium MSCBM.

[0089] Example 5

[0090] Flow cytometry testing was performed by a third-party medical laboratory, Jilin KingMed Medical Laboratory.

[0091] Table 1 Expression of surface markers detected by flow cytometry

[0092]

[0093] The results showed (Table 1 and Figures 1 to 7 ) Under a sterile state, the umbilical cord mesenchymal stem cell isolation and culture method provided by the present invention can achieve a cell viability of more than 98%, and a cell purity: CD34, CD45, and HLA-DR are all less than 0.5%, and CD73, CD90, and CD105 are all greater than 99.5%.

[0094] In order to further compare the technical effects of the present invention, the proliferation ability and multidirectional differentiation potential of the umbilical cord mesenchymal stem cells isolated in Example 2 were tested. The specific test methods and results are as follows:

[0095] Example 6

[0096] Identification of multipotential differentiation

[0097] 1. Adipogenesis

[0098] 1.1 Adipogenic differentiation of human umbilical cord mesenchymal stem cells

[0099] (1) Add 1 mL of 0.1% gelatin to a six-well plate and shake well to evenly cover the bottom of each well.

[0100] (2) Place a six-well plate covered with 0.1% gelatin on a clean bench or CO 2 Incubate for at least 30 minutes;

[0101] (3) After 30 minutes, remove the gelatin and use it to inoculate cells, or wait for the six-well plate to dry before inoculating;

[0102] (4) The stem cells to be induced were divided into 2×10 4 cells / cm 2 The cells were seeded at a density of 1.54 μg / mL in a six-well plate, and 2 mL of complete medium was added to each well;

[0103] (5) The cells were placed at 37°C and 5% CO 2 , saturated humidity CO 2 Culture in an incubator;

[0104] (6) When the cell confluence reaches 100%, carefully remove the complete medium in the well and add 2 mL of differentiation induction medium to the six-well plate;

[0105] (7) After 3 days of induction, add 2 mL of differentiation induction medium;

[0106] (8) After 1 day of maintenance, the medium was changed for induction;

[0107] (9) The cell status needs to be observed daily during this period;

[0108] (10) Repeat the induction and maintenance process until sufficient lipid droplets of appropriate size appear and then prepare for staining.

[0109] 1.2 Adipogenic staining of human umbilical cord mesenchymal stem cells

[0110] (1) After adipogenic differentiation was completed, the culture medium in the six-well plate was aspirated and the plate was gently washed 2 to 3 times with 1× PBS;

[0111] (2) Add 2 mL of 4% paraformaldehyde solution (or 10% formalin) to each well and fix for 30 min at room temperature;

[0112] (3) Prepare the working solution according to the ratio of Oil Red O stock solution to distilled water in a ratio of 3:2. Mix well and centrifuge at 250 × g for 4 min. Use the supernatant.

[0113] (4) Aspirate the fixative and gently wash 2 to 3 times with 1× PBS to ensure that the fixative is thoroughly washed away;

[0114] (5) Add 2 mL of Oil Red O dye working solution to each well and stain at room temperature for 30 min;

[0115] (6) Aspirate the Oil Red O staining solution and gently wash 2 to 3 times with 1× PBS to fully wash away the staining solution;

[0116] (7) Add 2 mL of PBS to each well and place the culture plate under a microscope to observe the adipogenic staining effect.

[0117] 2. Osteogenesis

[0118] 2.1 Osteogenic differentiation of human umbilical cord mesenchymal stem cells

[0119] (1) Add 1 mL of 0.1% gelatin to a six-well plate and shake well to evenly cover the bottom of each well.

[0120] (2) Place a six-well plate covered with 0.1% gelatin on a clean bench or CO 2 Incubate for at least 30 minutes;

[0121] (3) After 30 minutes, remove the gelatin and use it to inoculate cells, or wait for the six-well plate to dry before inoculating;

[0122] (4) Human umbilical cord mesenchymal stem cells to be induced were cultured at a rate of 2×10 4 cells / cm 2The cells were seeded at a density of 1.54 μg / mL in a six-well plate, and 2 mL of culture medium was added to each well;

[0123] (5) The cells were placed at 37°C and 5% CO 2 , saturated humidity CO 2 Culture in an incubator;

[0124] (6) When the cell confluence reaches 70%, carefully remove the culture medium from the wells and add 2 mL of culture medium to the six-well plate;

[0125] (7) Change the medium every 3 days;

[0126] 8. After 2 to 4 weeks of induction, the cells were stained with Alizarin Red depending on their morphological changes and growth.

[0127] 2.2 Osteogenic staining of human umbilical cord mesenchymal stem cells

[0128] (1) After osteogenic differentiation was completed, the culture medium in the six-well plate was aspirated and the plate was gently washed 2 to 3 times with 1× PBS;

[0129] (2) Add 2 mL of 4% paraformaldehyde solution (or 10% formalin) to each well and fix at room temperature for 30 min;

[0130] (3) Aspirate the fixative and gently wash 2 to 3 times with 1× PBS to ensure that the fixative is thoroughly washed away;

[0131] (4) Add 2 mL of Alizarin Red working solution to each well and stain for 5 to 10 minutes at room temperature;

[0132] (5) Aspirate the alizarin red staining solution and gently wash 2 to 3 times with 1× PBS to fully wash away the excess staining solution;

[0133] (6) Add 2 mL of 1× PBS to each well and place the culture plate under a microscope to observe the osteogenic staining effect.

[0134] 3. Cartilage

[0135] 3.1 Chondrogenic differentiation of human umbilical cord mesenchymal stem cells

[0136] (1) 3 to 4 × 10 5 Transfer the cells to be induced into a 15 mL centrifuge tube and centrifuge at 20°C, 250 × g for 4 min;

[0137] (2) Aspirate the supernatant, add 0.5 mL of premixed solution, resuspend the precipitate obtained by centrifugation in the previous step, and centrifuge at 150 g for 5 min at 20°C;

[0138] (3) Repeat step (2) and wash the cells again;

[0139] (4) Add the corresponding volume of premix to prepare complete culture medium and mix gently by pipetting;

[0140] (5) Resuspend the cell pellet obtained in step 3 with 0.5 mL of complete chondrogenic differentiation medium and centrifuge at 150 × g for 5 min at 20°C;

[0141] (6) Loosen the cap of the centrifuge tube to facilitate gas exchange and place it upright at 37°C and 5% CO 2 , saturated humidity CO 2 Culture in an incubator;

[0142] (7) When the cells begin to aggregate (usually after 24 or 48 hours, depending on the actual situation), gently tap the bottom of the centrifuge tube to detach the chondrocytes from the bottom of the tube and suspend them in the liquid;

[0143] (8) Starting from the time of inoculation, replace the culture medium with new medium every 2 to 3 days, about 0.5 L per tube;

[0144] (9) Continue induction until a cartilage ball with a diameter of 1.5 to 2 mm is formed in the tube, and then prepare for sectioning and staining.

[0145] 3.2 Human umbilical cord mesenchymal stem cell chondrogenesis staining

[0146] (1)Fixed:

[0147] The induced chondrocytes were washed with 1× PBS, immersed in 4% paraformaldehyde solution (or 10% formalin solution) and fixed for more than 30 min;

[0148] (2) Dehydration:

[0149] The fixed cartilage balls were dehydrated using graded concentrations of alcohol as follows, with each stage lasting 30 min.

[0150] 50% alcohol ◆ 70% alcohol ◆ 80% alcohol → 95% alcohol ◆ anhydrous alcohol;

[0151] (3) Transparency:

[0152] Since alcohol and paraffin are incompatible, they need to be transferred through xylene after dehydration;

[0153] Here is how:

[0154] ① Mix xylene and anhydrous alcohol in a volume ratio of 1:1. Soak the cartilage balls in it for 2 hours;

[0155] ② Soak the cartilage balls in pure xylene for 1.5 hours;

[0156] ③ Replace with new xylene and continue soaking for 1 hour;

[0157] (4) Wax dipping:

[0158] In order to remove the transparent agent in the cartilage ball and allow the paraffin to penetrate into the interior for embedding, wax immersion is required;

[0159] ① Mix xylene and paraffin in a volume ratio of 1:1. Soak the cartilage balls in it and place them in a 40℃ oven for 40 minutes;

[0160] ② Soak the cartilage balls in pure paraffin and place them in a 55℃ oven for 30 minutes;

[0161] (5) Embedding:

[0162] Take out the cartilage ball, place it in a mold, pour in paraffin, let it stand and cool, wait until the paraffin is fully cooled and formed, take it out, and trim the wax block;

[0163] (6) Slicing:

[0164] Serial sections, each slice thickness 3 μm;

[0165] (7) Adhesive sheet:

[0166] The cartilage ball slices were adhered to a glass slide with adhesive and dried in an oven at 35°C;

[0167] (8) Dewaxing:

[0168] ① Soak the slices in pure xylene for 15 minutes, then replace with new xylene and soak the slices for 10 minutes;

[0169] ② Mix xylene and anhydrous alcohol in a volume ratio of 1:1 and soak the slices for 10 minutes;

[0170] ③ Soak the slices in 95%, 85%, 70%, and 50% alcohol for 10 minutes in sequence and let them dry;

[0171] (9) Dyeing:

[0172] ① Add alcian blue to the dried slices and stain at 37℃ for 1h;

[0173] ② Rinse the slide with tap water for 5 minutes and dry it;

[0174] The Alcian blue staining effect was observed under a microscope. The Alcian blue staining part showed the acidic mucopolysaccharide in the cartilage tissue.

[0175] By dyeing Figures 8 to 10 It can be seen that the umbilical cord mesenchymal stem cells obtained by separation and culture have good proliferation ability and multidirectional differentiation potential. The umbilical cord mesenchymal stem cells obtained by separation and culture can be rapidly induced to differentiate into osteoblasts, adipocytes and chondrocytes under the induction differentiation medium.

Claims

1. A method for separating and culturing umbilical cord mesenchymal stem cells in a culture medium, characterized in that: The steps include: (1) Cleaning of umbilical cord tissue: Place the umbilical cord in a biosafety cabinet, remove the umbilical cord with sterile hook-tooth forceps, place it in a 10 cm petri dish containing saline solution prepared in advance, wash it 2-3 times, clean the blood stains on the surface of the tissue, cut the umbilical cord into small pieces with surgical scissors, and place it in a 10 cm petri dish containing saline solution and continue washing it 2-3 times; (2) Umbilical cord tissue stripping: Use sterile scissors to cut the umbilical cord parallel to the vein, then use hook-toothed forceps to tear off the venous membrane along the cut side, then spread the umbilical cord flat and find the two arteries; Use hook-toothed forceps to remove the two arteries in the umbilical cord. The remaining part is Wharton's jelly and the amniotic membrane on the surface of the umbilical cord. Use hook-toothed forceps to gently peel off the Wharton's jelly, put it in a 25ml glass beaker, and cut it into 1cm pieces with scissors. 2 Small tissue masses; (3) Primary cell inoculation: Suspend the tissue block with the prepared culture medium and inoculate it in a 10 cm culture dish, about 3 to 4 ml per dish; Shake it evenly to make it spread evenly on the ground, and put it into the incubator for cultivation, which is recorded as P0; (4) Primary cell collection: On the 10th to 12th day of cell inoculation, observe the growth of MSCs in the dish, and perform P0-P1 subculture operations according to the growth of cell colonies; The upper culture medium was discarded, and the cells were washed twice with physiological saline, digested with Accutase enzyme solution, and the cell suspension was collected into a 50 ml centrifuge tube, centrifuged at 200g for 5 min, and the cells were collected and washed once with physiological saline at 200g for 5 min; Add pre-warmed culture medium to the 50 ml centrifuge tube containing cells, resuspend the cells, and calculate the cell number according to 1.0*10 6 The cells were inoculated in a 15 cm culture dish, and each dish was filled with pre-warmed culture medium to 20 ml, and placed in a carbon dioxide incubator for culture, which was recorded as P1; (5) Cell passaging: Observe the growth of MSCs every day, and carry out subculture expansion when the cell density is ≥85%. Perform P1 to P2 subculture expansion. According to the cell count, the number of cells is 1.0*10 6 cells were seeded in 15 cm culture dishes at a density of 10; (6) Cell cryopreservation: Observe the growth status of the cells to be collected. When the cell density reaches more than 85%, add physiological saline to the culture dish, gently shake the culture bottle, and discard the physiological saline; Add Accutase to the culture flask or dish, place at 37°C, and observe the cell digestion under a microscope; When most of the cells are detached from the wall, collect the cell suspension into a 50ml centrifuge tube and centrifuge at 200g for 5min to collect the cells. After centrifugation, discard the supernatant, resuspend the cells with 50ml of normal saline, take 50ul of sample for counting, and wash once at 200g for 5min. According to the number of cells, add an appropriate amount of pre-warmed freezing solution to adjust the cell suspension density to 2×10 6 / ml, 1ml per tube, and marked P2. The cells were placed in a -80℃ refrigerator overnight and transferred to -196℃ liquid nitrogen for long-term storage the next day. (7) Cell counting: The P1 cell collection count can reach 1.5-2.5*10 8 The cell density was 2×10 6 / ml for freezing, 70 to 100 tubes can be frozen; The P2 generation cell collection count can reach 7.0~8.0*10 8 The cell density was 2×10 6 / ml for freezing, 300 to 400 tubes can be frozen; (8) Cell detection: Cell surface marker detection and stem cell tri-lineage differentiation function detection were performed before cell freezing. Sterility testing, activity testing, purity testing. The culture medium comprises the following components: basic culture medium, vitamin C, vitamin E, Centella asiatica extract, protein fiber, L-glycine, L-arginine, tyrosine, lanthanum nitrate and cerium nitrate.

2. The method for isolating and culturing umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The umbilical cord is cut into small segments of 3 to 5 cm using surgical scissors as described in step (1).

3. The method for isolating and culturing umbilical cord mesenchymal stem cells according to claim 1, characterized in that: During the subculturing operation described in step (4), the cell density is between 85% and 90%.

4. The method for isolating and culturing umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The basic culture medium is MSCBM culture medium.

5. The method for isolating and culturing umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The final concentrations of the vitamin E and vitamin C are the same, both 8.2-10.4 μg / L.

6. The method for isolating and culturing umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The final concentration of the Centella asiatica extract is 3.6-5.2 μg / L.

7. The method for isolating and culturing umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The final concentration of the protein fiber is 10.3-11.1 μg / L.

8. The method for isolating and culturing umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The final concentrations of L-glycine, L-arginine and tyrosine are 20.3-21.3 ng / L, 12.5-13.7 ng / L and 8.2-8.8 ng / L respectively.

9. The method for isolating and culturing umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The final concentrations of the lanthanum nitrate and cerium nitrate are 6.3-8.3 μg / L and 5.1-7.1 μg / L, respectively.

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