Preparation method of human umbilical cord mesenchymal stem cell injection and product thereof

Abstract

The invention relates to the technical field of medicine, in particular to a preparation method of a human umbilical cord mesenchymal stem cell injection and a product thereof, and the preparation method at least comprises the following steps: obtaining P0-generation secondary human umbilical cord mesenchymal stem cells; and carrying out inoculated culture on the P0-generation human umbilical cord mesenchymal stem cells, carrying out cell digestion, centrifugation and washing, and then carrying out resuspension by using an auxiliary material solution to obtain the human umbilical cord mesenchymal stem cell injection. The human umbilical cord mesenchymal stem cells obtained by screening and culturing the primary separation, the culture medium, the inoculation density, the centrifugal parameters, the culture container, the digestive juice, the washing conditions and the like have higher cell viability and more stable cell growth characteristics. According to the preparation method of the human umbilical cord mesenchymal stem cell injection provided by the invention, the high-quality and high-activity cell injection can be prepared, and a reliable guarantee is provided for clinical application.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a method for preparing a human umbilical cord mesenchymal stem cell injection solution and its product. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell with self-renewal and multi-lineage differentiation potential, widely found in various human tissues such as bone marrow, adipose tissue, and umbilical cord. Among them, human umbilical cord-derived MSCs have become a hot topic in stem cell research and application due to their wide availability, convenient sourcing, low immunogenicity, and strong proliferative capacity. Human umbilical cord MSCs not only possess multi-lineage differentiation potential, capable of differentiating into various cell types such as bone, cartilage, adipose tissue, cardiomyocytes, and nerve cells, but also exhibit various biological characteristics such as immunomodulation, anti-inflammation, and anti-oxidative stress, showing broad application prospects in tissue repair, organ regeneration, and the treatment of autoimmune diseases.

[0003] For example, Chinese invention patent application CN117643570A discloses the preparation and application of a human umbilical cord mesenchymal stem cell injection solution. The method involves isolating human umbilical cord mesenchymal stem cells and culturing them to passage P4, measuring their surface markers, and requiring CD73+, CD90+, CD105+ ≥95%, and CD34+ ≤5%. The human umbilical cord mesenchymal stem cells are mixed with physiological saline to prepare an injection solution, with each milliliter of the injection solution containing 1×10⁻⁶ cells / mL. 5 Personal umbilical cord mesenchymal stem cells. However, the current preparation process of human umbilical cord mesenchymal stem cells and their injections suffers from problems such as low primary isolation efficiency, poor culture medium compatibility, cell viability easily affected by process parameters, and difficulty in controlling impurity residues. These issues lead to insufficient product quality stability, limiting their clinical translational applications. Therefore, there is an urgent need to establish a standardized and efficient preparation method to ensure product quality. Summary of the Invention

[0004] To address the problems in the prior art, this invention provides a method for preparing a human umbilical cord mesenchymal stem cell injection solution, comprising at least the following steps: P0 generation human umbilical cord mesenchymal stem cells were obtained; The P0 generation human umbilical cord mesenchymal stem cells were seeded and cultured. After cell digestion, centrifugation, washing, and resuspending in an excipient solution, the human umbilical cord mesenchymal stem cell injection solution was obtained.

[0005] In one embodiment, the P0 generation human umbilical cord mesenchymal stem cells are obtained by umbilical cord tissue isolation and culture via direct seeding.

[0006] In one embodiment, the direct inoculation method includes: receiving a fresh umbilical cord; removing and discarding 2-3 cm sections of tissue from both ends of the umbilical cord in a biosafety cabinet; washing the tissue three times with sterile tissue washing solution to remove bloodstains and impurities; cutting the umbilical cord into 2-3 cm pieces and removing the umbilical artery and vein before inoculating it at T150cm. 2 Human umbilical cord mesenchymal stem cells (P0 generation) were harvested when the cells were cultured in a cell culture flask at 37°C and CO2 concentration of 5.0% until the cell confluence was not less than 60%.

[0007] In one embodiment, the obtained P0 generation human umbilical cord mesenchymal stem cells are subjected to cell digestion, centrifugation, and cell counting before being seeded and cultured.

[0008] In one embodiment, after centrifugation, the supernatant is discarded, and the cell pellet is resuspended in culture medium. The suspension is collected in a sterile square bottle. Culture medium is added to wash the centrifuge tube, and all the washings are transferred to the sterile square bottle and mixed by pipetting. 100-200 μL of cell suspension is taken into a 1.5 mL centrifuge tube for cell counting. 20 μL of 0.4% trypan blue is mixed with 20 μL of cell suspension and injected into a cell counting chamber. The cells are counted using a CountStar cell counter, and the average value is taken. The total number of viable cells is calculated based on the cell counter.

[0009] In one embodiment, the culture medium used in the inoculation culture includes phenol-free red human umbilical cord mesenchymal stem cell serum-free basal medium and HC-UC02F basal medium additive.

[0010] In a preferred embodiment, the volume ratio of the phenol-free red human umbilical cord mesenchymal stem cell serum-free basal culture medium to the HC-UC02F basal culture medium additive is (20-25):1.

[0011] In a more preferred embodiment, the volume ratio of the phenol-free red human umbilical cord mesenchymal stem cell serum-free basal culture medium to the HC-UC02F basal culture medium additive is 20:1.

[0012] In one embodiment, the culture medium is sourced from Suzhou Huachen Biotechnology Co., Ltd.

[0013] In one embodiment, the inoculation density in the inoculation culture is 3000-8000 cells / cm². 2 .

[0014] In one embodiment, the culture container for the inoculation culture is a Corning-CellBIND-T150 culture flask, a Jet-T175 culture flask, a Corning-TC-T150 culture flask, or a T3180 cm culture flask. 2 Any of the culture flasks.

[0015] In one embodiment, the digestive solution for the cell digestion step includes a recombinant trypsin digestive solution.

[0016] In one embodiment, the recombinant trypsin digest solution is sourced from Shanghai Yuanpei Biotechnology Co., Ltd.

[0017] In one embodiment, the cell digestion step includes: adding recombinant trypsin digestion solution to a culture flask and transferring it to a 37.0°C incubator for digestion until the cells detach from the bottom of the culture flask, appear round and clear, and then adding sodium chloride injection solution to terminate the digestion.

[0018] In one embodiment, the centrifugation parameters are a centrifugal force of 300-400g and a time of 5min.

[0019] In one embodiment, the precipitate is resuspended after centrifugation to obtain a suspension, and the suspension is washed twice.

[0020] In one embodiment, the centrifuged precipitate is resuspended in a phenol-free red human umbilical cord mesenchymal stem cell serum-free basal culture medium to obtain a suspension; the second and third washes are performed using a mixed solution of human serum albumin injection and sodium chloride injection as the washing solution.

[0021] In one embodiment, the volume ratio of the human serum albumin injection to the sodium chloride injection is 1:9.

[0022] In one embodiment, after each wash, the cell density is adjusted to 3.00E+06 cells / mL-1.00E+07 cells / mL based on the wash count results.

[0023] In one embodiment, the resuspension includes: after centrifugation, discarding the supernatant, adding phenol-free red human umbilical cord mesenchymal stem cell serum-free basal culture medium to resuspend the precipitate, and collecting the suspension in a sterile bottle; adding washing buffer to rinse once again, transferring all the washing buffer to the sterile bottle, and then storing it temporarily at 2-8°C. Take 100-200 μL of cell suspension into a 1.5 mL centrifuge tube for cell counting; mix 20 μL of 0.4% trypan blue with 20 μL of cell suspension and inject into a cell counting chamber, count using a CountStar cell counter, and take the average value; after counting, adjust the cell density to 3.00E+06 cells / mL-1.00E+07 cells / mL, aliquot all the cell suspension into centrifuge tubes, balance, centrifuge at 400g for 5 min, and perform an 8% increase and 4% decrease.

[0024] In one embodiment, the first washing includes: after centrifugation, discarding the supernatant, adding a mixed solution of human serum albumin injection and sodium chloride injection to resuspend the cell pellet, and collecting the suspension in a sterile bottle; adding washing solution again to rinse once more, transferring all the washing solution to the sterile bottle, adjusting the cell density to 3.00E+06 cells / mL-1.00E+07 cells / mL based on the first washing count results, and mixing all the suspension by pipetting to make up to a final volume. Take 100-200 μL of cell suspension into a 1.5 mL centrifuge tube for cell counting; mix 20 μL of 0.4% trypan blue with 20 μL of cell suspension and inject into a cell counting chamber, count using a CountStar cell counter, and take the average value; after counting, aliquot all the cell suspension into centrifuge tubes, balance, centrifuge at 400 g for 5 min, with an elevation of 8 and a descent of 4.

[0025] In one embodiment, the second washing repeats the first washing step, and after centrifugation, the supernatant is removed to obtain a precipitate containing human umbilical cord mesenchymal stem cells.

[0026] In one embodiment, the precipitate is resuspended in an excipient solution to obtain a human umbilical cord mesenchymal stem cell injection solution. The human umbilical cord mesenchymal stem cell injection solution is then aliquoted for subsequent use and cryopreservation.

[0027] In one embodiment, the excipient solution comprises, by mass percentage, 5-10% dimethyl sulfoxide, 5-25% human serum albumin injection, and the remainder being compound electrolyte injection.

[0028] The second aspect of the present invention provides a method for preparing a human umbilical cord mesenchymal stem cell injection solution and the resulting product.

[0029] Beneficial effects 1. The number of cells harvested per unit mass using the direct inoculation method of this invention is significantly higher than that using the block method, and the direct inoculation method is simpler to operate.

[0030] 2. This invention uses phenol-free red human umbilical cord mesenchymal stem cell serum-free basal medium and HC-UC02F basal medium additive as the inoculation culture medium. The morphology of cells cultured at low, medium, and high passages showed no significant differences. At the high passage (P16), the cells adhered to the culture wall, exhibiting spindle-shaped or irregular triangular growth in a whorled arrangement. The culture supernatant was clear, with no obvious cell debris. Furthermore, the cell expansion stability was better. The quality inspection results of the cultured cells met the STP-QC-045 "Quality Standard for Umbilical Cord Mesenchymal Stem Cell Injection".

[0031] 3. This invention controls the inoculation density to be 3000-8000 cells / cm². 2The cells have a higher PDL (population density), and all cells adhere to the wall, are spindle-shaped, and do not detach. After 3-4 days of culture, the confluence can reach 85% or more.

[0032] 4. This invention uses Corning-CellBIND-T150 culture flasks, Jete-T175 culture flasks, Corning-TC-T150 culture flasks, and T3180 cm⁻¹ culture flasks. 2 Any of the culture flasks can be used as a culture container, resulting in high stability for cell expansion.

[0033] 5. The present invention provides better uniformity of the total number of cells obtained after digestion with recombinant trypsin digestion solution, and has no visible effect on the biomarkers of cells that have been digested, dissociated and passaged.

[0034] 6. The centrifugation parameters controlled by this invention are 300-400g centrifugal force and 5min time, which can maintain the balance between cell viability and cell loss rate.

[0035] 7. The present invention can significantly reduce digestive fluid residue by washing twice. Taking into account both production efficiency and cell quality, the present invention performs two more washes after the first wash. Attached Figure Description

[0036] Figure 1 The images show the adherent growth status of P0 (low passage), P6 (medium passage), and P12 (high passage) cells cultured in medium 1. In the images, a, b, and c are the adherent growth status of P0 (low passage), P6 (medium passage), and P12 (high passage) cells under a 4X magnification microscope, respectively; d, e, and f are the adherent growth status of P0 (low passage), P6 (medium passage), and P12 (high passage) cells under a 10X magnification microscope, respectively.

[0037] Figure 2 The images show the adherent growth status of P0 (low passage), P6 (medium passage), and P12 (high passage) cells cultured in medium 2. In the images, a, b, and c are the adherent growth status of P0 (low passage), P6 (medium passage), and P12 (high passage) cells under a 4X magnification microscope, respectively; d, e, and f are the adherent growth status of P0 (low passage), P6 (medium passage), and P12 (high passage) cells under a 10X magnification microscope, respectively.

[0038] Figure 3Images show the adherent growth status of P0 (low passage), P6 (medium passage), and P12 (high passage) cells cultured in medium 3. In the images, a, b, and c are images of the adherent growth status of P0 (low passage), P6 (medium passage), and P12 (high passage) cells under a 4X magnification microscope, respectively; d, e, and f are images of the adherent growth status of P0 (low passage), P6 (medium passage), and P12 (high passage) cells under a 10X magnification microscope, respectively.

[0039] Figure 4 The images show the adherent growth status of high-passage (P14, P15) cells cultured in medium 3; in the figures, a and b are the adherent growth status of P14 cells under 4X and 10X magnification, respectively; and c and d are the adherent growth status of P15 cells under 4X and 10X magnification, respectively. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents, biological materials, and detection kits described are commercially available unless otherwise specified.

[0041] This example provides a method for preparing a human umbilical cord mesenchymal stem cell injection solution, including the following steps: Upon receiving a fresh umbilical cord, remove and discard 2-3 cm sections from both ends of the cord within a biosafety cabinet. Clean the cord three times with sterile tissue washing solution to remove blood and impurities. Cut the umbilical cord into 2-3 cm pieces, remove the umbilical artery and vein, and then inoculate it at T150cm. 2 Human umbilical cord mesenchymal stem cells (P0 generation) were harvested when the cells were cultured in a cell culture flask at 37°C and CO2 concentration of 5.0% until the cell confluence was not less than 60%. The P0 generation human umbilical cord mesenchymal stem cells were digested and centrifuged. After centrifugation, the supernatant was discarded, and the cell pellet was resuspended in culture medium. The suspension was collected in a sterile bottle. Culture medium was added to wash the centrifuge tubes, and all washings were transferred to the sterile bottle and mixed thoroughly. 100-200 μL of the cell suspension was transferred to a 1.5 mL centrifuge tube for cell counting. 20 μL of 0.4% trypan blue was mixed with 20 μL of the cell suspension and injected into a cell counting chamber. The cells were counted using a CountStar cell counter, and the average value was taken. The total number of viable cells was calculated based on the cell counter data. The cells were then incubated at 150 cm⁻¹. 2 Cell culture flasks were filled with 0.5 × 10⁻⁶ cells. 4 cells / cm 2The inoculation density was incubated at 37.0℃ in a 5.0% CO2 incubator for 3-5 days; after cell digestion, centrifugation, washing, and resuspending in an excipient solution, the human umbilical cord mesenchymal stem cell injection solution was obtained.

[0042] The cell digestion steps are as follows: add recombinant trypsin digestion solution to the culture flask and transfer to a 37.0°C incubator for digestion until the cells detach from the bottom of the culture flask, appear round and clear, and then add sodium chloride injection solution to terminate the digestion.

[0043] After centrifugation, the precipitate is resuspended to obtain a suspension, which is then washed twice: The resuspension process is as follows: After centrifugation, the supernatant is discarded, and the cell pellet is resuspended in washing buffer, serum-free basal culture medium for human umbilical cord mesenchymal stem cells, and the suspension is collected in a sterile bottle; washing buffer is added again to rinse once, and all washing buffer is transferred to the sterile bottle and then temporarily stored at 2-8℃. Take 150 μL of cell suspension into a 1.5 mL centrifuge tube for cell counting; mix 20 μL of 0.4% trypan blue with 20 μL of cell suspension and inject into a cell counting chamber, count using a CountStar cell counter, and take the average value; after counting, adjust the cell density to 3.00E+06 cells / mL, aliquot all the cell suspension into centrifuge tubes, balance, centrifuge at 400g for 5 min, and perform an 8°C upsampling and 4°C downsampling.

[0044] The first wash was performed as follows: After centrifugation, the supernatant was discarded, and the cell pellet was resuspended in a mixture of human serum albumin injection and sodium chloride injection. The suspension was collected in a sterile bottle. The cell pellet was rinsed again with washing solution, and all the washing solution was transferred to the sterile bottle. Based on the cell count results of the first wash, the cell density was adjusted to 3.00E+06 cells / mL. The suspension was then mixed thoroughly and the volume was adjusted. Take 150 μL of cell suspension into a 1.5 mL centrifuge tube for cell counting; mix 20 μL of 0.4% trypan blue with 20 μL of cell suspension and inject into a cell counting chamber, count using a CountStar cell counter, and take the average value; after counting, aliquot all the cell suspension into centrifuge tubes, balance, centrifuge at 400 g for 5 min, with an elevation of 8 and a descent of 4.

[0045] The second washing process was the same as the first washing process. After centrifugation, the supernatant was removed to obtain a precipitate containing human umbilical cord mesenchymal stem cells.

[0046] The culture medium consisted of 500 mL of phenol-free red human umbilical cord mesenchymal stem cell serum-free basal medium and 25 mL of HC-UC02F basal medium additive, sourced from Suzhou Huachen Biotechnology Co., Ltd.

[0047] The recombinant trypsin digest solution was obtained from Shanghai Yuanpei Biotechnology Co., Ltd.

[0048] The volume ratio of the human serum albumin injection to the sodium chloride injection is 1:9.

[0049] The precipitate was resuspended in an excipient solution to obtain a human umbilical cord mesenchymal stem cell injection solution. The human umbilical cord mesenchymal stem cell injection solution was aliquoted for subsequent use and cryopreservation.

[0050] The excipient solution, by mass percentage, includes 5% dimethyl sulfoxide, 25% human serum albumin injection, and the remainder is compound electrolyte injection.

[0051] The second aspect of this example provides a method for preparing a human umbilical cord mesenchymal stem cell injection solution and the resulting product.

[0052] Performance testing 1. An investigation into the effects of direct implantation and graft methods on the obtained P0 passage human umbilical cord mesenchymal stem cells: Umbilical cord tissue from donor CUC-24004 was isolated using the implantation method and seeded into 100 mm cell culture dishes; umbilical cord tissue from donor CUC-24005 was isolated using the direct seeding method and seeded into T150 cm cells. 2 Cell culture flasks. Umbilical cord tissues isolated by the different isolation methods were placed in the same culture medium and under the same culture conditions (37℃, 5.0% CO2) and cultured until the cell confluence was not less than 60%. P0 generation cells were harvested and counted. The P0 generation cell emergence time, culture time, and harvest count results are detailed in Table 1.

[0053] Table 1

[0054] Table 1 shows that there were no significant differences in cell emergence time, culture duration, cell viability, and cell diameter between the two isolation methods for P0 passage cells. However, CUC-24004, after being chopped, weighed 33.028 g. When seeded into 100 mm cell culture dishes using the block method, the total number of cells harvested was 1.85E+07 cells, with a cell count per unit mass of 5.60E+05 cells / g. CUC-24005, after being chopped, weighed 42.635 g. When seeded into T150 cm² cell culture flasks using the direct inoculation method, the total number of cells harvested was 4.58E+07 cells, with a cell count per unit mass of 1.07E+06 cells / g. The cell count per unit mass harvested using the direct inoculation method used in this application was significantly higher than that using the block method.

[0055] 2. Investigation into the effect of culture medium on cell culture: Investigate the effects of different manufacturers' culture media on cell culture as shown in Table 2: 1) Cell counting results: Table 2

[0056] In this experiment, the umbilical cord tissue of donor CUC-24008 was isolated using the direct inoculation method. The relevant cell sources are detailed in Table 3.

[0057] Table 3

[0058] Primary cells isolated by the direct inoculation method, donor number CUC-24008, were inoculated into culture media 1-5. Culture parameters: 37.0℃, 5.0% CO2. PDL was calculated based on the cell count results of continuous passage culture, and the results are shown in Table 4.

[0059] Table 4

[0060] Table 4 shows that five different culture media were used for continuous passage culture of human umbilical cord mesenchymal stem cells from the primary culture stage. Culture media 4 and 5 were discarded due to their low amplification values ​​in the early stages. Based on the RSD of the PDL, the remaining three culture media showed that culture medium 3 exhibited better cell amplification stability than culture media 1 and 2.

[0061] 2) Cell adhesion diagram Cells from batch CUC-24008-P0 were passaged continuously in five different culture media. Since media 4 and 5 were discarded during passage, adhesion diagrams are not included in the table below. Cell morphology comparisons were performed for P0 (low passage), P6 (medium passage), and P12 (high passage) cells cultured in the remaining three media; see details below. Figure 1-3 For a detailed diagram of the adherent growth status of high-passage cells cultured in medium 3, please see [link to diagram]. Figure 4 .

[0062] As shown in the figure, cells cultured in medium 2 at low, medium, and high passages gradually exhibited a slender, spindle-shaped morphology, with significant changes in morphology. The supernatant from high passages contained a large amount of cell debris. Cells cultured in medium 1 showed lateral edge lifting and detachment. Cells cultured in medium 3, used in this application, showed no significant difference in morphology across low, medium, and high passages. Furthermore, at the high passage (P15), cells adhered to the culture wall, growing in a spindle or irregular triangular shape, arranged in a whorl pattern. The supernatant was clear, with no obvious cell debris. Moreover, cells cultured in medium 3 showed better expansion stability than those cultured in the other media.

[0063] 3. Inoculation density test: 1) Cell counting results: Cells were cultured using medium 1. After thawing from passage P4, cells were seeded at different densities in monolayer and quinary layers. The quinary layers were divided into two groups: one group was supplemented with 700 mL of medium per bottle, and the other group was supplemented with 900 mL of medium per bottle. Cells were cultured at 37°C and 5.0% CO2. Passage P5 cells were harvested and counted. The harvested P5 cells were then cultured under the same seeding and culture conditions as passage P4, and passage P6 cells were harvested and counted. The cell counting results after cryopreserved and fresh cell expansion cultures are shown in Table 5.

[0064] Table 5

[0065] 2) Cell adhesion diagram: Cells were cultured using OptiVitro® MSC serum-free amplification kit ME 0 1. The cell adhesion and growth status of P3 and P4 generations harvested at different seeding densities are shown in Table 6.

[0066] Table 6

[0067] As shown in Tables 5 and 6 above, there were no significant differences in cell viability and cell diameter among cells cultured at different seeding densities. (Cell seeding density: 3000-8000 cells / cm²) 2 When cells are cultured for the same duration in culture dishes or monolayer flasks, and then harvested, the cell PDL is 9000-1000 cells / cm². 2 The inoculation density is even higher, ranging from 3000 to 8000 cells / cm². 2 At this time, there was no obvious cell detachment, and when the inoculated cell density was 9000-10000 cells / cm³, 2 At that time, cell shedding was obvious.

[0068] 4. Culture container screening test: After digesting and dissociating the freshly cultured cells, they were separated at a cell density of (6.0-8.0) × 10⁻⁶. 3 cells / cm 2 Cells were seeded into cell culture flasks of different brands and cultured in a CO2 incubator at 37.0℃ using Zhong Sheng's medium (Nuwacell®ncMission hMSC Medium + Nuwacell®ncMission Supplement (21×)). The cell counts at each passage are shown in Tables 7-8.

[0069] Table 7

[0070] Table 8

[0071] Table 7-8 shows that, among single-layer flasks, the order of priority for cell expansion stability and cell expansion capacity at each passage is: Corning-CellBIND-T150 > Jet-T175 > Corning-TC-T150. Among five-layer flasks, the Corning-5-layer flask exhibits better cell expansion stability at each passage than the Nest-5-layer flask.

[0072] 5. Digestive fluid screening test: Cells were cultured in Zhong Sheng's medium (Nuwacell® ncMission hMSC Medium + Nuwacell® ncMissionSupplement (21×)) for experiments (donor number CUC-24006, cell passage P4, cell batch number CUC24006M2404001-P2). After recovery at passage P2, cells were digested using two different digestion solutions (Gibco digestion dissociation enzyme and Shanghai Yuanpei recombinant trypsin digestion solution used in this application) and three digestion states (50% representing under-digestion, 100% representing normal digestion, and 150% representing over-digestion). A total of six groups were formed. After inoculation, each group was cultured at 37℃ and 5.0% CO2, and continuously passaged to P5. The cells after recovery were designated as passage P3. Since passage P3 cells were not dissociated using digestion solutions, they were not used for evaluation. Experimental data were recorded from passage P4 onwards. The cell count results after cell expansion culture are shown in Tables 9-10.

[0073] Table 9

[0074] Table 10

[0075] The data in the table above shows that there was no significant difference in cell viability after passage culture from cells digested with different digestion solutions. For cells of the same passage number, there was no significant difference in the total number of cells harvested after digestion with different degrees of digestion using Yuanpei digestion solution. However, the total number of cells harvested after digestion with different degrees of digestion using Gibco digestion solution fluctuated significantly. These phenomena indicate that the total number of cells harvested after digestion with Yuanpei digestion solution is more uniform than that harvested with Gibco digestion solution. Considering the actual production process, the transfer time of cell culture flasks fluctuates within a certain range from the addition of digestion solution to the termination of digestion; therefore, selecting a digestion solution with stable digestion capacity is particularly important. Furthermore, considering the results of cell biomarker analysis after digestion and passage with both digestion solutions (no significant differences in cell biomarker test data between different cell groups; according to STP-QC-041 "R809 Cell Injection Solution Quality Standard", all cell biomarker test data met the predetermined acceptance criteria), and from the perspective of cost reduction and efficiency improvement, the digestion solution from Shanghai Yuanpei Biotechnology was selected as the digestion solution for this application.

[0076] 6. Centrifugation parameter screening experiment: Centrifugation is an efficient method for separating cell debris and dead cells to obtain high-purity live cells. This application tested centrifugation parameters for both cryopreserved cell resuscitation and fresh cell resuscitation; details of the cell sources are shown in Table 11.

[0077] Table 11

[0078] 1) CUC-24005-P2 and CUC-24001-P2 cryopreserved cells were revived in a water bath. After revival, the cells were centrifuged twice at 200g, 300g, and 400g for 5 minutes, with centrifugation parameters of 8°C for both elevation and 4°C for depression. After each centrifugation, the cells were resuspended and counted. The number of viable cells, cell viability, and cell diameter were measured. The results are shown in Tables 12-13.

[0079] Table 12

[0080] Table 13

[0081] As shown in Tables 12-13, the cell viability was above 90% after centrifugation under different conditions, with no significant differences in cell diameter and aggregation rate. When the centrifugation force was set to 400g, cell loss from different donors was less than under other centrifugation conditions.

[0082] 2) Resuscitate CUC-24004-P2 cells in a water bath and inoculate them into culture. Digest and dissociate freshly cultured CUC-24004-P3 cells, aliquot them, and bring them to the same volume. Centrifuge them at 300 g, 400 g, and 500 g for 5 minutes with an 8:4 gradient. After centrifugation, count the cells. After counting, bring the volume to the same level with sodium chloride injection solution. Repeat the centrifugation, resuspending, and counting steps 5 times. Observe the number of viable cells, cell viability, and cell diameter. The results are detailed in Table 14.

[0083] Table 14

[0084] Table 14 shows that when freshly cultured cells were centrifuged at centrifuges with centrifugation forces of 300 g, 400 g, and 500 g, the cell viability was above 90% within five centrifugations, but below 90% after the sixth centrifugation. There was no significant change in cell diameter after six centrifugations. The clumping rate did not change trend with increasing centrifugation count. The amount of cell loss increased with increasing centrifugation count.

[0085] During scale-up production, fresh cells are typically centrifuged no more than three times. Therefore, analysis of the data from the first three centrifugations in Table 14 shows that the average cell loss rate of 11.2% at a centrifugal force of 500g is significantly higher than that at 300g (6.5%) or 400g (5.2%). Therefore, centrifugation at 300-400g is recommended for fresh cells.

[0086] 7. Washing condition screening test: Suitable washing conditions can effectively remove process-related and product-related impurities, ensuring the safety and efficacy of the cell injection solution. Therefore, experiments were conducted to test washing conditions and to investigate and determine the composition of the washing solution, the number of washes, and the washing density. The composition of each washing solution is shown in Table 15.

[0087] Table 15

[0088] See Table 16 for cell sources.

[0089] Table 16

[0090] 1) Washing solution screening: Cells were resuspended in Liji Biotechnology's culture medium (serum-free basal medium for human umbilical cord mesenchymal stem cells (phenol red-free) + serum-free additive for human umbilical cord mesenchymal stem cells, with a volume ratio of basal medium to additive of 9:1) to confirm cell viability of not less than 90%. The cells were mixed and divided into 8 groups. After centrifugation, the supernatant was discarded, and the cell pellet was resuspended in equal volumes of washing buffer 1, washing buffer 2, washing buffer 3, and washing buffer 4. Two groups of cells were resuspended in each washing buffer. One group was incubated at 18-26℃, and the other group was incubated at 2-8℃. Cells were counted using a cell counter at 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 18h, 20h, and 24h. Counting was stopped when the cell viability was below 80%. The cell count results at different time points are shown in Table 17.

[0091] Table 17

[0092] As can be seen from the table above, the cell suspensions resuspended in each washing solution showed a decreasing trend in viable cell density with increasing storage time when placed at 2-8℃ and 18-26℃.

[0093] Comparing cell counts after the same washing solution and the same storage time under different storage temperature conditions, the viable cell density was higher under the storage condition of 2-8℃ than that under the storage condition of 18-26℃.

[0094] Under the same placement time with different washing solutions, groups 3 and 4 had higher viable cell densities than the other two groups. Within 5 hours of placement, there was no significant difference in viable cell densities between the two groups. From 6 to 24 hours of placement, the viable cell density in washing solution 3 was higher than that in washing solution 4.

[0095] Cell viability was above 85.00% for all groups of cell suspensions within 24 hours of incubation at 2-8℃. Under incubation conditions of 18-26℃, cell viability decreased from 97.18% to 79.94% after 5 hours in washing buffer 2; however, cell viability was above 85.00% for washing buffers 1, 3, and 4 within 8 hours of incubation.

[0096] Meanwhile, the cell adhesion of each group was observed 72 hours after cell inoculation. Cells at the same inoculation density adhered and grew in a spindle shape, with no significant differences.

[0097] In summary, when human umbilical cord mesenchymal stem cells are resuspended in the same washing solution and placed for the same period of time, the cell density and viability are higher and more stable under the 2-8℃ storage condition than under the 18-26℃ storage condition. To ensure cell condition, if washed and resuspended cells need to be temporarily stored, 2-8℃ is preferred.

[0098] During pilot production, subsequent washes after the initial wash are typically performed at room temperature (18-26℃) for approximately 3 hours. Comparing the results at 18-26℃ for 3 hours, wash solutions 3 and 4 showed higher and more stable viable cell density and viability compared to other groups. Within 5 hours, there was no significant difference in viable cell density and viability between the two groups. Considering cost, wash solution 3 consists of HSA and compound electrolyte injection, while wash solution 4 consists of HSA and sodium chloride injection. Therefore, wash solution 4 is recommended for subsequent washes after the initial wash.

[0099] 2) Washing frequency screening: Cell sources are detailed in Table 16. Cells were resuspended in Liji Biotechnology's basal culture medium (human umbilical cord mesenchymal stem cell serum-free basal culture medium (phenol red-free)) to confirm cell viability of at least 90%. The cells were then mixed thoroughly and divided into two groups. After centrifugation, the supernatant was discarded. The cell suspension was brought to a final volume of 5.0E+06 cells / mL using washing buffers 3 and 4. Cell counts were performed, and cell viability and viable cell density were recorded after washing. After counting, at least 30mL of the supernatant was collected for digestion residue testing. This step was repeated, and the cells from the last wash were used for cell marker testing. Cell count data after multiple washes are shown in Table 18.

[0100] Table 18

[0101] After washing the cells seven times with either washing buffer 3 or washing buffer 4, the viable cell density, total cell density, and viability of both groups fluctuated slightly, with no significant differences between the groups. After seven washes, the cell viability of both groups remained above 90% and showed no significant downward trend, failing to meet the predetermined condition for stopping washing: cell viability below 80.00%. Considering the operability of the process and production efficiency, washing was discontinued in this experiment.

[0102] The test results of residual digestive fluid in the washing supernatant of each group are shown in Table 19: Table 19

[0103] The test results in Table 19 show that the residual digestive fluid in the supernatant of both groups of MSC cells decreased with increasing washing cycles, and after the second wash, the residual digestive fluid was below 10 ng / g, or 10 ppb. Subsequent washes resulted in a slower decrease in residual amount, and washing twice was more efficient. Considering process efficiency and product quality, in this application, after resuspension counting, a maximum of two washes are allowed.

[0104] Furthermore, after the final washing test of cell markers, the cells were washed 7 times with washing solution 3 and washing solution 4 respectively, and the cell markers all met the STP-QC-041 "R809 Cell Injection Solution Quality Standard" with no significant differences.

[0105] 3) Washing density screening: The density of each test is shown in Table 20.

[0106] Table 20

[0107] See Table 21 for cell sources.

[0108] Table 21

[0109] CUC-24006-P4-240514-LJ cells were resuscitated and cultured continuously until passage P6. Before each passage, fresh cells were resuspended in Liji Biotechnology's basal medium (serum-free basal medium for human umbilical cord mesenchymal stem cells (phenol red-free)) and counted. Based on the counting results, washing buffer 4 was added to adjust the cell density to 3.0E+06 cells / mL, 5.0E+06 cells / mL, 7.0E+06 cells / mL, and 1.0E+07 cells / mL, respectively. After washing, the cells were centrifuged twice, and the supernatant from each wash was sent for analysis to remove residual digestive fluid. Relevant test results are shown in Tables 22-23.

[0110] Table 22

[0111] Table 23

[0112] The data in Table 23 above show that the cell yield after one wash with washing buffer 4 compared to the initial cell yield, and the cell yield after two washes with washing buffer 4 compared to the initial cell yield, were both higher than 100%. It is speculated that this phenomenon may be due to the fact that the cells resuspended in the basal medium were in a worse state than those resuspended in washing buffer 4, leading to some of the poorly functioning cells being counted as dead cells during trypan blue staining. Meanwhile, Table 22 shows that the cell viability at harvest P5 was 91.04%. Under different washing densities, the cell viability increased to over 95.00% after washing with washing buffer 4, indicating that some dead cells were removed and the number of viable cells increased, suggesting that some cells recovered and were counted as viable cells. Therefore, the cell yields after one wash with washing buffer 4 compared to the initial cell yield, and the cell yields after two washes with washing buffer 4 compared to the initial cell yield, are only for reference and not for evaluation. This analysis focuses on the cell yield after two washes with washing buffer 4 compared to one wash, calculated from the number of viable cells measured in the same buffer system (washing buffer 4). Table 23 shows that after washing twice with washing solution 4, the cell yield was more than 85.00% higher than after washing once. Under the washing density of 3.00E+06, the cell yields of P5 and P6 passages were 92.11% and 94.39%, respectively, both remaining stable above 90%, which was more stable than other groups.

[0113] Meanwhile, the supernatant of each group was tested for residual digestive fluid. The results showed that when washing cells at densities of 3.0E+06 cells / mL, 5.0E+06 cells / mL, 7.0E+06 cells / mL, and 1.0E+07 cells / mL, the residual digestive fluid in the supernatant after washing with washing solution 4 once met the acceptable standard (1 ppm).

Claims

1. A method for preparing a human umbilical cord mesenchymal stem cell injection, characterized by, At least comprising the following steps: Obtaining P0 generation human umbilical cord mesenchymal stem cells; Inoculating and culturing the P0 generation human umbilical cord mesenchymal stem cells, resuspending the cells after cell digestion, centrifugation and washing with auxiliary solution to obtain the human umbilical cord mesenchymal stem cell injection.

2. The preparation method of the human umbilical cord mesenchymal stem cell injection according to claim 1, characterized in that, The P0 generation human umbilical cord mesenchymal stem cells are obtained by direct inoculation method for umbilical cord tissue separation and culture.

3. The method for preparing the human umbilical cord mesenchymal stem cell injection solution according to claim 1, characterized in that, The culture medium in the inoculation culture includes phenol red-free human umbilical cord mesenchymal stem cell serum-free basic medium and HC-UC02F basic medium additive.

4. The method for preparing the human umbilical cord mesenchymal stem cell injection solution according to claim 1, characterized in that, The inoculation density in the inoculation culture is 3000-8000 cells / cm 2 .

5. The method for preparing the human umbilical cord mesenchymal stem cell injection solution according to claim 3, characterized in that, The culture vessel for the inoculum culture is any one of a Corning-CellBIND-T150 flask, a Jet-T175 flask, a Corning-TC-T150 flask, a T3180 cm 2 flask.

6. The method for preparing the human umbilical cord mesenchymal stem cell injection solution according to claim 1, characterized in that, The digestion solution in the cell digestion step includes recombinant trypsin digestion solution.

7. The method for preparing the human umbilical cord mesenchymal stem cell injection solution according to claim 1, characterized in that, The centrifugal parameter is centrifugal force 300-400g for 5min.

8. The method for preparing the human umbilical cord mesenchymal stem cell injection solution according to claim 1, characterized in that, The precipitate after centrifugation is resuspended to obtain a suspension, and the suspension is washed twice.

9. The method for preparing the human umbilical cord mesenchymal stem cell injection solution according to claim 8, characterized in that, The precipitate after centrifugation is resuspended to obtain a suspension using phenol red-free human umbilical cord mesenchymal stem cell serum-free basic medium; the second and third washing use a mixed solution of human albumin injection and sodium chloride injection as the washing liquid.

10. The method for preparing the human umbilical cord mesenchymal stem cell injection solution according to claim 9, characterized in that, The volume ratio of human albumin injection and sodium chloride injection is 1:

9.

11. The method for preparing the human umbilical cord mesenchymal stem cell injection solution according to claim 8, characterized in that, After each washing, the cell density is adjusted to 3.00E+06 cells / mL-1.00E+07 cells / mL according to the washing count result.

12. The method for preparing the human umbilical cord mesenchymal stem cell injection solution according to claim 1, characterized in that, The auxiliary solution includes 5-10% dimethyl sulfoxide, 5-25% human albumin injection, and the balance is compound electrolyte injection, according to the mass percentage.

13. A product prepared by the preparation method of human umbilical cord mesenchymal stem cell injection according to any one of claims 1-12.

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