A method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank

Abstract

The present invention discloses a preparation method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank, which belongs to the field of stem cell engineering and includes the following steps: S1, primary culture of umbilical cord mesenchymal stem cells, S2, culture of P0-P1 generations of umbilical cord mesenchymal stem cells, S3, culture of P1-P2 generations, S4, harvesting, subculturing / cryopreserving of P2 generation umbilical cord mesenchymal stem cells, S5, recovery culture of P2 generation umbilical cord mesenchymal stem cells, S6, harvesting, subculturing of P3 generation umbilical cord mesenchymal stem cells, and S7, harvesting and freezing of P4 generation umbilical cord mesenchymal stem cells. The method of the present application has high repeatability and standardization, and can provide uniform cell sample quality. The present application uses improved umbilical cord mesenchymal stem cell complete culture medium to culture the obtained cells, and the surface marker detection results thereof are consistent with the biological characteristics of umbilical cord mesenchymal stem cells, and the safety and effectiveness of umbilical cord mesenchymal stem cells are guaranteed.

Description

Technical Field

[0001] The invention belongs to the field of stem cell engineering, and specifically relates to a preparation method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank. Background Art

[0002] Mesenchymal stem cells (MSCs) are multipotent adult stem cells derived from the mesoderm. They are widely found in bone marrow, umbilical cord tissue, umbilical cord blood, peripheral blood, fat and other tissues. They have the ability to self-renew and have multiple differentiation potentials. Under specific induction conditions in vivo or in vitro, they can differentiate into a variety of tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, endothelium, etc., and they still have multidirectional differentiation potentials after continuous subculture and cryopreservation. MSCs have good chemotactic properties and can penetrate the blood vessel wall. They can automatically chemotaxis to the damaged tissue site after intravenous or local injection. They are a "practical stem cell" that can treat multiple systemic diseases and have high clinical research and application value. According to data, more than 1,000 clinical research projects on MSCs have been registered worldwide.

[0003] Umbilical cord-derived mesenchymal stem cells (UC-MSCs) refer to a type of multifunctional stem cells found in the umbilical cord tissue of newborns. They have stronger self-renewal, high proliferation and multidirectional differentiation potentials. Due to the immaturity of immune cells, they have low immunogenicity and can be used allogeneically without causing immune rejection. All newborn umbilical cords contain a large number of stem cells, which are of high quality and abundant in quantity. Approximately 1.5 million mesenchymal stem cells can be isolated from every 1 cm of umbilical cord tissue. The total amount of cell growth factors secreted is also very high, which facilitates amplification and passage. The material is highly safe and does not involve ethical issues. It is called the "golden resource of stem cells" and is very suitable for clinical research and application. In the future, it is expected to replace other stem cells and become the main body of future stem cell anti-aging treatment.

[0004] However, the current umbilical cord MSCs culture method still has some shortcomings, including the following aspects: the umbilical cord sources are different, and there are differences between donors, which leads to the uncontrollable time of cell harvesting after the umbilical cord Wharton's jelly is peeled off, and it takes a long time for the Wharton's jelly tissue block to crawl out a sufficient number of primary cells, which is not conducive to arranging production plans; in order to meet the requirements of clinical applications, this stage of ex vivo expansion should be completed under the culture conditions that meet the requirements of good tissue culture practice and good manufacturing practice, and no heterologous components are introduced during the MSCs expansion process to ensure the safety of MSCs products. Therefore, the FBS added to the culture medium is gradually replaced by serum substitutes (a method for culturing mesenchymal stem cells and its application patent number: CN 117701500 A). In addition, MSCs have a certain limited expansion capacity during in vitro culture, and long-term culture may lead to cell aging, loss of function and decreased genetic stability. Therefore, it is an urgent problem to establish an in vitro expansion system of MSCs with stable quality and stronger biological functions on the basis of ensuring no xenobiotic contamination. Summary of the invention

[0005] The purpose of the present invention is to provide a method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank, which has high repeatability and standardization, and can provide uniform cell sample quality. The cells obtained by culturing with improved umbilical cord mesenchymal stem cell complete medium have surface marker detection results that conform to the biological characteristics of umbilical cord mesenchymal stem cells, and the safety and effectiveness of umbilical cord mesenchymal stem cells are guaranteed.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is:

[0007] A method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank comprises the following steps:

[0008] S1: Primary culture of umbilical cord mesenchymal stem cells:

[0009] After the outer packaging of the umbilical cord is disinfected with alcohol, it is transferred to the sterile preparation area and placed in a culture dish in a biological safety cabinet. After cleaning with 0.9% sodium chloride injection, it is immersed in alcohol for disinfection. After disinfection, the umbilical vein, umbilical artery and adventitia are removed, and the peeled Wharton's jelly is cut into pieces of about 1mm 3 of small pieces for inoculation.

[0010] Each T182 culture flask was filled with about 2 ml of Wharton's jelly and supplemented with culture medium to 18 ml. The culture flask was placed flat so that the tissue blocks were evenly distributed on the entire bottom surface. On the 5th day of inoculation, the culture supernatant was discarded and the medium was replaced with a full volume. On the 12th day of inoculation, the culture supernatant was discarded and the medium was replaced with a half volume. The cells were cultured until the cell confluence reached 80%.

[0011] S2: Culture of umbilical cord mesenchymal stem cells P0 to P1:

[0012] Tap the primary cell culture flask with a cell confluence of 80%, collect the culture medium and Wharton's jelly that is not attached to the wall, and centrifuge for later use; use 10ml of sodium chloride injection to gently rinse the cell bottle 1-2 times; use recombinant enzyme to digest the cells until 80% of the cells change from spindle shape to round and fall off, and add the supernatant after centrifugation to terminate the digestion; centrifuge all the cell suspensions, discard the supernatant, add MSC complete culture medium and mix well, and inoculate at a density of 1.0-1.2*10^6 / flask; after culturing for 3-4 days, subculture from P1 to P2 generations can be carried out.

[0013] S3: Cultivation of P1 to P2 generations

[0014] When the P1 cell fusion degree reaches more than 85%, it is harvested. Collect the culture medium in the culture bottle, reserve the culture medium required for neutralization and digestion, and recycle the rest into the waste bottle. Gently rinse the culture bottle once with 10-15ml sodium chloride injection. Use recombinant enzyme to digest until 80% of the cells change from spindle shape to round and fall off under an inverted microscope, and add the culture supernatant reserved in advance to terminate the digestion; centrifuge all the cell suspensions, discard the supernatant, add MSC complete culture medium and mix well, inoculate at a density of 1.0~1.2*10^6 / bottle, with a final volume of 25ml, and culture for 3-4 days before subculturing or freezing.

[0015] S4: Harvest, passage and cryopreservation of P2 umbilical cord mesenchymal stem cells

[0016] Observe the cell morphology and confluence of P2, and harvest when the cell confluence reaches more than 85%. The cell digestion, neutralization, collection and passage methods are the same as those of S3.

[0017] After harvest, the P2 cells can be partially or completely frozen and stored as MSCs seeds in a liquid nitrogen bank. All cell suspensions are centrifuged and collected, and the cell concentration is adjusted to 1.1-1.3×10^7 cells / ml using a fully prepared freezing solution. Each cryotube is added with 1ml of cell suspension containing freezing solution for freezing.

[0018] S5: Umbilical cord mesenchymal stem cell P2 recovery culture

[0019] Quickly take out the P2 cell cryopreservation tube from the liquid nitrogen tank and immediately put it into a 37°C water bath. Keep the cryopreservation tube cover above the water surface and shake gently until the cryopreservation solution is completely melted; transfer the cell suspension in the cryopreservation tube to a 50ml centrifuge tube and wash it with 0.9% sodium chloride injection. The number of viable cells inoculated in each T182 cell bottle is 0.82-1.1*10^6 / bottle, the final volume is 25ml, and the cells are harvested and passaged on the 3rd-4th day after culture.

[0020] S6: Harvest and cryopreservation of P4 umbilical cord mesenchymal stem cells

[0021] Observe the cell morphology and confluence of the P4 generation, and harvest when the cell confluence reaches more than 85%. Centrifuge and collect all cell suspensions, adjust the cell concentration to 1.1-1.3×10^7 cells / ml using a fully prepared freezing solution, and add 1ml of cell suspension containing freezing solution to each freezing tube for freezing.

[0022] A further improvement of the technical solution of the present invention is that the recombinase is balanced to room temperature before use.

[0023] The further improvement of the technical solution of the present invention is that in S1, the total volume of the Wharton's jelly block and the umbilical cord mesenchymal stem cell complete culture medium is 20 ml, which facilitates the adhesion of the tissue block to the wall and the mesenchymal stem cells in the tissue block to crawl out, while ensuring that the tissue block is given sufficient nutrition. Due to individual differences between umbilical cords, the time it takes for cells to crawl out varies. If the amount of cells that crawl out is small on the 17th day after the tissue block is inoculated, half of the medium can be replaced again until the cell fusion reaches 80%.

[0024] A further improvement of the technical solution of the present invention is that in S2, the amount of recombinant enzyme added is 5 ml, the growth density of the cells that have just crawled out is uneven, and the digestion time of the area with higher density is longer. During the digestion process, 5 ml of recombinant enzyme is convenient to shake, which is convenient for digestion of the area with high cell density; the Wharton's gel blocks attached to the wall fall off into the cell suspension after digestion by the recombinant enzyme, so all the cell suspensions after neutralization are filtered using a 100 μm cell sieve to ensure higher cell purity and more accurate culture density.

[0025] The further improvement of the technical solution of the present invention is that: in S3, the culture medium in the culture bottle is collected into a 50ml centrifuge tube, and after washing with saline, 10ml of recombinase is used to rinse 3-4 bottles of cells, and a small amount of residual recombinase in each bottle is digested, and the excess recombinase is discarded. Digestion is performed until 80% of the cells change from spindle shape to round and fall off under an inverted microscope; for every 3-4 bottles of digested cells, 10ml of the collected culture medium is used to terminate the digestion, and the cell bottle after collecting the suspension is washed with physiological saline, and the washed saline and cells are recovered and centrifuged together to maximize the recovery of cells.

[0026] The further improvement of the technical solution of the present invention is that: in S4, the umbilical cord mesenchymal stem cell cryopreservation solution includes 10% DMSO, 30% human serum albumin, and 60% MSC complete culture medium according to the volume fraction, and is placed at 4°C for precooling after preparation. The cryopreservation solution has simple ingredients, a simple and easy-to-operate preparation process, no serum, no animal components, and is not easy to introduce exogenous components, which is beneficial to the culture of umbilical cord MSCs after cryopreservation and resuscitation and direct clinical use.

[0027] A further improvement of the technical solution of the present invention is that: in S5, the number of live cells in each T182 cell bottle culture inoculated cell is 0.82-1.1*10^6 / bottle, and the cells are harvested and passaged 4 days after inoculation. The growth state of cells after cryopreservation and resuscitation is different from that of freshly digested cells. It takes a period of recovery to proliferate better, and simply increasing the number of culture days makes it easy for cells to grow too densely at the time of harvest. The inoculation density is slightly reduced, and the culture time is increased at the same time. The harvest time is controllable and the harvest density can reach a relatively ideal state; the culture system is 25ml, which makes it easier for cells to cover the bottom of the bottle, avoiding the uneven bottom of the bottle or the uneven partition of the incubator. The cells in a certain area are not covered with culture medium, resulting in a reduction in the amount of cells. At the same time, the total content of nutrients is increased to minimize the waste of culture medium while ensuring adequate nutrition for the cells.

[0028] The further improvement of the technical solution of the present invention is that in S6, the freshly digested and collected MSCs have good adhesion and proliferation after passage, and the number of viable cells inoculated in each T182 cell bottle is 1.0-1.2*10^6 / bottle, the final volume is 25ml, and the cells are harvested and frozen on D3 day. The passage batches of the application library are strictly limited to ensure the functionality and genetic stability of MSCs.

[0029] A further improvement of the technical solution of the present invention is that the MSC complete culture medium includes a basal culture medium, platelet lysate, a serum substitute, and sodium polyacrylate.

[0030] The further improvement of the technical solution of the present invention is that the concentration of platelet lysate is 1-5%, the concentration of serum substitute is 0.5-2%, the addition amount of sodium polyacrylate is 5-20 μg / ml, and the remainder is basal culture medium.

[0031] A further improvement of the technical solution of the present invention is that: the culture medium also includes a basal culture medium, and the basal culture medium includes but is not limited to MEMα culture medium.

[0032] The further improvement of the technical solution of the present invention is that the MSC complete culture medium includes 1-5% platelet lysate, 0.5-2% serum substitute, 5-20 μg / ml sodium polyacrylate and the remainder basal culture medium.

[0033] Due to the adoption of the above technical solution, the technical progress achieved by the present invention is:

[0034] The invention discloses a method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank, which provides an improved version of a complete umbilical cord mesenchymal stem cell culture medium, which does not contain heterologous substances such as serum and antibiotics, improves the adhesion effect and expansion effect of mesenchymal stem cells by selecting a serum-free culture medium, a serum substitute and sodium polyacrylate, and enables serum substitutes and platelet lysates to obtain better culture results at lower concentrations.

[0035] At the same time, the inoculation volume and culture days after cell recovery were improved. The number of live cells inoculated in each T182 cell bottle was 0.82-1.1*10^6, and the cells were harvested and passaged 4 days after inoculation. The growth state of cells after cryopreservation and recovery is different from that of freshly digested cells. It takes a period of recovery to proliferate better. Simply increasing the number of culture days makes it easy for cells to grow too densely at the time of harvest. By slightly reducing the inoculation density and increasing the culture time, the harvest time can be controlled and the harvest density can reach a relatively ideal state.

[0036] MSC cells are easy to digest, and the digestion method is changed to 10ml of trypsin for 3-4 bottles of cells, digested until 80% of the cells fall off; for every 3-4 bottles of digested cells, 10ml of the collected culture medium is used to terminate the digestion. This method reduces the amount of trypsin and culture medium used, is convenient for operation, and shortens the operation time overall, thereby reducing the overall storage time of cells at room temperature. At the same time, the frequency of use of operating items and reagents is reduced, and the frequency of placing items in the biosafety cabinet and entering and exiting the biosafety cabinet during operation is reduced, thereby reducing the risk of contamination.

[0037] The cells obtained by culturing the improved umbilical cord mesenchymal stem cell complete culture medium used in the present invention have surface marker detection results that conform to the biological characteristics of the umbilical cord mesenchymal stem cells, and the safety and effectiveness of the umbilical cord mesenchymal stem cells are guaranteed.

[0038] After the umbilical cord Wharton's jelly is stripped, the MSCs are cultured to the P2 generation and then cryopreserved, which saves liquid nitrogen storage space while facilitating subsequent culture. It only takes one week to culture the P2 generation cryopreserved cells to the P4 generation for harvesting, so the required cells can be quickly obtained, which is convenient for arranging production plans and conducting other experimental studies.

[0039] The present invention controls the appropriate seeding density when culturing mesenchymal stem cells in vitro, increases the clone quantity after passage and ensures higher cell activity.

[0040] The present invention uniformly manages cells of different batches, distinguishes between a P2 generation cell seed bank and a P4 generation cell application bank, strictly limits the passage batches of the application bank, and ensures the functionality and genetic stability of MSCs.

[0041] The present invention adopts the umbilical cord MSCs cryopreservation solution provided by the present application to cryopreserve cells, so that the umbilical cord MSCs after cryopreservation and resuscitation have higher cell viability, good cell morphology, good cell surface marker expression and good proliferation ability. In addition, the cryopreservation solution has simple ingredients, a simple and easy-to-operate preparation process, is serum-free, animal-free, and does not introduce exogenous ingredients, which is beneficial to the culture of umbilical cord MSCs after cryopreservation and resuscitation or direct clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is the effect of different sample culture media on cell proliferation ability;

[0043] Figure 2 is the effect of 1# medium on the expression of cell surface markers;

[0044] Figure 3 This is the effect of 3# culture medium on the expression of cell surface markers. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with embodiments:

[0046] Embodiment 1:

[0047] Effects of different doses of platelet lysate, serum supplements, and sodium polyacrylate on in vitro culture of human umbilical cord mesenchymal stem cells, including the expansion efficiency and the effect of maintaining stem cell characteristics.

[0048] The sample culture media are:

[0049] Medium 1# (MEMα medium with 4.7% platelet lysate and 1.9% serum replacement, without sodium polyacrylate)

[0050] Medium 2# (MEMα medium supplemented with 4.0% platelet lysate, 1.7% serum replacement, and 10 μg / ml sodium polyacrylate)

[0051] Medium #3 (MEMα medium supplemented with 3.0% platelet lysate, 1.4% serum replacement, and 10 μg / ml sodium polyacrylate)

[0052] Medium 4# (MEMα medium supplemented with 2.0% platelet lysate, 0.8% serum replacement, and 10 μg / ml sodium polyacrylate)

[0053] Medium #5 (MEMα medium supplemented with 1.5% platelet lysate, 0.5% serum replacement, and 10 μg / ml sodium polyacrylate)

[0054] Medium #6 (MEMα medium supplemented with 2.0% platelet lysate, 0.8% serum replacement, and 10 μg / ml sodium polyacrylate)

[0055] Medium #7 (MEMα medium supplemented with 2.0% platelet lysate, 0.8% serum replacement, and 10 μg / ml sodium polyacrylate)

[0056] Medium #8 (MEMα medium supplemented with 2.0% platelet lysate, 0.8% serum replacement, and 20 μg / ml sodium polyacrylate)

[0057] Medium 9# (MEMα medium with 1.5% platelet lysate and 0.5% serum replacement, without sodium polyacrylate)

[0058] The serum replacement was PALL serum replacement.

[0059] Example 2

[0060] Preparation of P2 seed bank of umbilical cord mesenchymal stem cells

[0061] Primary culture of S1 umbilical cord mesenchymal stem cells

[0062] After the outer packaging of the umbilical cord is disinfected with alcohol, it is transferred to the sterile preparation area and placed in a culture dish in a biological safety cabinet. After cleaning with 0.9% sodium chloride injection, it is immersed in alcohol for disinfection. After disinfection, the umbilical vein, umbilical artery and outer membrane of the umbilical cord are removed, and the peeled Wharton's jelly is cut into pieces of about 1mm 3 of small pieces.

[0063] Wash with physiological saline and centrifuge, fill each T182 culture bottle with about 2 ml of Wharton's jelly, and add the sample culture medium in Example 1 to 18 ml. Place the culture bottle horizontally so that the tissue blocks are evenly distributed on the entire bottom surface as much as possible, and culture in a 37.0°C, 5.0% CO2 incubator.

[0064] According to the cell growth, the culture supernatant was discarded and the medium was replaced in full volume on the 5th day of inoculation, and the culture supernatant was discarded and the medium was replaced in half volume on the 12th day of inoculation.

[0065] S2: Culture of umbilical cord mesenchymal stem cells P0 to P1:

[0066] Observe the cell fusion degree under the microscope and it is about 80%, then proceed to the subculture operation. Gently tap the primary cell culture bottle, collect the culture medium and the unattached Wharton's jelly in the culture bottle, centrifuge for use; use 10ml of sodium chloride injection to gently rinse the cell bottle once; use 5ml of recombinant enzyme to digest the cells until 80% of the cells change from spindle shape to round and fall off, add the supernatant after centrifugation to terminate the digestion; centrifuge all the cell suspension, discard the supernatant, add the sample culture medium in Example 1 and mix well, inoculate at a density of 1.0-1.2*10^6 / bottle, and culture in a 37.0℃, 5.0% CO2 incubator.

[0067] S3: Cultivation of P1 to P2 generations

[0068] When the fusion degree of P1 cells reaches more than 85%, they are harvested. Collect the culture medium in the culture bottle, set aside the culture medium required for neutralization and digestion, and recycle the rest into the waste liquid bottle. Gently rinse the culture bottle once with 10-15ml sodium chloride injection. Use 10ml of recombinant enzyme to rinse 3-4 bottles of cells, and digest a small amount of residual recombinant enzyme in each bottle, and discard the excess recombinant enzyme. Digest until 80% of the cells change from spindle shape to round and fall off under an inverted microscope; for every 3-4 bottles of digested cells, use 10ml of collected culture medium to terminate digestion, and wash the cell bottle after collecting the suspension with physiological saline, and recover the washed saline and cells together by centrifugation to maximize the recovery of cells; centrifuge all the cell suspensions, discard the supernatant, add the sample culture medium in Example 1 and mix well, inoculate at a density of 1.0-1.2*10^6 / bottle, and culture for 3-4 days before passage or cryopreservation.

[0069] S4: Harvest, passage and cryopreservation of P2 umbilical cord mesenchymal stem cells

[0070] Observe the cell morphology and confluence of P2, and harvest when the cell confluence reaches more than 85%. The cell digestion, neutralization, collection and passaging methods are the same as those of S3. After the cells are fixed to volume, they are counted and detected by flow cytometry.

[0071] After harvest, the P2 cells can be partially or completely frozen and stored as MSCs seeds in liquid nitrogen. All cell suspensions are centrifuged and collected, and the cell concentration is adjusted to 1.1-1.3×10^7 cells / ml using a complete cryopreservation solution (including 10% DMSO, 30% human albumin, and 60% MSC complete culture medium). Each cryopreservation tube is added with 1ml of cell suspension containing cryopreservation solution for cryopreservation.

[0072] Example 3

[0073] S1 umbilical cord mesenchymal stem cells P2 resuscitation culture

[0074] The cells frozen in Example 2 were quickly taken out from the liquid nitrogen tank and immediately placed in a 37°C water bath, with the cryopreservation tube lid kept above the water surface and gently shaken until the cryopreservation solution was completely melted; the cell suspension in the cryopreservation tube was transferred to a 50 ml centrifuge tube and washed with 0.9% sodium chloride injection. The number of viable cells inoculated in each T182 cell bottle was 0.82 to 1.1*10^6 / bottle, and the sample culture medium in Example 1 was used, with a final volume of 25 ml. The cells were cultured to the 4th day for harvesting and subculture.

[0075] S2 umbilical cord mesenchymal stem cells harvested and subcultured at P3

[0076] The cell morphology and confluence of the P3 generation were observed, and the cells were harvested when the confluence reached 85% or more. The cell digestion, neutralization, collection and passage were the same as those in Example 2S2.

[0077] The number of viable cells inoculated in each T182 cell flask was 1.0-1.2*10^6 / flask, with a final volume of 25 ml. The cells were harvested and frozen on D3.

[0078] Harvest and cryopreservation of P4 umbilical cord mesenchymal stem cells

[0079] The cell morphology and confluence of the P4 generation were observed, and the cells were harvested when the confluence reached 85% or more. The cell digestion, harvesting, and freezing methods were the same as those in Example 2S4.

[0080] Test content includes

[0081] 1. Cell morphology evaluation: Observe the morphology of P4 cells in each group under an inverted microscope and record the results.

[0082] 2. Cell harvesting, cell number and viability: The cells obtained by P4 digestion were counted using the AO / PI method, and the cell viability was recorded.

[0083] 3. Cell expansion multiples: P4 cells were inoculated at a concentration of 1.0 to 1.2*10^6 / bottle, cultured using the sample culture medium in Example 1, and harvested on D3 day. The cells obtained by digestion were counted, and the expansion multiples of each sample culture medium were compared.

[0084] 4. Cell surface antigen determination: The umbilical cord mesenchymal stem cells harvested from the P4 generation were labeled with CD73, CD90, CD105, CD45, CD34, and HLA-DR+, and the positive rate of cell surface markers was detected.

[0085] result:

[0086] 1. Effect of culture media with different sodium polyacrylate contents on cell morphology: After being cultured in different sample culture media, except for 9# culture medium, the fusion rate of P4 cells cultured in other groups of culture media can reach more than 85%, and the cell morphology is good.

[0087] 2. The cell viability of P4 cells cultured in different sample culture media reached more than 95% except for 9# culture medium. The number of cells harvested from 2# to 8# culture media with sodium polyacrylate added was higher than that without sodium polyacrylate added, as shown in Table 1.

[0088] 3. Effects of different sample culture media on cell proliferation ability: Results are as follows Figure 1 As shown, the cell proliferation multiples of medium 2# to 8# were higher than those of the group without sodium polyacrylate. 9# did not add sodium polyacrylate and had the worst proliferation ability in reducing serum substitutes and platelet lysates.

[0089] 4. Effects of different sample culture media on the expression of cell surface markers: The results are shown in Table 2. Figure 2-3 As shown, mesenchymal stem cells were cultured in sample culture media 1# to 9# to P4, and the harvested cells all showed the expression characteristics of mesenchymal stem cell surface markers, with the expression rates of CD73+ / CD90+ / CD105+ all higher than 95%, and CD45+ / CD34+ / HLA-DR+ all lower than 2%.

[0090] Conclusion: Under the premise that serum-free culture medium is gradually becoming a trend, the present invention improves the adhesion and expansion effects of mesenchymal stem cells by adding platelet lysate, serum substitute and sodium polyacrylate for radical culture, and well maintains the expression characteristics of surface markers of mesenchymal stem cells. By adding sodium polyacrylate, the amount of platelet lysate and serum substitute used is reduced, and a better culture effect is obtained.

[0091] Table 1

[0092]

[0093] Table 2

[0094]

Claims

1. A method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank, characterized in that: The following steps are involved: S1: Primary culture of umbilical cord mesenchymal stem cells: After disinfection, the umbilical cord is removed from the umbilical vein, umbilical artery and adventitia. The peeled Wharton's jelly is cut into pieces of about 1 mm 3 small pieces for inoculation; S2: Culture of umbilical cord mesenchymal stem cells P0-P1: tap the primary cell culture bottle with a cell confluence of 80%, collect the culture medium and Wharton's jelly that is not attached to the wall, and centrifuge for later use; use recombinant enzyme to digest the cells until 80% of the cells change from spindle shape to round and fall off, and add the supernatant after centrifugation to terminate the digestion; centrifuge all the cell suspensions, discard the supernatant, add MSC complete culture medium and mix well, and inoculate at a density of 1.0-1.2*10^6 / bottle; after 3-4 days of culture, subculture from P1 to P2 can be carried out; S3: Cultivation of P1 to P2 generations; harvest the P1 cells when their fusion degree reaches more than 85%, collect the culture medium in the culture bottle, digest with recombinant enzyme until 80% of the cells change from spindle shape to round shape and fall off under an inverted microscope, and add the culture supernatant reserved in advance to terminate the digestion; centrifuge all the cell suspensions, discard the supernatant, add MSC complete culture medium and mix well, inoculate at a density of 1.0~1.2*10^6 / bottle, and culture for 3-4 days before subculturing or freezing; S4: Harvest, passage and cryopreservation of P2 umbilical cord mesenchymal stem cells; observe the cell morphology and confluence of P2 cells, and harvest when the cell confluence reaches more than 85%. The cell digestion, neutralization, collection and passage methods are the same as those of S3. After harvest, P2 cells can be partially or completely cryopreserved and stored in a liquid nitrogen bank as MSCs seeds; S5: P2 resuscitation culture of umbilical cord mesenchymal stem cells; quickly take out the P2 cell cryopreservation tube from the liquid nitrogen tank for thawing, transfer the cell suspension in the cryopreservation tube to a 50ml centrifuge tube, wash with 0.9% sodium chloride injection, the number of live cells inoculated in each T182 cell bottle is 0.82~1.1*10^6 / bottle, the final volume is 25ml, and the culture is harvested and subcultured on the 3rd-4th day; S6: Umbilical cord mesenchymal stem cells P3 were harvested and subcultured; the cell morphology and confluence of the P3 generation were observed, and the cells were harvested when the confluence reached more than 85%. The cell digestion, neutralization, collection and subculture methods were the same as those of S3; S7: Harvest and cryopreservation of P4 umbilical cord mesenchymal stem cells: Observe the morphology and confluence of P4 cells and harvest them when the confluence reaches more than 85%.

2. The method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank according to claim 1, characterized in that: The MSC complete culture medium comprises a basal culture medium, platelet lysate, serum replacement, and sodium polyacrylate.

3. The method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank according to claim 2, characterized in that: The concentration of platelet lysate in the MSC complete culture medium is 1-5%, the concentration of serum substitute is 0.5-2%, the addition amount of sodium polyacrylate is 5-20 μg / ml, and the remainder is basal culture medium, and the basal culture medium includes MEMα culture medium.

4. The method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank according to claim 1, characterized in that: In step S4, all cell suspensions are centrifuged and collected, and the cell concentration is adjusted to 1.1-1.3×10^7 cells / ml using a fully prepared freezing solution. 1 ml of cell suspension containing freezing solution is added to each freezing tube for freezing.

5. The method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank according to claim 1, characterized in that: The thawing method in step S5 is to quickly take out the P2 cell cryopreservation tube from the liquid nitrogen tank and immediately put it into a 37°C water bath, and keep the cryopreservation tube cover above the water surface and shake it gently until the cryopreservation liquid is completely thawed.

6. The method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank according to claim 1, characterized in that: In step S6, the number of viable cells in each T182 cell bottle cultured and inoculated is 1.0~1.2*10^6 / bottle, the final volume is 25 ml, and the cells are harvested and frozen after culture until D3 day.

7. The method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank according to claim 1, characterized in that: In step S7, all cell suspensions are centrifuged and collected, and the cell concentration is adjusted to 1.1-1.3×10^7 cells / ml using a fully prepared freezing solution. 1 ml of cell suspension containing freezing solution is added to each freezing tube for freezing.

8. The method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank according to claim 1, characterized in that: The recombinant enzyme was equilibrated to room temperature before use.

9. The method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank according to claim 1, characterized in that: The freshly digested and collected MSCs in step S6 have good adhesion and proliferation after passaging. The number of viable cells inoculated in each T182 cell flask is 1.0~1.2*10^6 / flask, the final volume is 25 ml, and the cells are harvested and frozen after culture until D3 day.

10. The method for efficiently obtaining a human umbilical cord mesenchymal stem cell bank according to claim 1, characterized in that: The umbilical cord mesenchymal stem cell freezing solution includes 10% DMSO, 30% human serum albumin, and 60% MSC complete culture medium. After preparation, it is pre-cooled at 4°C.

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