A mesenchymal stem cell large-scale expansion protocol

Abstract

The application discloses a mesenchymal stem cell large-multiple expansion scheme and belongs to the technical field of biology. The method uses cryopreserved umbilical cord mesenchymal stem cells as seeds (including P1 / P2 / P3 / P4 / P5), rapidly recovers the seed cells, uses a mixture of platelet lysate and serum substitute as a culture medium additive to carry out culture, and achieves the method of large-multiple expansion of mesenchymal stem cells. Compared with a traditional single culture medium culture mode, the method has the advantages of different culture systems, can achieve large-multiple expansion of cells, improves production efficiency, has low cell damage, has stable and uniform cell diameters, has slow aging speed of cell growth, can still keep the cell state and the cell survival rate at a high level after multiple passage expansion, and can avoid the problems of insufficient quantity of mesenchymal stem cells in a single batch and uneven quality between multiple batches, and thus influences clinical application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a scheme for large-scale expansion of mesenchymal stem cells. Background Technology

[0002] Human umbilical cord mesenchymal stem cells (MSCs) are a type of stem cell with self-renewal, proliferation, and multi-lineage differentiation potential. Compared with MSCs from other sources, they have many advantages, including wide availability, ease of collection, preservation, and transportation, no allogeneic rejection, and avoidance of ethical controversies. With increasing clinical research on umbilical cord mesenchymal stem cells, the demand for stem cell quantities is growing. To ensure the accuracy of clinical data, the required cell quantity is extremely high, and the uniformity and stability of cell quality are also crucial.

[0003] To improve cell culture efficiency, increase single-batch cell yield, and ensure quality and high stability, existing culture systems and methods are improved to address industry pain points. Summary of the Invention

[0004] To address the aforementioned problems, the present invention proposes the following technical solution: a method for large-scale expansion of mesenchymal stem cells, comprising the following steps: S1. Umbilical cord seed cell resuscitation: Take out the cryovials of seed cells frozen at -196℃, place them in a water bath at 37℃-40℃, thaw them quickly, and then transfer them to centrifuge tubes containing pre-cooled saline in a biosafety cabinet. Centrifuge at 200g-300g for 10-15min, remove the supernatant, resuspend the cells in an appropriate amount of culture medium, and take samples for counting. S2. Preparation of high-multiplication amplification medium: Thawed platelet lysate was added to type 1 culture medium, and thawed serum substitute was added to type 2 culture medium. The high-amplification culture medium used for culture was a mixture of type 1 and type 2. S3, Expanding mesenchymal stem cells: Add the resuspended seed cells to a T75 culture flask at a density of 5000-10000 / cm³. 2 Add 10-15 ml of high-expansion culture medium to each T75 culture flask; shake the culture flask well, place it horizontally in a CO2 incubator with a CO2 concentration of 5% and a temperature of 37℃; incubate for 3-5 days. S4. Collect cryopreserved cells: When cell confluence reaches 80%-90%, collect cells in a biosafety cabinet; discard the culture supernatant, add an appropriate amount of saline to wash the culture surface, discard the saline, add an appropriate amount of trypsin digestion solution to infiltrate the cells, and incubate for digestion for 30-60 seconds. Observe that most cells become rounded and detach, then add an appropriate amount of high-glucose DMEM to terminate digestion; collect cells into centrifuge tubes and centrifuge at 200g-300g for 5-10 minutes; discard the washing supernatant, resuspend the cells in an appropriate amount of saline, and count the cells; centrifuge again at 200g-300g for 5-10 minutes; discard the washing supernatant, add an appropriate amount of cell cryopreservation solution, and add the cells to 1.8ml cryovials at a density of 1-2.4*102. 7 / ml; transfer to a programmed cooling box, freeze overnight at -80℃, and finally transfer to a liquid nitrogen tank.

[0005] Preferably, the high-expansion culture medium used for mesenchymal stem cells is a mixture of prepared type I and type II culture media in a 1:1 ratio.

[0006] Preferably, the cryopreservation solution for mesenchymal stem cells is a prepared type 2 culture medium, DMSO, and human serum albumin in a ratio of 8:1:1.

[0007] Preferred method for preparing mesenchymal stem cell cryopreservation solution is as follows: Take the prepared type 2 culture medium pre-cooled at 2-8℃, gradually add DMSO, mix evenly, then add human serum albumin, mix evenly, and place at 2-8℃ for pre-cooling and use.

[0008] The beneficial effects of this invention are: This invention addresses two existing cell culture methods: 1) basal culture medium + platelet lysis buffer, and 2) basal culture medium + serum substitute. Both methods, when used individually, have limited cell expansion (10-15 times), longer culture cycles, and generally lower efficiency. This invention combines the advantages of both methods, shortening the culture cycle, increasing expansion to 20-25 times, reducing costs, minimizing cell damage, ensuring stable and uniform cell diameter, and slowing cell aging after growth, thus guaranteeing cell quality and meeting the requirements of clinical research applications. Attached Figure Description

[0009] Figure 1 This is a schematic diagram of P3 generation cells cultured according to the mesenchymal stem cell large-scale expansion scheme of the present invention.

[0010] Figure 2 This is a schematic diagram of P3 generation cells cultured using other methods.

[0011] Figure 3 This is a flow cytometry result of a mesenchymal stem cell culture using a high-multiplication scheme according to the present invention.

[0012] Figure 4This is a flow cytometry result of a mesenchymal stem cell culture using a high-multiplication scheme according to the present invention.

[0013] Figure 5 This is a flowchart of a mesenchymal stem cell expansion scheme according to the present invention. Detailed Implementation

[0014] This invention is achieved through the following method: A protocol for large-scale expansion of mesenchymal stem cells: S1 and P2 generation seed cell resuscitation Take out the cryovials of P2 generation seed cells frozen at -196℃, place them in a water bath at 37℃-40℃, thaw them quickly, and then transfer them to centrifuge tubes containing pre-cooled saline at 2-8℃ in a biosafety cabinet. Centrifuge at 200g-300g for 10-15min, remove the supernatant, and resuspend the cells in an appropriate amount of culture medium.

[0015] S2, Preparation of high-multiplication amplification medium Thawed platelet lysate was added to type 1 culture medium, and thawed serum substitute was added to type 2 culture medium. The high-amplification culture medium used for culture was a mixture of type 1 and type 2 in a 1:1 ratio.

[0016] S3, mesenchymal stem cell passage expansion Add the resuspended seed cells to a T75 culture flask at a density of 5000-10000 / cm³. 2 Add 8-12 ml of high-expansion culture medium to each T75 culture flask. Shake the flask well and place it horizontally in a CO2 incubator at 37°C and a CO2 concentration of 5%. Incubate for 3-5 days.

[0017] S4. Collect cryopreserved cells Observe the cells. When the cell confluence reaches 80%-90%, collect the cells in a biosafety cabinet. Discard the culture supernatant, add an appropriate amount of saline to wash the culture surface, discard the saline, add an appropriate amount of trypsin digestion solution to infiltrate the cells, and incubate for digestion for 30-60 seconds. Observe that most cells become rounded and detach, then add an appropriate amount of high-glucose DMEM to stop the digestion. Collect the cells into centrifuge tubes and centrifuge at 200g-300g for 5-10 minutes. Discard the washing supernatant, resuspend the cells in an appropriate amount of saline, collect samples, count, and send for testing. Centrifuge again at 200g-300g for 5-10 minutes. Discard the washing supernatant, add an appropriate amount of cell cryopreservation solution, and add the cells to 1.8ml cryovials at a density of 1-2.4*103. 7 / ml. Transfer to a programmed cooling box, freeze overnight at -80°C, and finally transfer to a liquid nitrogen tank.

[0018] The cell cryopreservation solution used above is as follows: Take the prepared type 2 culture medium pre-cooled at 2-8℃, gradually add DMSO, mix well, and place at 2-8℃ for pre-cooling before use.

[0019] Biological characteristics of umbilical cord mesenchymal stem cells 1. Morphological observation of umbilical cord mesenchymal stem cells Cells prepared using the above method were observed under a microscope. The cells were tightly packed in a spindle shape and grew in a monolayer adherent state. Approximately 2 hours after inoculation, most cells adhered to the wall, gradually changing from a round shape. As the cells divided and multiplied, their number gradually increased, and they became tightly packed, maintaining a spindle shape and becoming increasingly uniform. With repeated passages, the cell morphology did not show significant changes.

[0020] 2. Cell number and viability Take the cell suspension prepared above, stain with trypan blue, and count the cells using a CountStar cell counter. Each bottle has a T75cm depth. 2 The culture flask can collect 1.1*10 cells. 7 The number of cells and the cell viability were greater than 95%, reaching 99.3%.

[0021] 3. Cell diameter measurement Countstar cell counter measurements showed that the cell diameter was 15-16 μm, indicating stability and uniformity.

[0022] 4. Flow cytometry analysis of umbilical cord mesenchymal stem cells Cells prepared using the above method were subjected to flow cytometry analysis. The results showed positive results for CD73, CD90, and CD105, and negative results for CD34, CD45, CD11b, CD19, and HLA-DR. The positive rates were 100% for CD73, 99.994% for CD90, 99.995% for CD105, 0.014% for CD34, 0.049% for CD45, 0.504% for CD11b, 0.187% for CD19, and 0.005% for HLA-DR.

[0023] 5. Induction of differentiation of umbilical cord mesenchymal stem cells The mesenchymal stem cells prepared as described above.

[0024] Adipogenic induction: P3 generation cells, mixed with 1*10 cells in complete culture medium. 5 One cell line was seeded into a six-well plate and placed at 37°C in 5% CO2. 2、 After 24 hours in a saturated humidity incubator, the medium was completely replaced, and 0.5 μm dexamethasone, 0.5 mm IBMX, and 10 μm insulin were added. The incubator was then placed at 37°C in 5% CO2. 2、Differentiation was induced in a saturated humidity incubator, and the medium was completely changed every 3 days thereafter. After two weeks, the formation of lipid droplets was identified by Oil Red O staining.

[0025] Results: Two weeks after induction, the cells were found to be filled with lipids, and the lipid droplets appeared red after Oil Red O staining.

[0026] Osteogenic induction: P3 generation cells, mixed with complete culture medium at a ratio of 1*102 5 One cell line was seeded into a six-well plate and placed at 37°C in 5% CO2. 2、 After 24 hours in a saturated humidity incubator, the medium was completely replaced, and 10 mg of β-glycerophosphate sodium, 1 μM dexamethasone, and 50 mg of vitamin C were added for continued incubation. The medium was then changed every 3 days. After two weeks of induction, alkaline phosphatase staining, bone morphogenetic protein, and calcified nodules were detected.

[0027] Results: After 2 weeks of induction, alkaline phosphatase staining showed a strong positive reaction.

[0028] Chondrogenic induction: P3 generation cells, mixed with complete culture medium at 2*103 5 Cells were seeded into 24-well plates, and each well was supplemented with 10 mM dexamethasone, 10 μg / L TGF-β, and 10 mM vitamin C. The plates were then incubated at 37°C in a 5% CO2 atmosphere. 2、 After two weeks of culture in a saturated humidity incubator, Liang Xu underwent type II collagen testing.

[0029] Two weeks after induction, type II collagen formation was observed by asin blue staining.

[0030] 6. Telomerase analysis It has telomerase activity.

[0031] 7. Karyotype analysis normal.

[0032] 8. Analysis of proto-oncogenes and tumor suppressor genes There were no significant changes in the expression of proto-oncogenes and tumor suppressor genes.

Claims

1. A protocol for large-scale expansion of mesenchymal stem cells, characterized in that, Includes the following steps: S1. Umbilical cord seed cell resuscitation: Take out the cryovials of seed cells frozen at -196℃, place them in a water bath at 37℃-40℃, thaw them quickly, and then transfer them to centrifuge tubes containing pre-cooled saline in a biosafety cabinet. Centrifuge at 200g-300g for 10-15min, remove the supernatant, resuspend the cells in an appropriate amount of culture medium, and take samples for counting. S2. Preparation of high-multiplication amplification medium: Thawed platelet lysate was added to type 1 culture medium, and thawed serum substitute was added to type 2 culture medium. The high-amplification culture medium used for culture was a mixture of type 1 and type 2. S3, Expanding mesenchymal stem cells: Add the resuspended seed cells to a T75 culture flask at a density of 5000-10000 / cm³. 2 Add 10-15 ml of high-expansion culture medium to each T75 culture flask; shake the culture flask well, place it horizontally in a CO2 incubator with a CO2 concentration of 5% and a temperature of 37℃; incubate for 3-5 days. S4. Collect cryopreserved cells: When cell confluence reaches 80%-90%, collect cells in a biosafety cabinet; discard the culture supernatant, add an appropriate amount of saline to wash the culture surface, discard the saline, add an appropriate amount of trypsin digestion solution to infiltrate the cells, and incubate for digestion for 30-60 seconds. Observe that most cells become rounded and detach, then add an appropriate amount of high-glucose DMEM to terminate digestion; collect cells into centrifuge tubes and centrifuge at 200g-300g for 5-10 minutes; discard the washing supernatant, resuspend the cells in an appropriate amount of saline, and count the cells; centrifuge again at 200g-300g for 5-10 minutes; discard the washing supernatant, add an appropriate amount of cell cryopreservation solution, and add the cells to 1.8ml cryovials at a density of 1-2.4*102. 7 / ml; transfer to a programmed cooling box, freeze overnight at -80℃, and finally transfer to a liquid nitrogen tank.

2. The solution according to claim 1, characterized in that: The high-expansion culture medium used for mesenchymal stem cells was prepared by mixing type I and type II culture media in a 1:1 ratio.

3. The solution according to claim 1, characterized in that: The cryopreservation solution for mesenchymal stem cells was prepared with type 2 culture medium, DMSO, and human serum albumin in a ratio of 8:1:

1.

4. The solution according to claim 1, characterized in that: The method for preparing mesenchymal stem cell cryopreservation solution is as follows: Take the prepared type 2 culture medium pre-cooled at 2-8℃, gradually add DMSO, mix well, then add human serum albumin, mix well, and place at 2-8℃ for pre-cooling and use.

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