Complete medium for efficient culture and amplification of human umbilical cord mesenchymal stem cells and culture and amplification method thereof

Abstract

A complete culture medium for efficient culture and amplification of human umbilical cord mesenchymal stem cells is characterized by comprising the following components: a basic culture medium accounting for 85%-93% of the complete culture medium; the addition proportion of the UltraGRO accounts for 3%-6% of that of the complete culture medium; the addition proportion of the KnockoutTM SR accounts for 3%-6% of that of the complete culture medium; the addition proportion of the L-alanyl-L-glutamine accounts for 1%-3% of that of the complete culture medium. According to the efficient culture and amplification method for the human umbilical cord mesenchymal stem cells, the complete culture medium for efficient culture and amplification of the human umbilical cord mesenchymal stem cells is used. When the complete culture medium with the components and the proportion is used for efficiently culturing and amplifying the human umbilical cord mesenchymal stem cells, the cell morphology of the human umbilical cord mesenchymal stem cells is still not obviously changed until the cell morphology of a P10 generation, the amplification multiple is not obviously changed after being stably passed to the P10 generation, the human umbilical cord mesenchymal stem cells are stably amplified, and the HGF secretion amount is also obviously increased; obviously, the culture amplification effect is obviously improved.

Description

Technical Field

[0001] The present invention relates to the technical field of cell therapy, and in particular to a complete culture medium for efficiently culturing and expanding human umbilical cord mesenchymal stem cells and a culture and expansion method thereof. Background Art

[0002] The culture medium currently used in the market is generally composed of complete medium 1 and complete medium 2. Complete medium 1 consists of basal medium, UltraGRO and L-alanyl-L-glutamine, and complete medium 2 consists of basal medium, Knockout TM SR and L-alanyl-L-glutamine. When the complete medium 1 and complete medium 2 are used for the efficient culture and expansion of human umbilical cord mesenchymal stem cells, the cell morphology becomes narrow and elongated, the distribution is uneven, and local shedding occurs at the P7 generation. In addition, the expansion multiple drops sharply after the P5 generation, and the expansion is basically stopped. Therefore, the culture and expansion effect is not ideal. Summary of the Invention

[0003] The present invention aims to solve the technical problem of providing a complete culture medium and a culture and expansion method thereof that can improve the efficient culture and expansion effect of human umbilical cord mesenchymal stem cells.

[0004] In order to solve the above technical problems, the technical solution of the present invention is:

[0005] A complete culture medium for efficient culture and expansion of human umbilical cord mesenchymal stem cells, characterized by comprising the following components in the following proportions:

[0006] Basal culture medium, the addition ratio of which accounts for 85%-93% of the complete culture medium;

[0007] UltraGRO, added in a ratio of 3%-6% to complete culture medium;

[0008] Knockout TM SR, which is added in a ratio of 3%-6% of the complete medium;

[0009] L-alanyl-L-glutamine is added in a ratio of 1% to 3% of the complete culture medium.

[0010] M-the basic medium is added in a proportion of 90% of the complete medium; the UltraGRO is added in a proportion of 5% of the complete medium; the Knockout TM The addition ratio of SR is 4% of the complete medium; the addition ratio of L-alanyl-L-glutamine is 2% of the complete medium.

[0011] A method for efficiently culturing and expanding human umbilical cord mesenchymal stem cells uses the above-mentioned complete culture medium for efficiently culturing and expanding human umbilical cord mesenchymal stem cells.

[0012] The technical effect achieved by the present invention is as follows: the complete culture medium of the present invention comprises a basal culture medium whose addition ratio accounts for 85%-93% of the complete culture medium, an UltraGRO whose addition ratio accounts for 3%-6% of the complete culture medium, and a Knockout TM SR is added in a proportion of 3%-6% of the complete culture medium, and L-alanyl-L-glutamine is added in a proportion of 1%-3% of the complete culture medium. The complete culture medium with the above components and proportions is used to efficiently culture and expand human umbilical cord mesenchymal stem cells. The cell morphology remains unchanged until the P10 generation, and the expansion multiple remains stable until the P10 generation without significant change, indicating very stable expansion. In addition, the HGF secretion amount is also significantly increased. Obviously, the culture and expansion effect of the present invention is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments:

[0014] Figures 1-8 The cell morphology of human umbilical cord mesenchymal stem cells at P0-P7 was photographed in sequence using complete medium 1 and complete medium 2.

[0015] Figures 9-19 These are photographs of the cell morphology displayed sequentially from P0 to P10 when human umbilical cord mesenchymal stem cells were cultured and expanded using the complete culture medium of the present invention. DETAILED DESCRIPTION

[0016] The present invention will be further described in detail below with reference to the accompanying drawings.

[0017] See Figures 1 to 19 .

[0018] A complete culture medium for efficient culture and expansion of human umbilical cord mesenchymal stem cells, comprising the following components in the following proportions:

[0019] Basal culture medium, the addition ratio of which accounts for 85%-93% of the complete culture medium;

[0020] UltraGRO, added in a ratio of 3%-6% to complete culture medium;

[0021] Knockout TM SR, which is added in a ratio of 3%-6% of the complete medium;

[0022] L-alanyl-L-glutamine is added in a ratio of 1% to 3% of the complete culture medium.

[0023] As a preference, the addition ratio of the basal medium is 90% of the complete medium, the addition ratio of UltraGRO is 5% of the complete medium, and the addition ratio of Knockout is 10%. TM The addition ratio of SR is 4% of the complete medium, and the addition ratio of L-alanyl-L-glutamine is 2% of the complete medium.

[0024] A method for efficiently culturing and expanding human umbilical cord mesenchymal stem cells uses the above-mentioned complete medium for efficiently culturing and expanding human umbilical cord mesenchymal stem cells. The specific method is as follows (the complete medium used in this method uses the above-mentioned complete medium for efficiently culturing and expanding human umbilical cord mesenchymal stem cells):

[0025] 1. Preparation of umbilical cord mesenchymal stem cells

[0026] The specific isolation method of human umbilical cord mesenchymal stem cells used in the experiment is as follows:

[0027] a) After obtaining the guardian's authorization and consent, remove the newborn's umbilical cord under sterile conditions, soak it in normal saline, and temporarily store it at 4°C;

[0028] b) Transfer the umbilical cord to a sterile container and rinse thoroughly twice with normal saline containing 1% penicillin / streptomycin, each rinse lasting 2 minutes;

[0029] c) Cut the umbilical cord into small pieces approximately 2-3 cm long, cut the cord open along the inner side of the umbilical vein, and use toothed forceps to remove the umbilical artery, umbilical vein, and umbilical cord capsule;

[0030] d) Mince the umbilical cord tissue, mix thoroughly with complete culture medium, and inoculate into a T75 cell culture flask. Culture the flask in a humidified, 5% CO2, 37°C incubator.

[0031] 2. Replace the culture medium every 72 hours, observe the cell morphology and density under an inverted microscope, and subculture and expand the cells after the local confluence reaches more than 80%;

[0032] 3. Passaging and expansion of human umbilical cord mesenchymal stem cells

[0033] a) After the primary cultured cells grow to 80% local confluence, remove the original complete culture supernatant and gently rinse the adherent cells twice with 10 mL of normal saline;

[0034] b) Add 3 mL of TrpLE digestion buffer and place the culture flask in a 37°C incubator for 3-5 minutes.

[0035] c) Observe the cells under a microscope. When the cells begin to become round and brighter, and the intercellular spaces gradually increase, quickly add complete culture medium to terminate the digestion.

[0036] d) Pipette the cells repeatedly and transfer the cell suspension to a new 50 mL centrifuge tube. Centrifuge at room temperature.

[0037] e) After discarding the supernatant, add an appropriate amount of complete medium to resuspend the cells, passage at a seeding concentration of (0.6-1.6)*10⁶, transfer 10-15 mL of the cell suspension to a new T75 culture flask, and continue culturing in a constant temperature incubator;

[0038] f) When the cell confluence reaches about 80% again, repeat the above steps for subculture;

[0039] 4. Cryopreservation of human umbilical cord mesenchymal stem cells

[0040] a) Pre-cool the cell freezing box in a 4°C refrigerator for at least 30 minutes;

[0041] b) Digest the cells as above, centrifuge, discard the supernatant, resuspend the cells in freezing buffer, and transfer them to a 2 mL cryovial;

[0042] c) Place the cryovials in a cell freezing box and store at -80°C overnight. Then transfer to a liquid nitrogen tank for long-term freezing.

[0043] 5. Resuscitation of human umbilical cord mesenchymal stem cells

[0044] a) Quickly remove the frozen cells from the liquid nitrogen tank and shake them rapidly and continuously in a 37-42°C water bath to thaw them as quickly as possible;

[0045] b) Transfer the frozen cell suspension to a new centrifuge tube containing complete culture medium prepared in advance and centrifuge;

[0046] c) After discarding the supernatant, resuspend the cells in complete medium, pipette evenly, and transfer to a T75 culture flask for further culture.

[0047] The following is a method for cell preparation (the complete culture medium used in this method also uses the above-mentioned complete culture medium for efficient culture and expansion of human umbilical cord mesenchymal stem cells):

[0048] 6. Processing of human umbilical cord mesenchymal stem cells

[0049] a) UC-MSCs were seeded into T75 culture flasks at a density of (0.6-1.6)*106 cells / well and cultured for 16-24 hours;

[0050] b) Prepare complete culture medium containing different cytokines in advance and add one or more of the following cytokines, where the final concentrations of each factor are: HGF: 20-50 ng / mL, TGF-β inhibitor: 1-10 μM;

[0051] c) adding complete medium containing cytokines after 16-24 hours and culturing for 36-72 hours;

[0052] d) collecting cells, making preparations, and performing tests;

[0053] 7. Preparation of Cell Preparations

[0054] a) Collect cells, wash them twice with saline, and then count them;

[0055] b) Resuspend the cells in human serum albumin (5%-10%) + compound electrolyte injection to a volume of 1 x 107 cells per unit, and dispense 20-25 ml into a blood transfusion bag;

[0056] 8. Cell Detection

[0057] a) Flow cytometry detection of mesenchymal stem cell-specific positive and negative surface markers such as CD73+ / CD90+ / CD105+, CD14- / CD34- / CD45- / CD79α- / HLA-DR-;

[0058] b) Testing for sterility, endotoxin, mycoplasma, etc.;

[0059] c) Detection of cytokines, mainly factors related to immune regulation. In the present invention, HGF is detected using an ELISA kit.

[0060] Specific comparative experimental operations:

[0061] 1. Cell generation division rules

[0062] a) Cells obtained from tissue blocks and cultured in vitro are P0 cells (primary cells);

[0063] b) The cells obtained after subculture of primary cells are called P1 cells. After each subculture, the cell generation number increases by one, and so on.

[0064] 2. Process flow

[0065]

[0066] 3 Process Description

[0067] 1. Extraction and culture of primary cells

[0068] 1) Umbilical cord receipt: Complete umbilical cord information, including donor informed consent, health questionnaire, collection record, transportation record, and receipt record;

[0069] 2) The umbilical cord quality meets the requirements, including the following: length ≥15cm, uniform thickness, no clots, no pinholes;

[0070] 3) Cleaning and disinfection: soak in 75% alcohol for 60 seconds, then rinse three times with 30ml of normal saline;

[0071] 4) Remove Wharton's jelly and cut the umbilical cord into 2-3 cm segments. Remove the amniotic membrane, two arteries, and one vein to obtain Wharton's jelly. Record the volume of Wharton's jelly.

[0072] 5) Breaking: Manually cut Wharton's jelly into small pieces with a diameter of about 2-3 mm;

[0073] 6) Inoculation: Inoculate 1 ml of minced Wharton's jelly into T75 culture flasks, add 7 ml of complete culture medium, and culture in a CO2 incubator at 37°C, 5% CO2, and saturated humidity.

[0074] 7) Change the medium. Perform the first medium change after 7 days of culture. Tilt the culture flask slightly and slowly aspirate the original medium with a pipette. Then add 6 ml of fresh medium. Change the medium every 3 days. Before each medium change, observe under a microscope to see if there are any cells crawling out and take pictures.

[0075] II. Cell Harvest

[0076] a) Obtaining P0 cells

[0077] When the cell confluence around the tissue block reached about 90% under the microscope, the P0 cells were harvested.

[0078] The specific operations are as follows:

[0079] 1) Shake the bottle to remove Wharton's jelly from the bottom of the bottle (if it is difficult to remove, use a cell scraper to peel it off). Discard the culture medium and tissue pieces, and add 10 ml of normal saline to rinse the remaining culture medium three times.

[0080] 2) Add 1.5 ml of trypsin pre-warmed to 37°C and observe the cells under a microscope. When the cells appear oval and approximately 50% have fallen off the bottom of the flask, add 5 ml of culture medium to terminate the digestion.

[0081] 3) Pour the cell suspension into a centrifuge tube, add 10 ml of normal saline to the culture flask and wash it once. Collect the remaining cells into a centrifuge tube and centrifuge at 300 g, room temperature, for 5 minutes.

[0082] 4) After centrifugation, discard the supernatant and resuspend the cell suspension to 5 ml with culture medium. After filtering through a 100 μm filter, measure the cell count and viability.

[0083] III. Harvesting of P1-P10 Cells

[0084] When the cell confluence reached about 90% under microscope, the cells were harvested.

[0085] The specific operations are as follows:

[0086] 1) Discard the culture medium and wash the bottle once with 10 ml of normal saline;

[0087] 2) Add 1.5 ml of trypsin pre-warmed to 37°C and observe the cells under a microscope. When the cells appear oval and approximately 50% have fallen off the bottom of the flask, add 10 ml of culture medium to terminate the digestion.

[0088] 3) Pour the cell suspension into a centrifuge tube, wash the bottle once with 10 ml of normal saline, collect the remaining cells into a centrifuge tube, and centrifuge at 300g at room temperature for 5 minutes;

[0089] 4) Discard the supernatant, resuspend the cells to 5 ml with culture medium, and measure the cell count and viability;

[0090] IV: Vaccination

[0091] Inoculate T75 culture flasks at 1×106 / flask, add complete medium to 12 ml, and place in a CO2 incubator at 37°C, 5% CO2 concentration, and saturated humidity.

[0092] Five: Cryopreservation

[0093] When the cells were passaged to P2, P5, and P10, they were frozen for subsequent research. The specific procedures are as follows:

[0094] 1) Preparation of cryopreservation solution: Mix complete culture medium and DMSO in a 4:1 ratio and store at 4°C until use.

[0095] 2) Resuspend the cell pellet in cryopreservation buffer and adjust the volume to 0.7*107 / ml;

[0096] 3) Aliquot into cryovials (1.5 ml / vial), place in a programmed cooling box, and cool at -80°C for 24 hours;

[0097] 4) Transfer to liquid nitrogen for storage.

[0098] VI: Recovery

[0099] 1) Place the frozen cells in a 38°C water bath until the clots loosen;

[0100] 2) Quickly transfer to the operating table, collect the cell suspension, add 10 ml of complete medium, mix thoroughly, and centrifuge at 300g for 5 minutes at room temperature. Repeat twice.

[0101] 3) Discard the supernatant and collect the cell pellet for the next step.

[0102] 7. Cell counting method

[0103] 1) Take 20 μl of sample and mix with an equal amount of trypan blue staining solution;

[0104] 2) Add 10 μl of the mixture to a cell counting chamber at a constant speed and let it sit for 60 seconds until the sample covers the entire chamber.

[0105] 3) Insert the counting slide into the counting instrument slot and start counting after the display becomes clear. Record the number of viable cells and cell viability and save the counting image.

[0106] 4) Repeat the counting twice, and the deviation of the results shall not exceed 10%;

[0107] 5) Calculation of cell number: measurement result * dilution volume.

[0108] 8. Collection and preservation of experimental data

[0109] 1) Data encoding rules:

[0110] a) Culture photo: umbilical cord number + passage + group + sequence number;

[0111] b) Test data: umbilical cord number + generation + grouping + serial number;

[0112] 2) Storage path: D drive - experiment - culture system - image / data

[0113] 3) Experimental data should be regularly stored in a dedicated hard disk for archiving

[0114] 9. Testing items and methods

[0115] Cell passage: P1-P10

[0116] The specific contents are as follows:

[0117]

[0118] 10. Treatment of remaining cells

[0119] The remaining cells were discarded.

[0120] The present invention obtains the following experimental data through the above experimental operation:

[0121] 1: The cell morphology of human umbilical cord mesenchymal stem cells cultured and expanded using complete medium 1 and complete medium 2, P0-P7 (reference Figures 1 to 8 );

[0122] The cell morphology of human umbilical cord mesenchymal stem cells cultured and expanded using the complete medium of the present invention is shown in the following images (refer to Figures 9-19 ).

[0123] 2: Comparative experimental data:

[0124] The experimental data of the existing complete culture medium are as follows:

[0125]

[0126] The experimental data of the complete culture medium of the present invention are as follows:

[0127]

[0128] The comparison of the above experiments shows that the complete medium of the present invention significantly improves the culture expansion effect and significantly increases the secretion of HGF. As the most preferred, the basal medium of the present invention accounts for 93% of the complete medium, the UltraGRO accounts for 3% of the complete medium, and the Knockout TM The addition ratio of SR is 3% of the complete culture medium, and the addition ratio of L-alanyl-L-glutamine is 1% of the complete culture medium. This ratio has low cost and significant effect.

Claims

1. A complete medium for efficient culture and expansion of human umbilical cord mesenchymal stem cells, characterized in that: Including the following proportions: Basal culture medium, the addition ratio of which accounts for 85%-93% of the complete culture medium; UltraGRO, added in a ratio of 3%-6% to complete culture medium; Knockout TM SR, which is added in a ratio of 3%-6% of the complete medium; L-alanyl-L-glutamine is added in a ratio of 1% to 3% of the complete culture medium.

2. A complete medium for efficient culture and expansion of human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The addition ratio of the basic culture medium is 90% of the complete culture medium; the addition ratio of the UltraGRO is 5% of the complete culture medium; the addition ratio of the Knockout TM The addition ratio of SR is 4% of the complete medium; the addition ratio of L-alanyl-L-glutamine is 2% of the complete medium.

3. A method for efficiently culturing and expanding human umbilical cord mesenchymal stem cells, characterized by: The complete culture medium for efficiently culturing and expanding human umbilical cord mesenchymal stem cells as described in claim 1 is used.

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