Preparation method of clinical-grade umbilical cord mesenchymal stem cell bank

Abstract

The invention discloses a preparation method of a clinical-grade umbilical cord mesenchymal stem cell bank. According to the preparation method, the necessity, safety and rationality of raw material use are fully considered in the preparation process, and the risks of exogenous factor pollution or immunogenicity and the like introduced by the raw materials are reduced to the maximum extent; during cell culture, passage and cryopreservation, a complete medium composed of a serum-free medium, a serum substitute and a glutamine additive is used. The clinical-grade umbilical cord mesenchymal stem cell bank is obtained by implementing quality control of the whole process of umbilical cord collection, transportation, separation and preparation, cell culture and cryopreservation and the like. The method has an important application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for preparing a clinical-grade umbilical cord mesenchymal stem cell bank. Background Art

[0002] There are many indications for umbilical cord mesenchymal stem cell transplantation, including more than a hundred common diseases, rare diseases, and refractory diseases, such as traumatic diseases including craniocerebral injury, spinal cord injury, etc.; ischemic diseases including acute myocardial infarction, myocardial ischemia, etc.; inflammatory diseases including local or systemic chronic inflammation, immune rejection after organ transplantation, acute inflammatory syndrome, etc.; autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, etc., as well as metabolic diseases, tissue defects, and so on. Therefore, umbilical cord mesenchymal stem cells have a broad market prospect, and the treatment research based on the above diseases will also be an important direction for future research.

[0003] Umbilical cord mesenchymal stem cells are derived from the umbilical cord of newborns and have the following characteristics: the umbilical cord is a medical waste, with rich sources and low cost, and will not cause new trauma to the donor; no tumorigenic activity and low immunogenicity; short in vitro doubling time and strong amplification ability.

[0004] At present, there are still some drawbacks in the method for preparing umbilical cord mesenchymal stem cell banks. For example, enzymes are introduced in the separation and preparation process, which not only causes damage to cells, but also the enzymes of animal origin introduce the risk of exogenous factor contamination; in the traditional methods for culturing mesenchymal stem cells, serum (bovine origin) and trypsin (porcine origin) are used. This animal-derived culture system increases the risk of users being infected with animal-derived viruses, the detection items are incomplete, and the quality control process is not rigorous enough, resulting in the final cells being unable to be used clinically and only being used for scientific research. Therefore, more and more researchers believe that using an animal-free culture system to culture mesenchymal stem cells has more clinical value. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing a clinical-grade umbilical cord mesenchymal stem cell bank, which not only ensures the quality of cells, but also ensures the safety of clinical applications.

[0006] The present invention first protects a culture medium. The culture medium may include a serum-free medium, a serum substitute, and a glutamine additive.

[0007] Preferably, the culture medium may specifically consist of a serum-free medium, a serum substitute, and a glutamine additive.

[0008] In any of the above-mentioned culture media, the volume ratio of the serum-free medium to the serum substitute may be (48 - 52):1 (such as (48 - 50):1, (50 - 52):1, 48:1, 50:1, or 52:1).

[0009] In any of the above-mentioned culture media, the amount of the glutamine additive can be 0.5 - 1.5% (such as 0.5 - 1.0%, 1.0 - 1.5%, 0.5%, 1.0% or 1.5%) by volume based on the total volume of the culture medium, that is, the glutamine additive is added in a ratio of 0.5 - 1.5% (such as 0.5 - 1.0%, 1.0 - 1.5%, 0.5%, 1.0% or 1.5%) by volume ratio. The glutamine additive needs to be added when the culture medium is used.

[0010] The present invention also protects a method for preparing any of the above-mentioned culture media, which may include the following steps:

[0011] (a1) Mix the serum-free culture medium and the serum substitute in a ratio of (48 - 52):1 (such as (48 - 50):1, (50 - 52):1, 48:1, 50:1 or 52:1) to obtain a mixture;

[0012] (a2) Add the glutamine additive to the mixture obtained in step (a1) to obtain the culture medium; the glutamine additive is added in a ratio of 0.5 - 1.5% (such as 0.5 - 1.0%, 1.0 - 1.5%, 0.5%, 1.0% or 1.5%) by volume based on the total volume of the culture medium. That is, the glutamine additive is added in a ratio of 0.5 - 1.5% (such as 0.5 - 1.0%, 1.0 - 1.5%, 0.5%, 1.0% or 1.5%) by volume ratio.

[0013] In the above (a2), the glutamine additive needs to be added when the culture medium is used.

[0014] In the above preparation method, after completing step (a1) and before performing step (a2), the following steps may also be included: filtering the mixture by suction and then storing it.

[0015] The application of any of the above-mentioned culture media in the culture, passage and / or cryopreservation of umbilical cord mesenchymal stem cells also belongs to the protection scope of the present invention.

[0016] In the above application, the umbilical cord mesenchymal stem cells may be a clinical-grade umbilical cord mesenchymal stem cell primary cell bank or a clinical-grade umbilical cord mesenchymal stem cell working cell bank.

[0017] The application of any of the above-mentioned culture media in the preparation of a clinical-grade umbilical cord mesenchymal stem cell primary cell bank and / or a clinical-grade umbilical cord mesenchymal stem cell working cell bank.

[0018] Any of the above-mentioned clinical-grade umbilical cord mesenchymal stem cell primary cell banks can be prepared according to the following method.

[0019] Any of the above-mentioned clinical-grade umbilical cord mesenchymal stem cell working cell banks can be prepared according to the following method.

[0020] The present invention also protects a method for preparing a clinical-grade umbilical cord mesenchymal stem cell bank, which may include step (1): preparing a primary cell bank of clinical-grade umbilical cord mesenchymal stem cells; the step (1) may sequentially include the following steps:

[0021] (1-1) Add any of the above-mentioned culture media to Wharton's jelly, culture to obtain P0 cells;

[0022] (1-2) Digest and collect the P0 cells, after centrifugation, resuspend the precipitate in any of the above-mentioned culture media, inoculate the cell suspension, and culture to obtain P1 cells;

[0023] (1-3) Digest and collect the P1 cells, after centrifugation, resuspend the precipitate in any of the above-mentioned culture media, inoculate the cell suspension, and culture to obtain P2 cells;

[0024] (1-4) Digest and collect the P2 cells, after centrifugation, resuspend the precipitate in the cryopreservation solution and store it to obtain a primary cell bank of clinical-grade umbilical cord mesenchymal stem cells;

[0025] The primary cell bank of clinical-grade umbilical cord mesenchymal stem cells is the clinical-grade umbilical cord mesenchymal stem cell bank.

[0026] In the above method, in the step (1-1), the Wharton's jelly can be isolated from the excised umbilical cord. The number of days of culture can be 14-18 days (such as 14-16 days, 16-18 days, 14 days, 15 days, 16 days, 17 days or 18 days), the first medium change is carried out after 6-8 days of culture, and then the medium is changed every 3-4 days; when the number of cell clusters > 5, P0 cells are obtained.

[0027] Any of the above-mentioned methods may further include step (2): after completing step (1), preparing a clinical-grade umbilical cord mesenchymal stem cell working cell bank; the step (2) may sequentially include the following steps:

[0028] (2-1) Resuscitate the primary cell bank of clinical-grade umbilical cord mesenchymal stem cells, then add any of the above-mentioned culture media, inoculate the cell suspension, and culture to obtain P3 cells;

[0029] (2-2) Digest and collect the P3 cells, after centrifugation, resuspend the precipitate in any of the above-mentioned culture media, inoculate the cell suspension, and culture to obtain P4 cells;

[0030] (2-3) Digest and collect the P4 cells, after centrifugation, resuspend the precipitate in the cryopreservation solution and store it to obtain a clinical-grade umbilical cord mesenchymal stem cell working cell bank;

[0031] The working cell bank of clinical-grade umbilical cord mesenchymal stem cells is the clinical-grade umbilical cord mesenchymal stem cell bank.

[0032] In any of the above methods, in the steps (1-1), (1-2), (1-3), (2-1) or (2-2), the cultivation can be carried out at 37±1°C and 5±0.5% CO2.

[0033] In any of the above methods, in the steps (1-2), (1-3), (2-1) or (2-2), the seeding concentration can be 8000-15000 cells / cm 2 (such as 8000-12000 cells / cm 2 , 12000-15000 cells / cm 2 , 8000 cells / cm 2 , 12000 cells / cm 2 or 15000 cells / cm 2 ).

[0034] In any of the above methods, in the steps (1-2), (1-3), (2-1) or (2-2), the cultivation can be carried out until the cell confluence rate reaches 80%-90% (such as 80%-85%, 85%-90%, 80%, 85% or 90%).

[0035] In any of the above methods, in the steps (1-2), (1-3), (1-4), (2-2) or (2-3), the centrifugation parameters can be 200-400g (such as 200-300g, 300-400g, 200g, 300g or 400g), 5-10 min (such as 5-8 min, 8-10 min, 5 min, 8 min or 10 min).

[0036] In any of the above methods, the cryopreservation solution can specifically be composed of 1 volume part of dimethyl sulfoxide and 9 volume parts of any of the above-mentioned culture media.

[0037] The preparation method of the clinical-grade umbilical cord mesenchymal stem cell bank established by the present invention fully considers the necessity, safety and rationality of the use of raw materials during the production process, and maximally reduces the risks of exogenous factor contamination or immunogenicity introduced by raw materials. The specific manifestations are as follows: 1. In the umbilical cord separation and preparation process, collagenase is not introduced, and mechanical crushing of the umbilical cord is not required, reducing the risk of introducing exogenous factors and avoiding damage to cells caused by mechanical shear force, thus ensuring the quality of the obtained cells; 2. During the umbilical cord collection and preservation process, antibiotics are not used, avoiding the allergy risk caused by antibiotic residues and improving the safety of cell clinical application; 3. When culturing, subculturing and cryopreserving cells, complete medium is used, which has the following advantages compared with serum-containing medium: (1) avoiding the safety risks that may be caused by animal-derived materials; (2) improving cell yield; (3) obtaining more stable cell quality; 4. When cryopreserving, the cryopreservation solution used is composed of 9 parts by volume of complete medium and 1 part by volume of DMSO, and the DMSO is of pharmaceutical grade, meeting the requirements of the quality standard of the Chinese Pharmacopoeia. The present invention adopts the method of two-level cell banks. Among them, P2 cells are selected for the primary cell bank, and P4 cells are selected for the working cell bank, which not only meets the production output requirements but also ensures the use of cells with as low a passage number as possible to guarantee the cell quality. The present invention conducts quality control on the whole process from umbilical cord collection, transportation, separation and preparation, cell culture and cryopreservation, meets the requirements of "Preparation and Quality Control of Animal Cell Substrates for the Production and Verification of Biological Products" in Part III of the Chinese Pharmacopoeia, and obtains a clinical-grade umbilical cord mesenchymal stem cell bank. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a schematic flow chart for preparing the primary cell bank of clinical-grade umbilical cord mesenchymal stem cells.

[0039] Figure 2 It is a schematic flow chart for preparing the working cell bank of clinical-grade umbilical cord mesenchymal stem cells.

[0040] Figure 3 It is the cell morphology of P2 cells, P4 cells and P10 cells in Example 2.4. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] The following further describes the present invention in detail in combination with the specific embodiments. The given embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0042] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0043] The detection items in the following examples refer to the "Technical Guidelines for Pharmaceutical Research and Evaluation of Human Stem Cell Products (Trial)", the third part of the "Chinese Pharmacopoeia 2020" "Preparation and Quality Control of Animal Cell Substrates for the Production and Verification of Biological Products", the "Technical Guidelines for Research and Evaluation of Cell Therapy Products (Trial)" and the "Guidelines for Quality Control and Preclinical Research of Stem Cell Preparations (Trial)". The relevant detection methods refer to the "Chinese Pharmacopoeia 2020" and the relevant detection item kits. The staining solutions, antibodies, kits, etc. used can be obtained from commercial sources.

[0044] The complete medium is obtained by mixing a serum-free medium (Lonza Group Ltd, product number BEBP12-725F), a serum substitute (Pall Corporation, product number 15950-017) and a glutamine additive (Thermo Fisher Scientific (China) Co., Ltd., product number 35050061); the volume ratio of the serum-free medium to the serum substitute is 50:1. After mixing the two, they are filtered with a suction flask and sub-packed for storage at 2-8°C. The glutamine additive is added at a ratio of 1% by volume when in use.

[0045] The serum-containing medium is obtained by mixing DMEM low-glucose medium (Gibco) and Australian special fetal bovine serum (Gibco); the volume ratio of DMEM low-glucose medium to Australian special fetal bovine serum is 10:1.

[0046] Example 1. Establishment of a method for a clinical-grade umbilical cord mesenchymal stem cell bank

[0047] The programmed cooling parameters in this embodiment are: Step 1: Wait at Chamber = 4.0°C until Sample = 5.0°C; Step 2: Hold at 4.0°C for 10.0 minutes; Step 3: Ramp at 1.0°C / min until Sample = -4.0°C; Step 4: Ramp at 25.0°C / min until Chamber = -40°C; Step 5: Ramp at 10.0°C / min until Chamber = -12.0°C; Step 6: Ramp at 1.0°C / min until Chamber = -40°C; Step 7: Ramp at 10.0°C / min until Chamber = -90°C; Step 8: End.

[0048] Through a large number of experiments, the inventors of the present invention have established a method for constructing a clinical-grade umbilical cord mesenchymal stem cell bank, which specifically includes the preparation of a primary cell bank of clinical-grade umbilical cord mesenchymal stem cells (hereinafter referred to as the primary cell bank) and a working cell bank of clinical-grade umbilical cord mesenchymal stem cells (hereinafter referred to as the working cell bank).

[0049] I. Preparation of the primary cell bank

[0050] Through a large number of experiments, the inventors of the present invention have prepared the primary cell bank. The schematic process diagram for preparing the primary cell bank is shown in Figure 1 .

[0051] The specific preparation steps are as follows:

[0052] 1. Screening of umbilical cord donors

[0053] Screen umbilical cord donors. The screening criteria for umbilical cord donors are as follows: The parents of the newborn have no genetic diseases and no history of infectious diseases; The maternal infectious disease test (the detection of pathogens in the infectious disease test must include HIV-1 / 2, HBV, HCV, syphilis) and the investigation of other general test conditions are all normal; The newborn has no congenital malformations and other congenital diseases; There are no complications in the mother and baby.

[0054] 2. Umbilical cord collection

[0055] After the umbilical cord is cut off from the newborn, the cut-off position is about 4 - 5 cm from the umbilical wheel of the newborn. Clamp the umbilical cord with a hemostatic forceps at about 2 - 3 cm from the placenta of the umbilical cord, tie the umbilical cord with a silk thread near the hemostatic forceps, and cut the umbilical cord with surgical scissors between the tied position and the hemostatic forceps; Clean and rinse the dirt such as vernix caseosa and amniotic fluid on the surface of the umbilical cord; After rinsing, vertically place the umbilical cord into an umbilical cord storage and transportation bottle filled with umbilical cord preservation solution (Shandong Sikejie Biotechnology Co., Ltd., product number: CR0013 - 500ML). At the same time, collect 5 ml of maternal peripheral blood for subsequent infectious disease testing.

[0056] 3. Umbilical Cord Transportation

[0057] Place the umbilical cord storage and transportation bottle containing the umbilical cord in a transportation box at 2 - 26°C for storage and transport it to the laboratory for acceptance inspection within 5 hours. A temperature recorder is used during transportation to record the temperature of the transportation box in real time.

[0058] 4. Umbilical Cord Acceptance

[0059] After arriving at the laboratory, it is necessary to check the temperature of the transportation box, attached documents, sample sealability, sample labels, etc.; at the same time, conduct human - derived virus detection on the maternal peripheral blood. The human - derived viruses to be detected include: human immunodeficiency virus type Ⅰ (HIV - 1), human hepatitis B virus (HBV), human hepatitis C virus (HCV), human cytomegalovirus (HCMV), human Epstein - Barr virus (EBV), human papillomavirus (HPV), human herpesvirus (HHV - 6). When performing umbilical cord separation, take the umbilical cord preservation solution for mycoplasma detection.

[0060] 5. Umbilical Cord Separation and Preparation

[0061] (1) Transfer the umbilical cord from the umbilical cord storage and transportation bottle to a culture dish, cut it into umbilical cord segments with a length of 4 - 8 cm, and wash with 75% (v / v) aqueous ethanol solution for 3 - 5 min.

[0062] (2) After completing step (1), wash the umbilical cord segments with PBS buffer for at least 3 times until the residual blood and dirt on the surface of the umbilical cord are removed.

[0063] (3) After completing step (2), use medical forceps to remove the amniotic membrane on the surface of the umbilical cord segments and 1 vein and 2 arteries inside the umbilical cord. The remaining substance is Wharton's jelly; cut the Wharton's jelly into pieces no larger than 2 mm 3 , estimate and record the volume of the collected Wharton's jelly pieces.

[0064] (4) After completing step (3), aliquot the cut Wharton's jelly into cell culture bottles (specifically T175 culture bottles) at a rate of 1 - 2 ml / bottle, add 30 ml of complete medium to each cell culture bottle, and culture at 37°C and 5% CO₂.

[0065] 6. Medium Replacement after Separation and Preparation

[0066] After culturing for 6 - 8 days in step 5 (4), perform the first medium change. Observe every 3 - 4 days after the first medium change for a total of 16 days (including 16 days): If the number of cell clusters > 5, record it as P0 cells and perform P0 passage operation; if the number of cell clusters ≤ 5, continue with the medium change operation and observe every 3 - 4 days; if the number of cell clusters ≤ 5 after 16 days (including 16 days) from separation and preparation, this batch of cells will be discarded.

[0067] The medium used for each medium change is complete medium.

[0068] 7. P0 Passage

[0069] (1) Before passage, take a photo to record the cell morphology of the P0 cells obtained in step 6.

[0070] (2) After completing step (1), take the cell culture flask, discard the medium, and then add no less than 20 ml of PBS buffer to each cell culture flask to wash the cell growth surface of the cell culture flask.

[0071] (3) After completing step (2), add 3 ml of recombinant trypsin (Thermo Fisher Scientific (China) Co., Ltd., product number 12605 - 010) to the cell culture flask, gently shake, and when visually observing that about 90% of the cells start to detach, collect the cell suspension into a storage bottle; add 10 ml of PBS buffer to each cell culture flask to wash the cell growth surface of the cell culture flask, and collect the washing solution into the storage bottle and combine it with the digested cell suspension; fully mix the cell suspension in the storage bottle to obtain cell suspension A.

[0072] (4) Take cell suspension A obtained in step (3), centrifuge at 200 g - 400 g for 5 min, and collect the precipitate; resuspend with PBS buffer to obtain cell suspension B. Take 1 ml of cell suspension B for cell counting and viability detection; take the remaining cell suspension B, centrifuge at 200 g - 400 g for 5 min, and collect precipitate A; according to the total number of live cells, resuspend precipitate A with an appropriate amount of complete medium and adjust the cell concentration to obtain cell suspension C with a concentration of 8000 - 15000 cells / cm 2 ².

[0073] (5) Take a cell culture flask, add cell suspension C, and culture at 37 °C and 5% CO₂ until the cell confluence rate reaches 80% - 90%, that is, P1 cells.

[0074] 8. P1 Passage

[0075] (1) Before passage, take a photo to record the cell morphology of the P1 cells obtained in step 7.

[0076] (2) After completing step (1), take the cell culture flask, discard the culture medium, and then add no less than 20 ml of PBS buffer to each cell culture flask to wash the cell growth surface of the cell culture flask.

[0077] (3) After completing step (2), add 3 ml of recombinant trypsin to the cell culture flask and gently shake. When visually observing that about 90% of the cells begin to detach, collect the cell suspension into a storage bottle; add 10 ml of PBS buffer to each cell culture flask to wash the cell growth surface of the cell culture flask, collect the washing solution into the storage bottle and combine it with the digested cell suspension; fully mix the cell suspension in the storage bottle to obtain cell suspension a.

[0078] (4) Take cell suspension a obtained in step (3), centrifuge at 200 g - 400 g for 5 min, and collect the precipitate; resuspend with PBS buffer to obtain cell suspension b. Take 1 ml of cell suspension b for cell counting and viability detection; take the remaining cell suspension b, centrifuge at 200 g - 400 g for 5 min, and collect precipitate B; according to the total number of live cells, resuspend precipitate B with an appropriate amount of complete culture medium and adjust the cell concentration to obtain cell suspension c with a concentration of 8000 - 15000 cells / cm 2 .

[0079] (5) Take a cell culture flask, add cell suspension c, and culture at 37 °C and 5% CO₂ until the cell confluence rate reaches 80% - 90%, that is, P2 cells.

[0080] 9. Cryopreservation of P2

[0081] (1) Before operation, take a photo to record the cell morphology of P2 cells obtained in step 8.

[0082] (2) After completing step (1), take the cell culture flask, discard the culture medium, and then add no less than 20 ml of PBS buffer to each cell culture flask to wash the cell growth surface of the cell culture flask.

[0083] (3) After completing step (2), add 3 ml of recombinant trypsin to the cell culture flask and gently shake. When visually observing that about 90% of the cells begin to detach, collect the cell suspension into a storage bottle; add 10 ml of PBS buffer to each cell culture flask to wash the cell growth surface of the cell culture flask, collect the washing solution into the storage bottle and combine it with the digested cell suspension; fully mix the cell suspension in the storage bottle to obtain cell suspension A.

[0084] (4) Take the cell suspension A obtained in step (3), centrifuge it at 200 g - 400 g for 5 min, and collect the precipitate; resuspend it with PBS buffer to obtain cell suspension B. Take 1 ml of cell suspension B for cell counting and viability detection; take the remaining cell suspension B, centrifuge it at 200 g - 400 g for 5 min, and collect precipitate C; according to the total number of viable cells, resuspend precipitate C with a cryopreservation solution (consisting of 9 parts by volume of complete medium and 1 part by volume of dimethyl sulfoxide) and adjust the cell concentration to obtain cell suspension C with a concentration of 4×10 6 cells / ml.

[0085] (5) Take cell suspension C, aliquot it with an electric pipettor, load 1 ml of cell suspension C into each cryopreservation tube, tighten the caps of the cryopreservation tubes, and perform programmed cooling to obtain P2 cryopreserved cells.

[0086] (6) Perform quality inspection on the P2 cryopreserved cells. If the P2 cryopreserved cells meet the standards in Table 1, then the P2 cryopreserved cells are qualified P2 cryopreserved cells.

[0087] The qualified P2 cryopreserved cells are the primary cell bank.

[0088] Table 1

[0089]

[0090]

[0091] II. Preparation of the working cell bank

[0092] The inventor of the present invention prepared a working cell bank through a large number of experiments. The schematic diagram of the preparation process of the working cell bank is shown in Figure 2 .

[0093] The specific preparation steps are as follows:

[0094] 1. P2 resuscitation

[0095] Quickly transfer the cryopreservation tube containing the qualified P2 cryopreserved cells to a water bath preheated to 37 ± 2°C, and shake continuously to quickly melt the P2 cryopreserved cells; then first add complete medium, and then transfer them together to a cell culture flask, and culture at 37°C and 5% CO2 until the cell confluence rate reaches 80% - 90%, that is, P3 cells.

[0096] 2. P3 passage

[0097] (1) Before passage, take a photo to record the cell morphology of the P3 cells obtained in step 1.

[0098] (2) After completing step (1), take the cell culture flask, discard the medium, and then add no less than 20 ml of PBS buffer to each cell culture flask to wash the cell growth surface of the cell culture flask.

[0099] (3) After completing step (2), add 3 ml of recombinant trypsin into the cell culture flask, gently shake it, and when visually observing that about 90% of the cells begin to detach, collect the cell suspension into the storage bottle; add 10 ml of PBS buffer into each cell culture flask, wash the cell growth surface of the cell culture flask, and collect the washing solution into the storage bottle to merge with the digested cell suspension; fully mix the cell suspension in the storage bottle to obtain cell suspension (A).

[0100] (4) Take the cell suspension (A) obtained in step (3), centrifuge it at 200 g - 400 g for 5 min, and collect the precipitate; resuspend it with PBS buffer to obtain cell suspension (B). Take 1 ml of cell suspension (B) for cell counting and viability detection; take the remaining cell suspension (B), centrifuge it at 200 g - 400 g for 5 min, and collect precipitate D; according to the total number of live cells, resuspend precipitate D with an appropriate amount of complete medium and adjust the cell concentration to obtain cell suspension (C) with a concentration of 8000 - 15000 cells / cm 2 .

[0101] (5) Take a cell culture flask, add cell suspension (C), and culture it at 37 °C and 5% CO₂ until the cell confluence rate reaches 80% - 90%, namely P4 cells.

[0102] 3. Cryopreservation of P4

[0103] (1) Before operation, take a photo to record the cell morphology of the P4 cells obtained in step 2.

[0104] (2) After completing step (1), take the cell culture flask, discard the medium, and then add no less than 20 ml of PBS buffer into each cell culture flask to wash the cell growth surface of the cell culture flask.

[0105] (3) After completing step (2), add 3 ml of recombinant trypsin into the cell culture flask, gently shake it, and when visually observing that about 90% of the cells begin to detach, collect the cell suspension into the storage bottle; add 10 ml of PBS buffer into each cell culture flask, wash the cell growth surface of the cell culture flask, and collect the washing solution into the storage bottle to merge with the digested cell suspension; fully mix the cell suspension in the storage bottle to obtain cell suspension H.

[0106] (4) Take the cell suspension H obtained in step (3), centrifuge it at 200 g - 400 g for 5 min, and collect the precipitate; resuspend it with PBS buffer to obtain cell suspension I. Take 1 ml of cell suspension I for cell counting and viability detection; take the remaining cell suspension I, centrifuge it at 200 g - 400 g for 5 min, and collect precipitate E. According to the total number of live cells, resuspend precipitate E with the cryopreservation solution (consisting of 9 parts by volume of complete medium and 1 part by volume of dimethyl sulfoxide) and adjust the cell concentration to obtain cell suspension J with a concentration of 4×10 6 cells / ml.

[0107] (5) Take cell suspension J, aliquot it with an electric pipettor, load 1 ml of cell suspension J into each cryopreservation tube, tighten the caps of the cryopreservation tubes, and perform programmed cooling to obtain cryopreserved P4 cells.

[0108] (6) Perform quality inspection on the cryopreserved P4 cells. If the cryopreserved P4 cells meet the standards in Table 2, then the cryopreserved P4 cells are qualified cryopreserved P4 cells.

[0109] The qualified cryopreserved P4 cells are the working cell bank.

[0110] Table 2

[0111]

[0112]

[0113] Example 2. Comparison of the preparation of clinical-grade umbilical cord mesenchymal stem cell banks and their detection results using complete medium and serum-containing medium respectively according to the method established in Example 1

[0114] 1. Take umbilical cord donors, and prepare the primary cell bank and the working cell bank using complete medium according to the method established in Example 1; the umbilical cord donors are Donor 1, Donor 2, or Donor 3. Among them, the quality inspection results of the primary cell bank are shown in Table 3, and the quality inspection results of the working cell bank are shown in Table 4.

[0115] Table 3

[0116]

[0117]

[0118] Note: The culture supernatant refers to the medium used for culturing cells after the resuscitation or subculture operation and before the next operation.

[0119] Table 4

[0120]

[0121]

[0122] Note: The culture supernatant refers to the culture medium used for culturing cells after the resuscitation or subculture operation and before the next operation.

[0123] 2. According to the method established in Example 1, replace the complete culture medium with the serum-containing culture medium, and the umbilical cord donors are Donor 1, Donor 2, or Donor 3. Keep other steps unchanged to prepare the primary cell bank and the working cell bank. Among them, the quality inspection results of the primary cell bank are shown in Table 5, and the quality inspection results of the working cell bank are shown in Table 6.

[0124] Table 5

[0125]

[0126]

[0127] Note: The culture supernatant refers to the culture medium used for culturing cells after the resuscitation or subculture operation and before the next operation.

[0128] Table 6

[0129]

[0130]

[0131] Note: The culture supernatant refers to the culture medium used for culturing cells after the resuscitation or subculture operation and before the next operation.

[0132] Compare the results in Table 5 with those in Table 3. The results show that all the detection indexes of the primary cell banks obtained with the two culture media are qualified and there is no obvious difference. Compared with the primary cell bank obtained with the complete culture medium, the cell viability of the primary cell bank obtained with the serum-containing culture medium after resuscitation decreases significantly compared with that before cryopreservation.

[0133] Compare the results in Table 6 and Table 4. The results show that all the indexes of the working cell banks obtained with the two culture media are qualified and there is no obvious difference. Compared with the working cell bank obtained with the complete culture medium, the decrease in the cell viability of the working cell bank obtained with the serum-containing culture medium after resuscitation is also more obvious compared with that before cryopreservation.

[0134] 3. Detect the yields of the P1 cells obtained in Step 1 and Step 2 respectively.

[0135] The detection results are shown in Table 7.

[0136] Table 7

[0137]

[0138]

[0139] As can be seen from Table 7, the number of P1 cells obtained by culturing in serum-containing medium is almost 1 / 2 of the number of P1 cells obtained by culturing in complete medium. Thus, it can be seen that culturing in serum-containing medium will reduce the yield of P1 cells.

[0140] 4. Photograph and record the morphologies of the P2 cells and P4 cells obtained in Steps 1 and 2 respectively. Continuously passage a part of the P4 cells to P10 cells according to the passage steps in Example 1, and photograph and record the cell morphologies.

[0141] The results of the cell morphologies are shown in Figure 3 . The results show that the cell morphologies obtained by culturing in both media are adherent cells and are in a relatively uniform spindle shape, growing in parallel arrangement or in a swirling pattern, both conforming to the morphology of human umbilical cord mesenchymal stem cells. The difference is that the cell morphology of the cells cultured in complete medium is more three-dimensional; in addition, for the P10 cells obtained by culturing in serum-containing medium and passaged to P10, the cell morphology becomes disordered, with more antennae, showing an aging state; while for the P10 cells obtained by culturing in complete medium and passaged to P10, the cell morphology does not significantly show an aging state.

[0142] As can be seen from Example 2 above, using complete medium for culturing can increase the cell yield, and the cell quality obtained may be more stable.

[0143] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. A culture medium, comprising a serum-free medium, a serum substitute and a glutamine additive.

2. The culture medium according to claim 1, wherein: In the said culture medium, the volume ratio of the serum-free medium to the serum substitute is (48 - 52):1; the amount of the glutamine additive is 0.5 - 1.5% by volume based on the total volume of the culture medium.

3. The method for preparing the culture medium according to claim 1 or 2, comprising the following steps: (a1) Mix the serum-free medium and the serum substitute in a ratio of (48 - 52):1 to obtain a mixture; (a2) Add the glutamine additive to the mixture obtained in step (a1) to obtain the said culture medium; the glutamine additive is added in a ratio of 0.5 - 1.5% by volume based on the total volume of the culture medium.

4. The application of the culture medium according to claim 1 or 2 is for A1) or A2): A1) For the culture, passage and / or cryopreservation of umbilical cord mesenchymal stem cells; A2) To prepare a clinical-grade umbilical cord mesenchymal stem cell primary cell bank and / or a clinical-grade umbilical cord mesenchymal stem cell working cell bank.

5. A method for preparing a clinical-grade umbilical cord mesenchymal stem cell bank, comprising step (1): preparing a clinical-grade umbilical cord mesenchymal stem cell primary cell bank; The said step (1) successively comprises the following steps: (1-1) Add the culture medium according to claim 1 or 2 to Wharton's jelly, culture to obtain P0 cells; (1-2) Digest and collect the said P0 cells, after centrifugation, resuspend the precipitate in the culture medium according to claim 1 or 2, inoculate the cell suspension, culture to obtain P1 cells; (1-3) Digest and collect the said P1 cells, after centrifugation, resuspend the precipitate in the culture medium according to claim 1 or 2, inoculate the cell suspension, culture to obtain P2 cells; (1-4) Digest and collect the said P2 cells, after centrifugation, resuspend the precipitate in the cryopreservation solution for storage to obtain a clinical-grade umbilical cord mesenchymal stem cell primary cell bank.

6. The method according to claim 5, wherein: In the said step (1-1), the Wharton's jelly is obtained by separating from an ex vivo umbilical cord; the number of days of culture is 14 - 18 days, the first medium change is carried out after 6 - 8 days of culture, and then the medium is changed every 3 - 4 days; when the number of cell clusters > 5, P0 cells are obtained.

7. The method according to claim 5, wherein: The said method further comprises step (2): after completing step (1), preparing a clinical-grade umbilical cord mesenchymal stem cell working cell bank; The said step (2) successively comprises the following steps: (2-1) Resuscitate the clinical-grade umbilical cord mesenchymal stem cell primary cell bank, then add the culture medium according to claim 1 or 2, inoculate the cell suspension, culture to obtain P3 cells; (2-2) Digest and collect the said P3 cells, after centrifugation, resuspend the precipitate in the culture medium according to claim 1 or 2, inoculate the cell suspension, culture to obtain P4 cells; (2-3) Digest and collect the said P4 cells, after centrifugation, resuspend the precipitate in the cryopreservation solution for storage to obtain a clinical-grade umbilical cord mesenchymal stem cell working cell bank.

8. According to the method according to any one of claims 5 to 7, characterized in that: In the steps (1-2), (1-3), (2-1) or (2-2), the inoculation concentration is 8,000 - 15,000 cells / cm 2 ; In the steps (1-2), (1-3), (2-1) or (2-2), the culturing is carried out until the cell confluence rate reaches 80%-90%.

9. The method according to any one of claims 5 to 7, characterized in that: In the steps (1-2), (1-3), (1-4), (2-2) or (2-3), the centrifugation parameters are 200-400 g for 5-10 min.

10. The method according to any one of claims 5 to 7, characterized in that: The cryopreservation solution is composed of 1 volume part of dimethyl sulfoxide and 9 volume parts of the culture medium according to claim 1 or 2.

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