Method and system for preparing clinical-grade human umbilical cord mesenchymal stem cells

Abstract

The invention relates to the field of cell therapy, in particular to a preparation method and system of clinical-grade human umbilical cord mesenchymal stem cells. The core preparation method comprises the following steps: dynamically screening raw materials according to the cell climbing condition in the 9th-11th day of culture; carrying out low-damage digestion by adopting a two-step method of combining low-temperature enzyme pre-incubation with physical shearing; amplification is carried out at the density of 8000 to 10000 pieces / cm; freezing the P3 generation cells according to the density of 5 * 10 < 6 > cells / mL to build a library; and process monitoring and decision making are carried out based on metabolic indexes such as the glutamine consumption rate. The invention also discloses a cell product which is prepared by the method and has excellent functions, and application of a pharmaceutical composition containing the product in treatment of graft versus host disease and other diseases. According to the scheme, standardization, datamation and intellectualization of cell production are realized, and uniform, stable and safe quality of a final product is ensured.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and cell therapy, and in particular to a method and system for preparing clinical-grade human umbilical cord mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) have shown great promise in regenerative medicine and the treatment of immune diseases due to their multi-lineage differentiation potential and immunomodulatory functions. Human umbilical cord-derived MSCs have advantages such as abundant source, painless collection, and strong proliferation capacity, making them ideal seed cells for cell therapy.

[0003] However, moving UC-MSCs from laboratory research to large-scale, standardized clinical application faces many challenges:

[0004] Unstable production processes: Traditional primary separation methods such as enzyme digestion are costly and cause significant cell damage, and the enzyme reagents used often do not meet clinical-grade production standards; while the tissue block method is gentler, it lacks objective raw material screening standards, resulting in large batch-to-batch variations.

[0005] Lagging quality control: Existing quality control relies heavily on the release inspection of the final product, such as viability and surface markers. It cannot predict and intervene in the cell state during the production process in the early stage. Once the inspection fails, all resources are wasted.

[0006] Cell damage and control: During passage digestion, the traditionally used trypsin and other enzymes, when used for extended periods at 37°C, can damage cell membrane proteins, affecting the cell's subsequent proliferative potential and function. The cryopreservation and thawing process can also lead to a decrease in cell viability.

[0007] Lack of functional evaluation: Existing standards mainly focus on cell identity and purity, but lack rapid and effective means of monitoring the functional potency that determines its therapeutic effect.

[0008] Therefore, there is an urgent need in this field to establish a complete industrial production system that integrates source control, process optimization, online monitoring and functional evaluation in order to prepare safe, effective and uniformly high-quality clinical-grade UC-MSCs. Summary of the Invention

[0009] The primary objective of this invention is to overcome the shortcomings of the prior art and provide a method and system for preparing clinical-grade human umbilical cord mesenchymal stem cells.

[0010] Another objective of this invention is to provide a quality control method based on metabolomics to enable early prediction and decision-making in the cell production process.

[0011] Another object of the present invention is to provide a low-damage cell digestion method.

[0012] Another object of the present invention is to provide a human umbilical cord mesenchymal stem cell product with specific functional indicators prepared by the said method, a production system comprising the product, and a pharmaceutical composition.

[0013] To achieve the above objectives, the present invention provides the following technical solution:

[0014] In a first aspect, the present invention provides a method for preparing clinical-grade human umbilical cord mesenchymal stem cells, characterized by comprising the following steps:

[0015] S1. Dynamic screening of raw materials: Primary mesenchymal stem cells are isolated from umbilical cord tissue using the tissue block adhesion method. If no mesenchymal stem cells are observed to emerge within 9 to 11 days of culture, the umbilical cord raw material is deemed unqualified and discarded.

[0016] S2. Modular low-damage digestion: For adherent cells, a two-step digestion method is used, including pre-incubation with low concentrations of non-animal-derived digestive enzymes at 2-8°C, discarding the digestive solution, and then applying fluid shear force to detach the cells.

[0017] S3. Precise density expansion: Digested cells are passaged and seeded at a density of 8,000 to 10,000 cells / cm².

[0018] S4. Functional library construction: Third-generation cells were cryopreserved using a chemically defined cryopreservation solution at a concentration of 5 × 10⁻⁶ cells / mL. 6 Cryopreservation was carried out at a density of cells / mL to establish a master cell bank;

[0019] S5. Process metabolic monitoring: After digestion in step S2 or after cryopreservation and thawing in step S4, metabolomics analysis is performed on the cell culture supernatant, and decisions are made on the production use of cell batches based on the levels of metabolic markers.

[0020] Preferably, in step S2, the two-step digestion method specifically involves: first, incubating the cells at 4°C for 10-15 minutes with a non-animal-derived recombinant trypsin substitute at a concentration of 0.2x-0.5x; then, discarding the digestive fluid; and finally, applying a fluid shear force of 50-200g to detach the cells.

[0021] In this embodiment, for example: the fluid shear force is achieved by gently blowing the bottom of the culture flask 10 times at a frequency of 1 time per second using a 10 mL pipette, or by placing the cell culture flask on a shaker platform and oscillating it at a speed of 100 rpm for 5 minutes.

[0022] Preferably, in step S5, the indicators of the metabolomics analysis include the lactate / glucose consumption ratio and / or the glutamine consumption rate; wherein, if the glutamine consumption rate is less than 30% within 24 hours after resuscitation, the batch of cells will not be used for expansion to produce clinical formulations.

[0023] Preferably, after step S1 and before step S3, a super seed bank construction step is also included: isolating and screening monoclonal cells with high proliferative potential and / or high immunosuppressive function from primary mesenchymal stem cells, and using the cells obtained therefrom to establish the master cell bank.

[0024] In a second aspect, the present invention provides a quality control method for evaluating the preparation process of mesenchymal stem cells, characterized in that, in the industrial preparation method described in the first aspect, the method includes:

[0025] After digestion in step S2 or after cryopreservation and thawing in step S4, cell culture supernatant is collected for metabolomics analysis.

[0026] Compare the measured value of at least one metabolic marker with a preset threshold;

[0027] Based on the comparison results, the subsequent expansion potential or functional output of this batch of cells is predicted, and a decision is made on whether to approve, downgrade, or eliminate the production use of this batch of cells.

[0028] Thirdly, the present invention provides a low-damage cell digestion method, characterized in that, in the industrial preparation method described in the first aspect, the method includes:

[0029] Pre-incubate cells at 2-8°C for 10-15 minutes using a non-animal-derived recombinant trypsin substitute at a concentration of 0.2x-0.5x.

[0030] Remove the digestive enzyme solution;

[0031] Cells are detached from the culture surface by applying a fluid shear force of 50-200g.

[0032] Fourthly, the present invention provides a human umbilical cord mesenchymal stem cell product, characterized in that the cell product is prepared by the method described in the first aspect, and within 24 hours after the human umbilical cord mesenchymal stem cell product is revived, the glutamine consumption rate in its culture supernatant is higher than 30%, and its immunosuppressive titer is not lower than 80% of the standard.

[0033] Fifthly, the present invention provides a human umbilical cord mesenchymal stem cell production system, characterized in that the system comprises:

[0034] A super seed bank, consisting of monoclonal mesenchymal stem cells screened and expanded according to the method described in the first aspect;

[0035] The master cell bank is established by expanding the cells of the super seed bank and then cryopreserving them at passage 3.

[0036] The process decision module is configured to execute the quality control method described in the second aspect;

[0037] The clinical formulation production unit is used to revive and passage cells from the master cell bank to the target passage, according to the instructions of the process decision module, in order to prepare clinical cell formulations.

[0038] In a sixth aspect, the present invention provides a pharmaceutical composition characterized in that it comprises the human umbilical cord mesenchymal stem cell product described in the fourth aspect and a pharmaceutically acceptable carrier.

[0039] In a seventh aspect, the present invention provides the use of the pharmaceutical composition described in the sixth aspect in the preparation of a medicament for treating graft-versus-host disease.

[0040] Compared with the prior art, the present invention has the following significant advantages:

[0041] By setting a clear time window for primary cell emergence from day 9 to day 11, early, rapid, and objective screening of umbilical cord raw materials was achieved, ensuring the uniformity of the quality of the starting materials for production from the source.

[0042] The two-step digestion method, which combines low-temperature pre-incubation and physical shearing, minimizes the chemical damage to cells caused by enzymes, effectively maintaining high cell viability, high cell adhesion, and strong subsequent proliferation potential after cell recovery.

[0043] By introducing metabolomics analysis into key nodes of the production process, a correlation model between cellular metabolic state and subsequent functional output was established, enabling a quality control approach that moves from post-production inspection to pre-production prediction and process decision-making, which greatly improves the production success rate.

[0044] By defining the final cell product through metabolic characteristics and functional potency, we ensure that the produced cells are not only correct but also possess the expected therapeutic function, providing a solid guarantee for clinical efficacy.

[0045] The constructed super seed bank-master cell bank-process decision-making-clinical formulation production system organically integrates various innovation links, forming a flexible, intelligent, and traceable closed-loop production system, providing a complete solution for the large-scale and standardized production of clinical-grade cell drugs.

[0046] The entire process uses non-animal-derived reagents with clearly defined chemical compositions that meet GMP standards (such as TrypLE™ Express digestive enzymes and CryoStor® CS10 cryopreservation solution), and has passed the inspection of the National Institutes for Food and Drug Control (NIFDC), which fully demonstrates the safety and stability of the cells prepared by this process and that they have direct clinical translational value. Attached Figure Description

[0047] Figure 1 These are comparative images of primary human mesenchymal stem cell migration in Example 1 of the present invention; where A represents a large number of cells that have migrated after 10 days of culture; B represents the cells in Image A that have reached 80% fusion after 3-5 days of subsequent culture; C represents the cells that have migrated without migration after 10 days of culture; and D represents the cells in Image C that have migrated without migration even after 15 days of subsequent culture.

[0048] Figure 2 This is a growth curve of cells at different inoculation densities in Example 2 of the present invention.

[0049] Figure 3 This is a growth curve of cells after different digestion times in Example 3 of the present invention.

[0050] Figures 4-8 This is a comprehensive inspection report from the China National Institutes for Food and Drug Control, confirming its compliance with regulations. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions in the art or as recommended by the manufacturer. Unless otherwise specified, all reagents and consumables used are commercially available products that meet clinical-grade or GMP standards. The following embodiments are merely illustrative and are not intended to limit the scope of protection of this invention.

[0052] Example 1: Separation and dynamic screening of primary UC-MSCs

[0053] This embodiment corresponds to step S1 in claim 1, and aims to establish raw material screening standards.

[0054] 1. Experimental Design:

[0055] The umbilical cords of healthy full-term cesarean section fetuses were collected and transported to the laboratory in 0.9% sodium chloride injection (Anhui Fengyuan Pharmaceutical Co., Ltd., National Drug Approval Number H34021876) at 2-8℃.

[0056] Under sterile conditions, the umbilical cord is cut into 3-4 cm segments. After cleaning the umbilical cord by pushing with forceps, it is bluntly peeled open and the veins, arteries, and outer skin are removed to separate Wharton's jelly.

[0057] Use sterile scissors to cut the tissue into pieces of approximately 1 mm³. Use a Pasteur pipette (Nest, 318314) to aspirate 0.5–1 mL of the tissue piece and evenly seed it into the bottom of a T75 cell culture flask (Thermo, 156499).

[0058] Add an appropriate amount of complete culture medium (α-MEM serum-free medium (Gibco, C12571500BT) supplemented with UltraGRO-Advanced (Helios, HPCFDCCL50)), and transfer the cell culture flask to a 37°C, 5% CO2 incubator. Change the culture medium every 5 days.

[0059] 2. Results and Analysis:

[0060] The results showed that a large number of primary mesenchymal stem cells could be observed crawling out of most umbilical cords during the second medium change (i.e., day 10 of culture). Figure 1 A), after 3-5 days of subsequent culture, cell confluence can reach approximately 80%. Figure 1 B). A small number of umbilical cords showed no cell migration by day 10 of culture ( Figure 1 C), continued culturing until day 15 (third medium change) still resulted in only a small number or no cells crawling out. Figure 1 D). Based on this, it is determined that... Figure 1 The umbilical cord materials shown in C and D are substandard and are discarded. This embodiment establishes a dynamic screening standard for raw materials with "day 9 to 11" (preferably day 10) as the key time node.

[0061] Example 2: Optimization of cell passaging seeding density

[0062] This embodiment corresponds to step S3 in claim 1, and aims to determine the optimal inoculation density for passage.

[0063] 1. Experimental Design:

[0064] P4 generation clinical-grade umbilical cord mesenchymal stem cells were digested and cell counting was performed.

[0065] According to different inoculation densities (A. 0.2250 × 10 4 Pieces / cm², B.0.4500×10 4 Pieces / cm², C.0.9000×10 4 Pieces / cm², D. 1.8 × 10 4 pcs / cm², E.3.6×10 4 Inoculate 10 cells / cm² into a 96-well plate (Corning, 3599).

[0066] OD values ​​of each well were measured using a CCK-8 assay kit (MedChemExpress, HY-K0301) 1-6 days after inoculation, and cell growth curves were plotted to assess cell proliferation capacity.

[0067] 2. Results and Analysis:

[0068] like Figure 2 As shown, the inoculation density was for group C (0.9000 × 10⁻⁶). 4 Cells with a density of 9000 cells / cm² (i.e., 9000 cells / cm²) exhibit the most ideal growth curve, with a short latency period and a relatively long logarithmic growth phase. Cells in groups A and B proliferate slowly; cells in groups D and E directly enter the logarithmic growth phase with a short plateau phase. Therefore, an optimal seeding density range of 8000 to 10000 cells / cm² was determined, with 9000 cells / cm² being the most suitable density.

[0069] Example 3: Optimization of cell digestion time

[0070] This embodiment corresponds to step S2 in claim 1 and the digestion method of the third aspect, and aims to determine the optimal time for low-damage digestion.

[0071] 1. Experimental Design:

[0072] P4 generation umbilical cord mesenchymal stem cells were digested using TrypLE™ Express (Gibco, catalog number: 12604021) at different digestion times (1 min, 3 min, 5 min, 7 min), and cell viability was assessed by counting. The recombinant trypsin substitute used was TrypLE™ Express (catalog number: 12604021) from Gibco.

[0073] All groups of cells were seeded into 96-well plates at a density of 9000 cells / cm².

[0074] Cell growth curves were plotted using the CCK-8 assay 1-6 days after inoculation.

[0075] 2. Results and Analysis:

[0076] As shown in Table 1, there was no significant difference in immediate cell viability at different digestion times (all >97%). However, as Figure 3 As shown, there were significant differences in their subsequent proliferation capacity. Cells digested for 1 min and 3 min exhibited the best proliferation capacity; cells digested for 5 min and 7 min showed significantly flattened growth curves, indicating severely impaired proliferation capacity. Therefore, the optimal digestion time was determined to be 1 minute, and should not exceed 3 minutes. This verifies the low-damage advantage of low-temperature short-time digestion combined with physical shedding.

[0077] Table 1. Cell number and viability at different digestion times

[0078]

[0079] Example 4: Optimization of cell cryopreservation solution and cryopreservation density

[0080] This embodiment corresponds to step S4 in claim 1, and aims to determine the optimal cryopreservation solution and cryopreservation density.

[0081] For example, the cryopreservation solution could be STEMCELL Technologies' CryoStor® CS10 (part number: 07930).

[0082] 1. Experimental Design:

[0083] Digested P4 generation clinical-grade umbilical cord mesenchymal stem cells, counted, and then divided into different densities (A: 1×10⁻⁶). 6 cells / mL, B: 5×10 6 Cells / mL, C: 1×10 7 The cells (per mL) were resuspended in StemCell cryopreservation buffer (CryoStor® CS10, STEMCELL, 07930) and Sansheng cryopreservation buffer (Nanjing Sansheng, YB090050) respectively, and then aliquoted into cryovials (Corning, 430488).

[0084] Cells were cryopreserved using a programmed cooling method and thawed on day 30 and day 90 after cryopreservation, respectively.

[0085] Cell viability was assessed after resuscitation, and cell adhesion rate was assessed 8 hours after plating (density: 9000 cells / cm²).

[0086] 2. Results and Analysis:

[0087] The results are shown in Tables 2 and 3. At the same cryopreservation density, cells cryopreserved using StemCell cryopreservation medium (CryoStor® CS10) showed significantly higher viability and adhesion rates at 30 and 90 days post-thaw compared to or equivalent to those using Sansheng cryopreservation medium, with less degradation over time. Considering both stability and ease of operation, StemCell cryopreservation medium with a clearly defined chemical composition was selected, and the density was increased to 5 × 10⁻⁶ cells / day. 6 P3 generation cells were constructed and cryopreserved at a density of cells / mL.

[0088] Table 2. Cell viability and adhesion rate after 30 days of cryopreservation in different cryopreservation solutions.

[0089] Cryopreservation density StemCell cryopreservation solution Sansheng cryopreservation solution

[0090]

[0091] Table 3. Cell viability and adhesion rate after 90 days of cryopreservation in different cryopreservation solutions.

[0092]

[0093] Example 5: Metabolomics-based process monitoring and product functionality definition

[0094] This embodiment corresponds to step S5 in claim 1, the quality control method of the second aspect, and the product definition of the fourth aspect.

[0095] 1. Experimental Design:

[0096] After the digestion step (S2) described in Example 3, and after the P3 generation cells frozen in Example 4 were thawed, cell culture supernatant was collected within 24 hours.

[0097] Untargeted metabolomics analysis was performed using liquid chromatography-mass spectrometry (LC-MS), with a focus on energy metabolism-related pathways.

[0098] The detected metabolites were correlated with subsequent cell expansion folds and immunosuppressive function (immunosuppressive titer was assessed by detecting the proportion of Treg cells induced or the IFN-γ inhibition rate through co-culture with PBMCs).

[0099] 2. Results and Analysis:

[0100] The study found a strong positive correlation between glutamine consumption rate and subsequent cell proliferation potential and immunosuppressive function within 24 hours post-resuscitation. When the glutamine consumption rate was below 30%, subsequent cell proliferation was slow, and the immunosuppressive titer was below 80% of the standard. Conversely, when the glutamine consumption rate was above 30%, the cells exhibited robust proliferative capacity and high immunosuppressive titer (not lower than 80% of the standard). This finding establishes a glutamine consumption rate >30% within 24 hours post-resuscitation as a decision threshold for process monitoring and a critical quality attribute (CQA) for cell products.

[0101] The 'standard product' mentioned in this invention is a fifth-generation human umbilical cord mesenchymal stem cell reference product that has passed the inspection and certification of the China National Institutes for Food and Drug Control, or a batch of cells from an internal master cell bank with known high immunosuppressive activity.

[0102] Example 6: Complete Production Process and Inspection by the National Institutes for Food and Drug Control

[0103] Integrating the optimized parameters of all the above embodiments, the complete preparation process is executed as follows: selecting qualified umbilical cords (S1) → primary culture → constructing a super seed bank (after S1) → employing a two-step low-damage digestion method (S2) → passage expansion at a density of 9000 cells / cm² (S3) → increasing P3 generation cells at a density of 5×10⁻⁶ cells / cm² 6Establish a master cell bank using CryoStor® CS10 cryopreservation at a density of cells / mL (S4) → Perform metabolic monitoring at key points (S5) → Revive P3 generation cells and expand to P5 generation to prepare clinical formulations.

[0104] The P5 generation clinical-grade human umbilical cord mesenchymal stem cells prepared using this process have passed comprehensive testing by the China National Institutes for Food and Drug Control and have obtained a test certificate. See details below. Figures 4-8 As shown, the results demonstrate that all indicators of the cells (including cell viability, purity, sterility, endotoxin content, immunophenotype, and function) meet clinical drug standards. This fully verifies the reliability, stability, and safety of the industrial-scale preparation process provided by this invention.

[0105] Example 7: Validation of the immunosuppressive function and pharmaceutical applications of human umbilical cord mesenchymal stem cell products

[0106] 1. Experimental Design:

[0107] To verify the immunosuppressive function of the P5 generation human umbilical cord mesenchymal stem cells (prepared according to Example 6) prepared by the method of the present invention, we conducted an in vitro lymphocyte proliferation inhibition experiment.

[0108] Experimental group: UC-MSCs in different ratios (e.g., 1:1, 1:5, 1:10) were co-cultured with human peripheral blood mononuclear cells (PBMCs) activated by phytohemagglutinin (PHA).

[0109] Control group: Contains only activated PBMCs, without the addition of UC-MSCs.

[0110] Detection method: After 72 hours of culture, the proliferation of PBMCs was detected by the CCK-8 assay, and the inhibition rate of UC-MSCs on lymphocyte proliferation was calculated.

[0111] 2. Results and Analysis:

[0112] The results, as shown in Table 4, demonstrate that the UC-MSCs prepared in this invention significantly inhibited lymphocyte proliferation in a dose-dependent manner. At a ratio of 1:5, the inhibition rate reached over 80%. These results fully demonstrate that the cell products of this invention possess potent immunosuppressive function, providing direct experimental evidence for their potential use in treating graft-versus-host disease (GvHD), rheumatoid arthritis, and other diseases characterized by excessive immune activation.

[0113] Inhibition rate of UC-MSCs on lymphocyte proliferation

[0114]

[0115] 2. Expanding the range of potential pharmaceutical applications

[0116] Based on the core functional data, its scope of application can be reasonably deduced.

[0117] 3. Description of potential pharmaceutical uses:

[0118] Based on the high immunosuppressive titer of UC-MSCs demonstrated in this invention, as well as their ability to secrete a variety of nutritional factors and antifibrotic factors, and confirmed by detecting factors such as HGF, PGE2, and TGF-β in their supernatant using an ELISA kit, this cell product is expected to be used not only for the treatment of graft-versus-host disease (GvHD), but also for the prevention or treatment of other diseases requiring immune regulation and tissue repair.

[0119] Including but not limited to:

[0120] Autoimmune diseases: such as systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, multiple sclerosis, autoimmune liver disease, etc.

[0121] Inflammatory diseases such as ulcerative colitis, Crohn's disease, acute pancreatitis, and acute respiratory distress syndrome (ARDS).

[0122] Tissue damage and repair: such as cardiac repair after myocardial infarction, nerve repair after ischemic stroke, reversal of liver fibrosis / cirrhosis, and healing of diabetic foot ulcers.

[0123] As a vector for gene therapy: it can be used to carry therapeutic genes for the treatment of certain hereditary diseases or cancer.

[0124] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0125] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing clinical-grade human umbilical cord mesenchymal stem cells, characterized in that, Includes the following steps: S1. Primary mesenchymal stem cells are isolated from umbilical cord tissue using the tissue block adhesion method. If no mesenchymal stem cells are observed to emerge within the predetermined culture time, the umbilical cord raw material is deemed unqualified and discarded. S2. For adherent cells, a two-step digestion method is used, including pre-incubation with low concentrations of non-animal-derived digestive enzymes, and then detachment of the cells by applying fluid shear force after discarding the digestive solution. S3. Passage the digested cells at a predetermined seeding density; S4. Cryopreserve the third-generation cells using cryopreservation solutions with clearly defined chemical compositions to establish a master cell bank; S5. After the cell digestion step and / or after cell cryopreservation and thawing, perform metabolomics analysis on the cell culture supernatant and make a decision on the production use of the cell batch based on the levels of metabolic markers.

2. The method according to claim 1, characterized in that, In step S2, the two-step digestion process specifically involves: First, incubate the cells at 4°C for 10-15 minutes with a non-animal-derived recombinant trypsin substitute at a concentration of 0.2x-0.5x. After discarding the digestive fluid, detach the cells by applying a fluid shear force of 50-200g.

3. The method according to claim 1, characterized in that, In step S5, the indicators of the metabolomics analysis include the lactate / glucose consumption ratio and / or the glutamine consumption rate. If the glutamine consumption rate is less than 30% within 24 hours after resuscitation, the batch of cells will not be used for expansion and production of clinical formulations.

4. The method according to claim 1, characterized in that, After step S1 and before step S3, a super seed bank construction step is also included: isolating and screening monoclonal cells with high proliferative potential and / or high immunosuppressive function from primary mesenchymal stem cells, and using the cells obtained from this expansion to establish the master cell bank.

5. A quality control method for evaluating the preparation process of mesenchymal stem cells, characterized in that, In the industrial preparation method according to claim 1, the method includes: After digestion in step S2 or after cryopreservation and thawing in step S4, cell culture supernatant is collected for metabolomics analysis. Compare the measured value of at least one metabolic marker with a preset threshold; Based on the comparison results, the subsequent expansion potential or functional output of this batch of cells is predicted, and a decision is made on whether to approve, downgrade, or eliminate the production use of this batch of cells.

6. A low-damage cell digestion method, characterized in that, In the method for preparing human umbilical cord mesenchymal stem cells according to claim 1 or 2, the method includes: Pre-incubate cells at 2-8°C for 10-15 minutes using a non-animal-derived recombinant trypsin substitute at a concentration of 0.2x-0.5x. Remove the digestive enzyme solution; Cells are detached from the culture surface by applying a fluid shear force of 50-200g.

7. A human umbilical cord mesenchymal stem cell product, characterized in that, The cell product is prepared by the method according to any one of claims 1 to 4; Within 24 hours after cryopreservation and thawing, the glutamine consumption rate in the culture supernatant of human umbilical cord mesenchymal stem cell products is higher than 30%, and their immunosuppressive titer is not lower than 80% of that of the standard.

8. A human umbilical cord mesenchymal stem cell production system, characterized in that, The system includes: The super seed bank consists of monoclonal primary mesenchymal stem cells screened and expanded according to the method described in claim 4; The master cell bank is established by expanding the cells of the super seed bank and then cryopreserving them at passage 3. The process decision module is configured to execute the quality control method as described in claim 5; The clinical formulation production unit is used to revive and passage cells from the master cell bank to the target passage, according to the instructions of the process decision module, in order to prepare clinical cell formulations.

9. A pharmaceutical composition, characterized in that, It includes the human umbilical cord mesenchymal stem cell product of claim 7 and a pharmaceutically acceptable carrier.

10. Use of the pharmaceutical composition of claim 9 in the preparation of a medicament for treating graft-versus-host disease.

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