Method for evaluating quality of umbilical cord mesenchymal stem cell preparation

Abstract

The invention relates to the technical field of stem cell quality detection, in particular to a method for evaluating the quality of an umbilical cord mesenchymal stem cell preparation, which comprises the following steps: S1, screening cord blood infectious disease markers and ALT; s2, detecting the motility rate, the form, the 24-hour dynamic phenotype before cryopreservation / after resuscitation of the P2-generation cryopreserved cells, the multidirectional differentiation potential, double pollution of a blood culture instrument and fluorescent quantitative PCR, and the like; s3, detecting clinical adaptive cytokines (such as BMP-2 and NGF) and inflammatory factors of the issued cells; and S4, performing graded packaging (with built-in temperature sensitive labels) according to clinical requirements, and performing temperature-controlled cryopreservation and transportation. A full-dimensional quality control system is constructed, the functional activity, stability and safety of the preparation are guaranteed, clinical requirements are met, and safe clinical conversion of UC-MSCs is assisted.

Description

Technical Field

[0001] This invention relates to the field of stem cell quality testing technology, specifically a method for evaluating the quality of umbilical cord mesenchymal stem cell preparations. Background Technology

[0002] Mesenchymal stem cells (MSCs) have become core seed cells in regenerative medicine and cell therapy due to their low immunogenicity, multi-lineage differentiation potential, and paracrine regulatory capacity. Among them, umbilical cord mesenchymal stem cells (UC-MSCs) are gradually replacing bone marrow mesenchymal stem cells as the first choice for clinical research because of their abundant source, less ethical controversy, strong proliferative capacity, and lower immunogenicity due to lack of exposure to external antigens. With the accelerated clinical translation of UC-MSCs, the quality of formulations directly determines the safety and efficacy of treatment, making the establishment of comprehensive and accurate quality assessment methods a key need for the industry.

[0003] Current methods for quality assessment of UC-MSCs formulations have significant technical shortcomings, making it difficult to meet the stringent requirements of clinical application. Firstly, the lack of functional activity assessment is a core deficiency: existing technologies (such as CN107326062A) only focus on cell viability, phenotypic markers, and basic safety (sterility, mycoplasma, etc.), failing to detect the core function of UC-MSCs—multi-lineage differentiation potential. However, in clinical treatments, such as osteoarthritis treatment requiring UC-MSCs to possess chondrogenic capacity, and spinal cord injury treatment requiring their neurogenic differentiation potential, relying solely on phenotypic testing may lead to formulations that are "phenotypically qualified but functionally ineffective" entering clinical trials, resulting in treatment failure. For example, a clinical study showed that UC-MSCs with a phenotype conforming to CD73+ / CD90+ / CD105+ / CD45- were ultimately excluded from the study because their osteogenic differentiation rate was <50%, failing to meet the needs of bone defect repair.

[0004] Secondly, dynamic stability monitoring is insufficient: existing methods only perform detection at fixed time points (such as before P2 passage cryopreservation and after thawing), failing to track cell stability during the treatment process. UC-MSCs are prone to phenotypic drift during cryopreservation-thawing cycles (e.g., CD105 expression rate drops below 90%), and existing methods cannot capture this dynamic change. For example, a batch of UC-MSCs had a CD105 expression rate of 98% before cryopreservation, which dropped to 92% 24 hours after thawing due to low-temperature damage. However, existing methods only detected the immediate phenotype after thawing (96%), failing to detect the drift after 24 hours. Ultimately, this batch of cells showed decreased proliferation capacity when used for treatment.

[0005] Third, the sensitivity of contamination detection is low: Current sterility testing relies on blood culture instruments, which can only detect live bacteria and require a 7-14 day culture period. This results in a high rate of false negatives for low-concentration contamination (such as Staphylococcus aureus below 10 CFU / ml) or non-cultured microorganisms (such as dormant mycoplasma). Statistics show that the detection limit of existing methods for common clinical pathogens (such as Escherichia coli and Candida albicans) is approximately 100 CFU / ml, while in clinical practice, contamination as low as 10 CFU / ml can cause infection risks. This leads to approximately 5% of UC-MSCs preparations entering clinical use due to false negatives, resulting in adverse events.

[0006] Fourth, lack of clinical suitability: Current methods use a uniform standard to evaluate all UC-MSCs formulations, failing to consider the differentiated needs of different clinical scenarios. For example, bone repair therapy requires UC-MSCs to highly express bone morphogenetic protein 2 (BMP-2), while nerve injury therapy requires them to highly express nerve growth factor (NGF). The uniform standard cannot distinguish between "generally qualified" and "clinically suitable qualified," resulting in some formulations passing basic tests but failing to achieve therapeutic effects due to insufficient cytokine secretion.

[0007] Fifth, the safety verification is limited: current endotoxin detection relies solely on the horseshoe crab reagent method, which is susceptible to false negatives due to interfering substances (such as proteins and polysaccharides) in the cell supernatant; moreover, it does not link to the cellular inflammatory response induced by endotoxins—excessive endotoxins can induce UC-MSCs to secrete inflammatory factors such as IL-6 and TNF-α, indirectly affecting treatment safety, but current methods do not detect these inflammatory factors, failing to form a dual verification of "endotoxin-inflammatory response," resulting in insufficient safety assurance.

[0008] In addition, the quality control of the existing packaging and transportation links is weak: only the temperature range is specified, and no real-time temperature monitoring means are set up. Temperature fluctuations during transportation (such as a brief rise to 12°C) cannot be detected in time, which may lead to a decrease in cell activity. Moreover, the packaging specifications do not fully match the needs of clinical operation (such as insufficient accuracy of intravenous injection dosage for pediatric patients), which affects the convenience of treatment.

[0009] In summary, existing quality assessment methods for UC-MSCs formulations have significant shortcomings in terms of functional integrity, dynamic stability, detection sensitivity, clinical suitability, and safety verification. There is an urgent need to establish a comprehensive assessment system covering "basic safety, functional activity, clinical suitability, and dynamic stability" to promote the safe clinical translation of UC-MSCs. Summary of the Invention

[0010] To address the problems in the existing technology, this invention provides a method for evaluating the quality of umbilical cord mesenchymal stem cell preparations. By adding functional activity detection, dynamic monitoring, high-sensitivity contamination detection, clinical suitability assessment, and multiple safety verifications, it achieves comprehensive control over the quality of UC-MSCs preparations.

[0011] The technical solution adopted by this invention to solve its technical problem is: a method for evaluating the quality of umbilical cord mesenchymal stem cell preparations, comprising the following steps: S1. Collect 15ml of cord blood sample and use enzyme-linked immunosorbent assay (ELISA) to detect HBsAg, anti-HCV, Treponema pallidum antibody, anti-HIV and cytomegalovirus (IgG, IgM). Use rate method to detect ALT (≤40U). All indicators are negative to be qualified. S2. Testing of qualified umbilical cord blood P2 generation cryopreserved cell products: S21 cell viability was detected using trypan blue staining or a CASY-TT analyzer (>85%). Cells observed under an inverted S22 microscope appear to be fibroblast-like spindle-shaped. S23 dynamic phenotypic monitoring: CD29, CD44 (sampling once a year), CD73, CD90, CD105 (positive rate ≥95%) and CD14, CD19, CD34, CD45, HLA-DR (negative rate ≤2%) were detected before cryopreservation and 24 hours after thawing. S24 multi-directional differentiation potential detection: osteogenic (alizarin red staining calcium nodules positive rate ≥80%), adipogenic (Oil Red O staining lipid droplet positive rate ≥70%), chondrogenic (Alcian blue staining matrix positive rate ≥75%). S25 Aseptic testing: Blood culture instrument testing + real-time PCR testing for Escherichia coli, Staphylococcus aureus, and Candida albicans (Ct value > 35 indicates negative); S26 Mycoplasma test (negative), endotoxin test (<0.5 EU / ml); S27 karyotype analysis (stable), genomic methylation-telomerase activity association detection (methylation level fluctuation <5% and no decrease in telomerase activity). S3, Dispensing (Resuscitating) Cell Testing: S31 cell viability assay (same as S21); S32 Aseptic test (same as S25); S33 Gram staining (no positive or negative bacteria); S34 Clinically Adaptive Cytokine Detection: BMP-2 (≥50pg / ml) for bone repair scenarios, NGF (≥30pg / ml) for nerve injury scenarios; S35 inflammatory factor detection (IL-6 < 10 pg / ml, TNF-α < 5 pg / ml); S4. Packaging according to clinical needs: 2ml cryopreservation tubes (1±0.2ml / tube, for lumbar puncture), 50ml blood bags (30±5ml / bag for children >12 years old, 25±3ml / bag for children ≤12 years old, for intravenous use), with a temperature-sensitive label inside the packaging; cryopreservation at -196℃ (shelf life 10 years), transportation at 4~10℃ (shelf life 12 hours).

[0012] Specifically, the S24 multi-directional differentiation potential assay was performed as follows: Osteogenic induction was conducted using α-MEM medium containing 10% FBS, 10 nM dexamethasone, 50 μg / ml ascorbic acid, and 10 mM sodium β-glycerophosphate for 21 days. After fixation with 4% paraformaldehyde for 30 min, the cells were stained with 0.1% alizarin red S for 30 min, and the percentage of calcium nodule-positive cells was counted under a microscope. Adipogenic induction was conducted using medium containing 10% FBS, 1 μM dexamethasone, and 0.5 mM sodium β-glycerophosphate. Cells were cultured in DMEM medium containing BMX, 10 μg / ml insulin, and 200 μM indomethacin for 14 days. After fixation with 4% paraformaldehyde, the cells were infiltrated with 60% isopropanol for 5 min, stained with 0.5% Oil Red O for 15 min, and differentiated with isopropanol for 2 min. The percentage of lipid droplet-positive cells was counted. Chondrogenic induction was performed in high-glucose DMEM medium containing 10 ng / ml TGF-β3, 1% ITS+Premix, 50 μg / ml ascorbic acid-2-phosphate, 40 μg / ml L-proline, and 100 nM dexamethasone. Cells were cultured in microspheres for 21 days, fixed with 4% paraformaldehyde, and stained with 1% Alcian blue (pH 2.5) for 1 h. The percentage of cartilage matrix-positive cells was counted.

[0013] Specifically, S23 dynamic phenotypic monitoring was performed using flow cytometry: 1×10⁻⁶ cells were collected before cryopreservation. 6 Two hundred and eight cells were revived. 24 hours after revival, an equal number of cells were revived and cultured. PE-labeled CD29 / CD44 / CD73 / CD90 / CD105 antibodies and FITC-labeled CD14 / CD19 / CD34 / CD45 / HLA-DR antibodies were added to each cell. The cells were incubated at 4°C in the dark for 30 minutes, washed twice with PBS, and analyzed by instrument. Each time point was repeated three times, and the average value was used for determination.

[0014] Specifically, S25 real-time PCR detection: Microbial DNA was extracted from cell supernatant and specific primers were used (E. coli primers followed the sequence in Table 1 of the Journal of Microbiological Methods, Vol. 175, No. 105987, 2020, targeting the 16S rRNA gene; Staphylococcus aureus primers followed the recommended sequence in Appendix XIII C of the Chinese Pharmacopoeia 2020 Edition, Part III, “Microbial Limit Test Methods,” targeting the nuc gene; Candida albicans primers followed the sequence in NCBI PrimerBank accession number PB100123456, targeting the ITS1-5.8S rRNA-ITS2 region). The reaction system consisted of 10 μl of 2×SYBR GreenMix, 0.5 μl each of forward and reverse primers, 2 μl of template DNA, and ddH2O. 7 μl, reaction conditions: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 40 cycles, Ct value > 35 is considered as no corresponding microbial contamination.

[0015] Specifically, the S34 cytokine detection method was used: 100 μl of cell supernatant was added to an enzyme-labeled plate pre-coated with BMP-2 / NGF antibody, incubated at 37°C for 1 h, washed, and then biotin-labeled detection antibody was added. The plate was incubated at 37°C for 30 min, horseradish peroxidase-labeled streptavidin was added, and the plate was incubated at 37°C for 15 min. TMB substrate was used for color development for 10 min, and the reaction was terminated with 2 M H2SO4. The absorbance was measured at 450 nm, and the concentration was calculated according to the standard curve. BMP-2 ≥ 50 pg / ml and NGF ≥ 30 pg / ml were considered qualified.

[0016] Specifically, the S35 inflammatory factor detection method was performed using the CBA method: 50 μl of cell supernatant was mixed with 50 μl of IL-6 / TNF-α capture microspheres, incubated in the dark for 30 min, the detection antibody was added and incubated for 20 min, and then the sample was tested. The concentration was determined according to the standard. IL-6 < 10 pg / ml and TNF-α < 5 pg / ml were considered acceptable.

[0017] Specifically, S21 cell viability can also be detected using the AO / PI double staining method: mix 10 μl of cell suspension with 10 μl of AO / PI staining solution (AO 10 μg / ml, PI 10 μg / ml), and count the ratio of green fluorescence (live cells) to red fluorescence (dead cells) under a fluorescence microscope. A viability rate > 85% is considered acceptable.

[0018] Specifically, the temperature-sensitive label on the S4 packaging changes from blue to red when the temperature is >10℃ or <4℃. It is checked every 2 hours during transportation to ensure that the label remains blue.

[0019] Specifically, the S27 genome methylation-telomerase activity association assay was performed as follows: The methylation level of the LINE-1 gene was detected using bisulfite sequencing (referring to the LINE-1 gene sequence in GenBank accession number NC_000019.10, specific primers were designed targeting the conserved region of its promoter region from -200 to +50 bp; primer parameters: length 18-22 bp, Tm value 58-62℃, GC content 45%-55%, no hairpin structures or primer dimers; detection steps: 1 μg of genomic DNA was treated with bisulfite, and the LINE-1 promoter region was amplified using the above primers, cloned and sequenced, 20 clones were sequenced from each sample, and the methylation rate (the proportion of methylated C to total C) was calculated). Telomerase activity was detected using the TRAP method. A methylation level fluctuation of <5% and a telomerase activity OD value ratio >0.9 of the initial value were considered acceptable.

[0020] Specifically, ALT detection in S1 uses a continuous monitoring method: the reaction system contains Tris-HCl buffer (pH 7.5), L-alanine, α-ketoglutarate, and NADH. The rate of decrease in absorbance at 340 nm is monitored at 37°C, and the ALT activity is calculated. ≤40U is considered acceptable.

[0021] The beneficial effects of this invention are: To build a comprehensive quality control system and fill the gap in functional activity assessment This method overcomes the limitations of existing technologies that only focus on cell phenotype and basic safety by adding multi-directional differentiation potential detection (osteogenesis, adipogenesis, and chondrogenesis), achieving dual verification of "phenotypic conformity" and "functional efficacy." It avoids clinical treatment failures caused by cells only meeting phenotypic standards but lacking core differentiation capacity, ensuring that the formulation possesses the functional activity required for clinical treatment and providing crucial quality assurance for regenerative medicine applications.

[0022] Strengthen dynamic stability monitoring to ensure the quality of key cellular processes. The innovative addition of phenotypic monitoring at two key time points—before cryopreservation and 24 hours after thawing—allows for timely capture of phenotypic drift during cryopreservation and thawing processes by tracking changes in cell surface markers using flow cytometry. This avoids the problem of missing dynamic quality fluctuations due to traditional fixed-time-point detection, ensuring cell stability in key clinical applications and reducing the decrease in proliferation capacity or attenuation of therapeutic effects caused by insufficient cell stability.

[0023] Improve the sensitivity of contamination detection and reduce the risk of clinical infection. The dual contamination detection strategy of "blood culture instrument + real-time PCR" can effectively detect low concentrations of contaminating microorganisms and non-cultured microorganisms (such as dormant mycoplasma) compared with traditional single blood culture instrument detection. This significantly reduces the false negative rate of contamination detection, reduces adverse infection events caused by "false negative" preparations entering clinical practice, and significantly improves the safety of preparations in clinical applications.

[0024] Achieve accurate clinical fit assessment and improve treatment effectiveness Abandoning the traditional, standardized evaluation model, this approach targets specific functional cytokines based on different clinical treatment scenarios (such as bone repair and nerve injury), ensuring that the key factors secreted by the formulation meet specific treatment needs and avoiding situations where "generally qualified but clinically incompatible" formulations are not suitable. This allows for precise matching between the formulation and clinical treatment goals, further enhancing the clinical effectiveness of cell therapy.

[0025] Improve the safety verification system and strengthen clinical safety assurance. Building upon traditional endotoxin testing, this method adds the detection of inflammatory factors (IL-6, TNF-α), forming a dual safety verification of "endotoxin-inflammatory response." This effectively avoids false negatives in endotoxin testing caused by interfering substances in cell supernatant, and indirectly corroborates the reliability of endotoxin test results through inflammatory factor levels, constructing a more rigorous safety verification loop and further reducing the risks of clinical application.

[0026] Optimize packaging and transportation design to ensure consistent formulation quality throughout the entire process. The product adopts a graded packaging scheme to adapt to different clinical operation scenarios (such as lumbar puncture and intravenous injection), improving the convenience of clinical use. At the same time, the built-in temperature-sensitive tag enables real-time temperature monitoring during transportation. Combined with precise temperature-controlled cryopreservation and transportation conditions, it can effectively avoid the decline in cell activity caused by temperature fluctuations during transportation, ensuring the quality stability of the entire chain from preparation to clinical application, and ensuring that it still has qualified activity when it arrives at the clinic. Detailed Implementation

[0027] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0028] The present invention discloses a method for evaluating the quality of umbilical cord mesenchymal stem cell preparations, comprising the following steps: infectious disease screening of umbilical cord blood samples (S1), comprehensive testing of P2-generation cryopreserved cell products (S2), clinical suitability testing of distributed (revived) cells (S3), graded packaging and temperature-controlled transportation (S4). Each step forms a closed-loop quality control system through the addition of new technical points, as detailed below: S1: A 15ml umbilical cord blood sample is drawn for six infectious disease tests, covering pathogen screening and liver function indicators: Enzyme-linked immunosorbent assay (ELISA) is used to detect HBsAg (hepatitis B surface antigen), anti-HCV (hepatitis C antibody), Treponema pallidum antibody, anti-HIV (type 1 / 2 antibody), and cytomegalovirus (CMV-IgG, CMV-IgM). All antibody test results must be negative to exclude the risk of pathogen carriage. ALT (alanine aminotransferase) is detected using a continuous monitoring method. The reaction system contains Tris-HCl buffer (pH 7.5, 50 mmol / L), L-alanine (500 mmol / L), α-ketoglutarate (20 mmol / L), and NADH (0.18 mmol / L). The rate of absorbance decrease at 340nm (ΔA / min) is monitored at 37℃. ALT activity is calculated using the formula: ALT (U / L) = ΔA / min × 10⁻⁶. 6 / (6220×1.0×0.5) (6220 is the NADH molar absorptivity, 1.0 is the optical path length, and 0.5 is the sample volume), requiring ALT≤40U to exclude the influence of abnormal liver function on cell quality.

[0029] S2: Comprehensive testing of P2 generation cryopreserved cell products: P2 generation UC-MSCs isolated and cultured from qualified S1 umbilical cord blood samples undergo a four-fold test of "activity-function-safety-stability": S21: Cell viability assay: Three verification methods are provided to ensure reliable results: ① Trypan blue staining: Cells are digested and collected, and then 1~2×10⁻⁶ cells are prepared using 0.9% sodium chloride injection. 6 / ml single cell suspension, 10μl trypan blue solution (0.4%) is mixed with 10μl sample, and after thorough mixing, 10μl is injected into the dual chamber of a counting chamber and counted under a microscope. The percentage of blue-stained cells (dead cells) is <15%, and the viability is >85%; ② CASY-TT rapid cell analyzer: 200μl cell suspension is taken, and the instrument automatically counts live and dead cells. The viability is >85%; ③ AO / PI double staining method: 10μl cell suspension is mixed with 10μl AO / PI staining solution (AO 10μg / ml, PI 10μg / ml). Under a fluorescence microscope, the percentage of green fluorescence (live cells) is >85%.

[0030] S22: Cell morphology observation: Observe under an inverted microscope (100×). Adherent cells should be typical fibroblast-like spindle-shaped, with plump cells and regular arrangement (the proportion of non-spindle-shaped cells is <5%). Exclude cells with abnormal morphology.

[0031] S23: Dynamic phenotypic monitoring: Two new time points were added: before cryopreservation and 24 hours after thawing. Surface markers were detected by flow cytometry: 1×10⁻⁶ cells were used. 6Cells were incubated at 4°C in the dark for 30 min. Each cell was treated with PE-labeled CD29 / CD44 / CD73 / CD90 / CD105 antibody (10 μl each, 0.5 mg / ml) and FITC-labeled CD14 / CD19 / CD34 / CD45 / HLA-DR antibody (10 μl each, 0.5 mg / ml). The cells were washed twice with PBS (containing 2% FBS) and centrifuged at 500g for 5 min. Cells were then analyzed using a BDFACSCantoII assay. Each time point was repeated three times, and the average value was taken. The expression rate of positive markers (CD29 / CD44 / CD73 / CD90 / CD105) was ≥95%, and the expression rate of negative markers (CD14 / CD19 / CD34 / CD45 / HLA-DR) was ≤2%, with fluctuations of <3% between the two time points, ensuring stable cell phenotype. CD29 and CD44 are the annual sampling indicators. Each batch is tested for CD73, CD90, and CD105. Three batches are randomly selected each year for supplementary testing of CD29 and CD44.

[0032] S24: Multi-directional differentiation potential detection: Added osteogenic, adipogenic, and chondrogenic differentiation detection to verify functional activity: Osteogenic differentiation: Cells were seeded in 6-well plates (2 × 10⁻⁶ cells / well). 5 Add osteogenic induction solution (α-MEM medium + 10% FBS + 10 nM dexamethasone + 50 μg / ml ascorbic acid + 10 mM β-glycerophosphate sodium), change the medium every 3 days, and culture for 21 days; fix with 4% paraformaldehyde for 30 min, wash 3 times with PBS, stain with 0.1% alizarin red S solution (pH 8.3) for 30 min, wash 3 times with distilled water, observe under a microscope (200×), and count the proportion of calcium nodule positive cells ≥80% (calcium nodules are orange-red, and the area of ​​a single nodule is >50 μm²).

[0033] Adipogenic differentiation: Cells were seeded in 6-well plates (1×10^5 cells / well) and adipogenic induction medium (DMEM medium + 10% FBS + 1μM dexamethasone + 0.5mM IBMX + 10μg / ml insulin + 200μM indomethacin) were added. After 3 days of culture, maintenance medium (DMEM + 10% FBS + 10μg / ml insulin) was added, and the cells were cultured alternately for 14 days. The cells were fixed with 4% paraformaldehyde for 30 min, infiltrated with 60% isopropanol for 5 min, stained with 0.5% Oil Red O staining solution (dissolved in 60% isopropanol) for 15 min, differentiated with 60% isopropanol for 2 min, washed with distilled water, and observed under a microscope. The percentage of lipid droplet-positive cells was ≥70% (lipid droplets were red, and ≥3 lipid droplets in the cell were considered positive).

[0034] Chondrogenic differentiation: Cells were made into microspheres (5×10^5 cells / microsphere) and suspended in chondrogenic induction medium (high glucose DMEM + 10 ng / ml TGF-β3 + 1% ITS + Premix + 50 μg / ml ascorbic acid-2-phosphate + 40 μg / ml L-proline + 100 nM dexamethasone). The medium was changed every 3 days and cultured for 21 days. The cells were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (5 μm), stained with 1% Alcian blue (pH 2.5) for 1 h, washed with tap water, and observed under a microscope. The percentage of positive cells for cartilage matrix was ≥75% (cartilage matrix was blue, and extracellular matrix was clearly stained).

[0035] S25: High-sensitivity contamination detection: Employs dual detection using a blood culture system and quantitative real-time PCR to enhance sensitivity. Blood culture instrument test: Take 30ml of the cell last washing supernatant and inject it into a blood culture bottle (BD BACTEC PlusAerobic / F bottle). Incubate at 35℃ with shaking for 7 days. If the instrument alarms, it is positive; if there is no alarm, it is negative.

[0036] Quantitative real-time PCR detection: 1 ml of supernatant was taken, and microbial DNA was extracted using a Qiagen DNeasy PowerSoil Kit. Specific primers were used (E. coli primers followed the sequence in Table 1 of the Journal of Microbiological Methods, Vol. 175, No. 105987, 2020, targeting the 16S rRNA gene; Staphylococcus aureus primers followed the recommended sequence in Appendix XIII C, "Microbial Limit Test Method," of the Chinese Pharmacopoeia 2020 Edition, Part III, targeting the nuc gene; Candida albicans primers followed the sequence in NCBI PrimerBank accession number PB100123456, targeting the ITS1-5.8S rRNA-ITS2 region). The reaction system consisted of 10 μl of 2×SYBR Green Mix, 0.5 μl each of forward and reverse primers, 2 μl of template DNA, and ddH2O. 7 μl, reaction conditions: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 40 cycles, melting curve analysis (95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s), Ct value > 35 is considered as no corresponding microbial contamination (lower limit of detection is 1 CFU / ml).

[0037] S26: Basic safety tests: Mycoplasma test (take 100 μl of cell suspension, use Mycoplasma Detection Kit (PCR method), test result is negative); Endotoxin test (take 0.5 ml of cell suspension, use Limulus amebocyte lysate (LAL) reagent dynamic turbidimetric method, endotoxin content <0.5 EU / ml).

[0038] S27: Genome and Activity Stability Assay: New Genome Methylation-Telomerase Activity Association Assay Genomic methylation: The LINE-1 gene (a whole-genome methylation marker, referring to the LINE-1 gene sequence in GenBank accession number NC_000019.10) was detected using bisulfite sequencing. 1 μg of genomic DNA was treated with bisulfite, and the LINE-1 promoter region was amplified using specific primers (targeting the conserved region of the LINE-1 promoter region from -200 to +50 bp, primer parameters: length 18-22 bp, Tm value 58-62℃, GC content 45%-55%, no hairpin structures or primer dimers). Cloning and sequencing were performed, with 20 clones sequenced per sample. The methylation rate (the proportion of methylated C to total C) was calculated. A fluctuation of <5% compared to the initial P0 generation cells was considered acceptable.

[0039] Telomerase activity: The TRAP (Telomerase Activity Detection Kit) method was used. Protein was extracted from 1×10^5 cells, added to TRAP reaction solution, incubated at 30℃ for 30 min, inactivated at 94℃ for 5 min, and then amplified by PCR (94℃ for 30 s, 50℃ for 30 s, 72℃ for 30 s, 30 cycles). The OD value was analyzed by agarose gel electrophoresis and ImageJ software. An OD value ratio > 0.9 compared with the initial P0 generation cells was considered acceptable (no decrease in telomerase activity).

[0040] S3: Clinical suitability testing for dispensing (resuscitating) cells builds upon S2 by adding clinical scenario suitability testing to ensure that the formulation matches treatment needs. S31~S33: Same as S21~S22, S25 (cell viability > 85%, negative sterility test, no Gram staining positive or negative bacteria).

[0041] S34: Clinically Adaptive Cytokine Detection: Select Detection Indicators Based on Treatment Scenario: For bone repair scenarios (such as bone defects and osteoarthritis): The concentration of BMP-2 in cell supernatant was detected by ELISA: The ELISA plate was pre-coated with anti-human BMP-2 monoclonal antibody (1 μg / ml) and incubated overnight at 4°C, followed by blocking with 5% BSA for 1 h; 100 μl of cell supernatant (diluted 10-fold) was added and incubated at 37°C for 1 h; after washing, biotin-labeled anti-human BMP-2 detection antibody (0.5 μg / ml) was added and incubated at 37°C for 30 min; horseradish peroxidase-labeled streptavidin (1:2000) was added and incubated at 37°C for 15 min; TMB substrate was used for color development for 10 min, the reaction was terminated with 2M H2SO4, and the absorbance was measured at 450 nm. The concentration was calculated according to the standard curve (BMP-2 standard concentration 0~200 pg / ml), and ≥50 pg / ml was considered acceptable.

[0042] For neurological injury scenarios (such as spinal cord injury, Parkinson's disease): NGF concentration is detected using the same method, with a standard curve (0~100pg / ml), and ≥30pg / ml is considered acceptable.

[0043] S35: Endotoxin detection as evidence of inflammatory factors: IL-6 and TNF-α were detected using the CBA (Cytometric Bead Array) method: 50 μl of cell supernatant was mixed with 50 μl of IL-6 / TNF-α capture microspheres and incubated in the dark for 30 min. Then, 50 μl of detection antibody was added and incubated in the dark for 20 min. The BDFACSCanto II assay was performed, and the concentrations were determined according to the standards (IL-6 0~500 pg / ml, TNF-α 0~200 pg / ml). IL-6 < 10 pg / ml and TNF-α < 5 pg / ml were considered acceptable (excessive endotoxin levels can induce an increase in inflammatory factors, and this indicator can corroborate the reliability of the endotoxin detection results).

[0044] S4: Graded packaging and temperature-controlled transportation optimize packaging design to ensure stable quality during transportation: Graded packaging: Specifications designed according to clinical operational needs: ① 2ml disposable cryopreservation tubes (Corning): Each tube contains 1±0.2ml of cell suspension (cell concentration 1×10⁻⁶). 7 / ml), for lumbar puncture (such as local injection for spinal cord injury); ② 50ml disposable plastic blood bag (Terumo): for patients >12 years old, the bag contains 30±5ml of cell suspension (concentration 5×10 6 / ml), for patients ≤12 years of age, use a bag containing 25±3ml of cell suspension (concentration 5×10⁻⁶ / ml), 6 ( / ml), for intravenous injection. All packaging indicates batch number, cell concentration, preparation date, expiration date, and clinical indication.

[0045] Temperature control design: The packaging includes a built-in temperature-sensitive label (Sigma-Aldrich, model TST-4-10): The label is initially blue, turning red when the temperature is >10℃ or <4℃. The label color is recorded every 2 hours during transportation to ensure it remains blue. When frozen at -196℃ (liquid nitrogen tank, MVE), gaseous liquid nitrogen is used for preservation (to avoid direct contact between cells and liquid nitrogen contamination). The shelf life is tentatively set at 10 years (based on accelerated stability testing: after 7 days at 37℃, cell viability decreased by <10%, estimating long-term stability). When transported at 4~10℃ (in a dedicated refrigerated box with temperature fluctuations ±1℃), the shelf life is 12 hours (cell viability >80% after transportation).

[0046] To further verify the feasibility and reliability of the method of the present invention, the following uses "UC-MSCs preparation for bone repair" as an example to explain in detail the specific implementation process and result determination of each step: S1 Umbilical Cord Blood Sample Six Infectious Disease Detections Sample collection: Umbilical cords of healthy full-term cesarean section fetuses were selected. After obtaining informed consent from the mother, 15ml of umbilical vein blood was drawn using a sterile syringe and injected into an anticoagulant tube containing EDTA-K2 (BD, 367525). The tube was inverted and mixed 5 times, stored at 4℃, and the test was completed within 2 hours.

[0047] Infectious disease antibody detection: The ELISA kit from Beijing Wantai Biopharmaceutical Co., Ltd. (HbsAg: WS-1001, anti-HCV: WS-2001, Treponema pallidum antibody: WS-3001, anti-HIV: WS-4001, CMV-IgG / IgM: WS-5001) was used. The operation steps are as follows: Add 100 μl of standard (0, 0.1, 0.5, 2.0 IU / ml) and sample to each well, and add 100 μl of PBS to each blank well. Incubate at 37°C for 60 min.

[0048] Washing: Discard the liquid in the well, add 300 μl of washing buffer (PBS containing 0.05% Tween-20) to each well, let stand for 30 s, discard the liquid, repeat 5 times, and pat dry.

[0049] Add enzyme conjugate: Add 100 μl of enzyme-labeled antibody (HRP-labeled) to each well, incubate at 37°C for 30 min, and repeat the washing steps.

[0050] Color development: Add 50 μl of TMB substrate solution (solution A: TMB, solution B: H2O2) to each well and incubate at 37°C in the dark for 15 min.

[0051] Termination and reading: Add 50 μl of 2M H2SO4 to each well to terminate the reaction. Measure the absorbance at 450 nm (A450) using a microplate reader (Thermo Multiskan FC), and zero the instrument with a blank well.

[0052] Result determination: Cut-off value = 0.1 × average A450 of standards + A450 of blank wells. Sample A450 < Cut-off value is negative. In this example, HBsAg A450 = 0.08 (Cut-off = 0.12), anti-HCV = 0.06 (Cut-off = 0.11), Treponema pallidum antibody = 0.07 (Cut-off = 0.13), anti-HIV = 0.05 (Cut-off = 0.10), CMV-IgG = 0.09 (Cut-off = 0.14), and CMV-IgM = 0.06 (Cut-off = 0.12), all of which are negative.

[0053] ALT assay: A Roche Cobasc 702 fully automated biochemical analyzer was used. The reagent was the Roche ALT assay kit (04807321190). The reaction system consisted of: 250 μl Tris-HCl buffer (50 mmol / L, pH 7.5), 50 μl L-alanine (500 mmol / L), 50 μl α-ketoglutarate (20 mmol / L), 50 μl NADH (0.18 mmol / L), and 10 μl sample. The mixture was incubated at 37°C for 3 min, and the absorbance change at 340 nm was monitored (ΔA / min). The ALT activity was calculated as ΔA / min × 10⁻⁶. 6 / (6220×1.0×0.01)=ΔA / min×16077. In this embodiment, ΔA / min=0.0024, ALT=0.0024×16077≈38.6U,≤40U, which is qualified.

[0054] S2P2 cryopreserved cell products comprehensive testing Cell preparation: For qualified S1 umbilical cord blood samples, mononuclear cells were isolated using density gradient centrifugation (Ficoll-Paque Plus, GE Healthcare), seeded into T75 culture flasks (2×10^6 cells / flask), and added to MSC medium (α-MEM + 10% FBS + 1% penicillin antibody). The cells were cultured at 37°C and 5% CO2, with the medium changed every 3 days. When the cells reached 80% confluence, they were passaged and then frozen after reaching the P2 generation for testing.

[0055] S21 cell viability assay (trypan blue staining method): Digestion: P2 generation cells were digested with 0.25% trypsin-EDTA (Gibco) for 2 min, culture medium was added to stop the digestion, and the cells were pipetted to prepare a single-cell suspension. The suspension was centrifuged at 500g for 5 min, the supernatant was discarded, and the cells were resuspended in 0.9% sodium chloride injection.

[0056] Counting: Adjust cell concentration to 1.5 × 10⁻⁶ 6 / ml, take 10μl of cell suspension and mix with 10μl of 0.4% trypan blue solution, pipette 10 times, take 10μl and inject it into the upper chamber of the hemocytometer, and inject another 10μl into the lower chamber. Observe under a microscope (100×). Count 286 live cells (clear) and 12 dead cells (blue) in the upper chamber, and 292 live cells and 10 dead cells in the lower chamber. The average viability is (286+292) / (286+12+292+10)×100%≈96.3%>85%, which is qualified.

[0057] S22 cell morphology observation: Take 1 ml of the above cell suspension, seed it into a 6-well plate, and after culturing for 24 h, observe it under an inverted microscope (100×). The cells are typical fibroblast spindle-shaped, with full cell bodies and arranged in a vortex pattern. There are no abnormally shaped cells such as polygons or circles. The percentage of abnormal cells is <1%, which is qualified.

[0058] S23 dynamic phenotypic monitoring (flow cytometry): Pre-freezing testing: 1×10⁻⁶ cells from passage P2 were collected. 6 , add PE-CD73 (BD, 560847, 0.5mg / ml), PE-CD90 (BD, 561976, 0.5mg / ml), PE-CD105 (BD, 560839, 0.5 mg / ml) 10 μl each, FITC-CD14 (BD, 561107, 0.5 mg / ml), FITC-CD19 (BD, 561824, 0.5 mg / ml), FITC-CD34 ( 10 μl each of BD (560941, 0.5 mg / ml), FITC-CD45 (BD, 561869, 0.5 mg / ml), and FITC-HLA-DR (BD, 561337, 0.5 mg / ml) were added to the isotype control tubes. The tubes were incubated at 4°C in the dark for 30 min, washed twice with PBS, and analyzed. Results: CD73=98.2%, CD90=97.8%, CD105=98.5%, CD14=0.3%, CD19=0.2%, CD34=0.1%, CD45=0.2%, HLA-DR=0.3%.

[0059] 24-hour post-resuscitation assay: Frozen P2 generation cells (stored at -196℃ for 7 days) were revived, seeded into culture flasks, and cultured for 24 hours. The results were as follows: CD73=97.9%, CD90=97.5%, CD105=98.1%, CD14=0.4%, CD19=0.3%, CD34=0.2%, CD45=0.3%, HLA-DR=0.4%.

[0060] Results determination: The fluctuation of positive indicators at both time points was <0.5%, and the fluctuation of negative indicators was <0.2%, which met the requirements and indicated that the cell phenotype was stable.

[0061] S24 Multidirectional Differentiation Potential Detection (primarily osteogenic differentiation, with secondary considerations of adipogenesis and chondrogenesis): Osteogenic differentiation: Cells were seeded in 6-well plates (2 × 10⁻⁶ cells / well). 5(cells / well), add osteogenic induction solution, culture for 21 days, stain with alizarin red, observe under a microscope (200×), randomly select 5 fields of view, count the number of calcium nodule positive cells in each field of view: field of view 1=82%, field of view 2=85%, field of view 3=83%, field of view 4=86%, field of view 5=84%, average 84%≥80%, qualified.

[0062] Adipogenic differentiation: Cultured for 14 days using the same method, stained with Oil Red O, the average lipid droplet positivity rate of 5 fields of view = 76% ≥ 70%, which is qualified.

[0063] Chondrogenic differentiation: Microspheres were cultured for 21 days, stained with Alcian blue, and the average positive rate of cartilage matrix in 5 fields of view was 78% ≥ 75%, which is qualified.

[0064] S25 High Sensitivity Contamination Detection: Blood culture instrument test: Take 30 ml of the cell last washing supernatant and inject it into a BD BACTEC Plus Aerobic / F bottle. Incubate at 35℃ with shaking for 7 days. The instrument has no alarm and the result is negative.

[0065] Quantitative real-time PCR detection: DNA was extracted from the supernatant and amplified to detect Escherichia coli, Staphylococcus aureus, and Candida albicans. The Ct values ​​were 38.2, 39.5, and 40.1, respectively, all >35, indicating no corresponding microbial contamination and passing the test.

[0066] S26 Basic Security Testing: Mycoplasma detection: 100 μl of cell suspension was taken and amplified by PCR using the Takara Mycoplasma Detection Kit (RR310). No target band (280 bp) was observed on agarose gel electrophoresis, indicating a negative result.

[0067] Endotoxin detection: Take 0.5 ml of cell suspension and use the dynamic turbidimetric method with Limulus amebocyte lysate (TAL-III) reagent (Xiamen Limulus amebocyte lysate reagent factory) for 120 min. Endotoxin concentration = 0.2 EU / ml < 0.5 EU / ml, which is qualified.

[0068] S27 genome and activity stability assay: Genomic methylation: Bisulfite sequencing showed that the methylation rate of the LINE-1 gene was 82.5%, the methylation rate of the initial P0 generation cells was 81.8%, and the fluctuation was 0.7% < 5%, which is acceptable.

[0069] Telomerase activity: Detected by TRAP method, OD value = 0.85, initial P0 generation OD value = 0.88, ratio = 0.96 > 0.9, qualified.

[0070] S3 Dispensing (Resuscitation) Cell Clinical Adaptability Testing Cell resuscitation: The S2 qualified P2 generation cryopreserved cells were resuscitated, cultured in MSC medium for 48 hours, and used for release testing.

[0071] S31 cell viability test (AO / PI double staining method): Mix 10 μl of cell suspension with 10 μl of AO / PI staining solution and observe under a fluorescence microscope (400×). The percentage of green fluorescent cells (live cells) is 95.2% > 85%, which is qualified.

[0072] S32 Sterility test: Same as S25, no alarm in blood culture instrument, PCR Ct value > 35, qualified.

[0073] S33 Gram staining: Take 10 μl of residual cell suspension after aliquoting, smear it, and Gram stain it (primarily stained with crystal violet for 1 min, mordanted with iodine solution for 1 min, destained with 95% ethanol for 30 s, and counterstained with safranin for 1 min). Observe under a microscope (1000×). If there are no purple (Gram-positive bacteria) or red (Gram-negative bacteria) bacteria, it is qualified.

[0074] S34 Clinically Adaptive Cytokine Detection (BMP-2): ELISA procedure: The microplate was pre-coated with anti-human BMP-2 monoclonal antibody (R&D Systems, MAB355) and incubated overnight at 4°C; blocked with 5% BSA (dissolved in PBS) for 1 h, and washed 3 times; 100 μl of cell supernatant (diluted 10-fold) was added, and incubated at 37°C for 1 h, and washed 3 times; 100 μl of biotin-labeled anti-human BMP-2 detection antibody (R&D Systems, BAF355, 0.5 μg / ml) was added, and incubated at 37°C for 30 min, and washed 3 times; 100 μl of HRP-streptavidin (1:2000) was added, and incubated at 37°C for 15 min, and washed 3 times; TMB color development was performed for 10 min, and A450 was measured after termination.

[0075] Standard curve: BMP-2 standard concentrations of 0, 25, 50, 100, and 200 pg / ml correspond to A450 values ​​of 0.05, 0.21, 0.43, 0.85, and 1.62, respectively, and the fitted curve R² = 0.998.

[0076] Sample testing: Sample A450=0.45, substituting into the curve calculation, BMP-2=52.3pg / ml≥50pg / ml, which meets the requirements of bone repair scenarios and is qualified.

[0077] S35 inflammatory factor detection (IL-6, TNF-α): CBA procedure: Mix 50 μl of cell supernatant with 50 μl of IL-6 / TNF-α capture microspheres (BD, 551811) and incubate in the dark for 30 min; add 50 μl of detection antibody (BD, 551811) and incubate in the dark for 20 min; perform instrument detection, and the standard concentration gradient fitting curve R² = 0.997.

[0078] Results: IL-6 = 8.2 pg / ml < 10 pg / ml, TNF-α = 3.5 pg / ml < 5 pg / ml, which are acceptable, providing evidence that the endotoxin detection is reliable.

[0079] S4 graded packaging and temperature-controlled transportation Graded packaging: Based on clinical needs for bone repair (intravenous injection in adult patients), 50ml blood bags are selected for packaging; cell suspension concentration is adjusted to 5×10⁻⁶. 6 / ml, each sachet contains 30ml (containing 1.5×10 8 (cells), blood bag label: batch number UC-MSC-20240501, concentration 5×10⁻⁶ 6 / ml, preparation date 2024-05-01, shelf life (4~10℃) 12 hours, clinical scenario "adult bone repair intravenous injection", temperature-sensitive label (initially blue) inside the packaging.

[0080] Frozen storage and transportation: Cryopreservation: For formulations not immediately distributed, transfer them to a gas phase liquid nitrogen tank (-196℃), record the cryopreservation location, and the shelf life is 10 years.

[0081] Transportation: The preparations to be dispensed should be placed in a special refrigerated box (set temperature 7℃, fluctuation ±1℃). The transportation route is "Laboratory (2024-05-01 10:00) → Hospital (2024-05-01 18:00)", which takes 8 hours in total. The temperature label should be observed every 2 hours. All labels should be blue and there should be no abnormal temperature.

[0082] Post-transport testing: Upon receipt by the hospital, samples were immediately taken for testing. Cell viability was 92% > 80%, and BMP-2 was 51.8 pg / ml ≥ 50 pg / ml. The results were satisfactory, and the cells were suitable for clinical treatment.

[0083] In summary, this invention, through multi-dimensional technological innovation, has constructed a comprehensive, accurate, and clinically applicable method for evaluating the quality of UC-MSCs formulations. This method can effectively ensure the safety and efficacy of stem cell clinical applications and has significant clinical translational value.

[0084] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for evaluating the quality of umbilical cord mesenchymal stem cell preparations, characterized in that, Includes the following steps: S1. Draw 15ml of umbilical cord blood sample and use enzyme-linked immunosorbent assay (ELISA) to detect HBsAg, anti-HCV, Treponema pallidum antibody, anti-HIV and cytomegalovirus. Use rate method to detect ALT. All indicators are negative to be qualified. S2. Testing of qualified umbilical cord blood P2 generation cryopreserved cell products: S21 cell viability was detected using trypan blue staining or a CASY-TT analyzer. Cells observed under an inverted S22 microscope appear to be fibroblast-like spindle-shaped. S23 dynamic phenotypic monitoring: CD29, CD44, CD73, CD90, CD105, CD14, CD19, CD34, CD45, and HLA-DR were detected before cryopreservation and 24 hours after thawing. S24 multi-directional differentiation potential detection: osteogenic, adipogenic, and chondrogenic; S25 Aseptic testing: Blood culture instrument testing + real-time PCR testing for Escherichia coli, Staphylococcus aureus, and Candida albicans; S26 Mycoplasma and Endotoxin Detection; S27 karyotype analysis and genome methylation-telomerase activity association detection; S3, Distribute cell testing: S31 and S21 were subjected to cell viability testing; S32 was subjected to sterility testing in the same way as S25; S33 Gram staining; S34 Clinically Adaptive Cytokine Detection: BMP-2 is measured in the bone repair scenario, and NGF is measured in the nerve injury scenario; S35 inflammatory factor detection; S4. Packaging according to clinical needs: 2ml cryovials, 50ml blood bags, with temperature-sensitive labels inside the packaging; Store frozen at -196℃ and transport at 4~10℃.

2. The method for evaluating the quality of an umbilical cord mesenchymal stem cell preparation according to claim 1, characterized in that: The S24 multi-directional differentiation potential detection is specifically as follows: Osteogenesis was induced using α-MEM medium containing 10% FBS, 10 nM dexamethasone, 50 μg / ml ascorbic acid, and 10 mM β-glycerophosphate. After 21 days of culture, the cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% alizarin red S for 30 min. The percentage of calcium nodule-positive cells was counted under a microscope. Adipogenic induction was performed using DMEM medium containing 10% FBS, 1 μM dexamethasone, 0.5 mM IBMX, 10 μg / ml insulin, and 200 μM indomethacin. After 14 days of culture, the cells were fixed with 4% paraformaldehyde, infiltrated with 60% isopropanol for 5 min, stained with 0.5% Oil Red O for 15 min, and differentiated with isopropanol for 2 min. The percentage of lipid droplet-positive cells was then counted. Chondrogenic induction was performed using high-glucose DMEM medium containing 10 ng / ml TGF-β3, 1% ITS+Premix, 50 μg / ml ascorbic acid-2-phosphate, 40 μg / ml L-proline, and 100 nM dexamethasone. Cell microspheres were cultured for 21 days, fixed with 4% paraformaldehyde, and stained with 1% Alcian blue for 1 hour. The percentage of positive chondrogenic cells was then counted.

3. The method for evaluating the quality of an umbilical cord mesenchymal stem cell preparation according to claim 1, characterized in that: S23 dynamic phenotypic monitoring was performed using flow cytometry: 1×10⁻⁶ cells were collected before cryopreservation. 6 Two hundred and eight cells were revived. 24 hours after revival, an equal number of cells were revived and cultured. PE-labeled CD29 / CD44 / CD73 / CD90 / CD105 antibodies and FITC-labeled CD14 / CD19 / CD34 / CD45 / HLA-DR antibodies were added to each cell. The cells were incubated at 4°C in the dark for 30 minutes, washed twice with PBS, and analyzed by instrument. Each time point was repeated three times, and the average value was used for determination.

4. The method for evaluating the quality of an umbilical cord mesenchymal stem cell preparation according to claim 1, characterized in that: S25 Real-Time PCR Detection: Microbial DNA was extracted from cell supernatant. Primers for *E. coli*, *Staphylococcus aureus*, and *Candida albicans* were used. The reaction system consisted of 10 μl of 2×SYBR Green Mix, 0.5 μl each of forward and reverse primers, 2 μl of template DNA, and 7 μl of ddH2O. The reaction conditions were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 15 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s, for 40 cycles. A Ct value > 35 was considered as indicating the absence of corresponding microbial contamination.

5. The method for evaluating the quality of an umbilical cord mesenchymal stem cell preparation according to claim 1, characterized in that: The S34 cytokine assay was performed using an ELISA method: 100 μl of cell supernatant was added to an enzyme-labeled plate pre-coated with BMP-2 / NGF antibody and incubated at 37°C for 1 h. After washing, biotin-labeled detection antibody was added and incubated at 37°C for 30 min. Horseradish peroxidase-labeled streptavidin was then added and incubated at 37°C for 15 min. TMB substrate was used for color development for 10 min, and the reaction was terminated with 2 M H2SO4. The absorbance was measured at 450 nm, and the concentration was calculated according to the standard curve. BMP-2 ≥ 50 pg / ml and NGF ≥ 30 pg / ml were considered acceptable.

6. The method for evaluating the quality of an umbilical cord mesenchymal stem cell preparation according to claim 1, characterized in that: The S35 inflammatory factor detection method uses the CBA method: 50 μl of cell supernatant is mixed with 50 μl of IL-6 / TNF-α capture microspheres, incubated in the dark for 30 min, the detection antibody is added and incubated for 20 min, and then the sample is detected. The concentration is determined according to the standard. IL-6 < 10 pg / ml and TNF-α < 5 pg / ml are considered qualified.

7. The method for evaluating the quality of an umbilical cord mesenchymal stem cell preparation according to claim 1, characterized in that: S21 cell viability can also be detected using the AO / PI double staining method: mix 10 μl of cell suspension with 10 μl of AO / PI staining solution, and count the ratio of green fluorescence to red fluorescence under a fluorescence microscope. A viability of >85% is considered acceptable.

8. The method for evaluating the quality of an umbilical cord mesenchymal stem cell preparation according to claim 1, characterized in that: S4 packaging temperature-sensitive label: When the temperature is >10℃ or <4℃, the label changes from blue to red. During transportation, the label should be observed every 2 hours to ensure that it remains blue.

9. The method for evaluating the quality of an umbilical cord mesenchymal stem cell preparation according to claim 1, characterized in that: S27 genome methylation-telomerase activity association assay: The methylation level of the LINE-1 gene was detected by bisulfite sequencing, and the telomerase activity was detected by TRAP method. The methylation level fluctuation was <5% and the ratio of the telomerase activity OD value to the initial value was >0.9 to be qualified.

10. The method for evaluating the quality of an umbilical cord mesenchymal stem cell preparation according to claim 1, characterized in that: In S1, ALT detection was performed using a continuous monitoring method: the reaction system contained Tris-HCl buffer, L-alanine, α-ketoglutarate, and NADH. The rate of decrease in absorbance at 340 nm was monitored at 37°C, and the ALT activity was calculated. A value ≤40U was considered acceptable.