Large-scale stem cell culture method

Abstract

The invention discloses a large-scale stem cell culture method, and relates to the technical field of stem cell culture, and the method comprises the following steps: stem cell pretreatment: taking P3-P5 generation mesenchymal or embryo dry and thin parts, digesting for 2-3 minutes at 37 + / -0.5 DEG C by using trypsin containing 0.01%-0.05% of EDTA (Ethylene Diamine Tetraacetic Acid), the pretreated cells are inoculated to a bioreactor containing a modified microcarrier, the microcarrier is a chitosan and gelatin composite material, the aperture is 50-100 microns, the particle size is 200-300 microns, RGD peptide is grafted, dynamic fed-batch culture is conducted, the concentration of glucose and lactic acid is detected in real time through an intelligent feedback system, harvesting and purification are conducted, 0.02%-0.04% of collagenase is added after culture is finished, incubation is conducted for 15-20 min at the temperature of 37 DEG C, and the concentration of glucose and lactic acid in the bioreactor is detected. The limitation of a traditional single-material microcarrier is broken through, the chitosan and gelatin composite carrier is adopted, RGD peptide is grafted, the cell attachment rate is increased, a three-dimensional growth space is provided for cells through specific aperture / particle size design, and the problem of poor cell uniformity in large-scale culture is solved.

Description

Technical Field

[0001] This invention relates to the field of stem cell culture technology, and in particular to a method for large-scale stem cell culture. Background Technology

[0002] With their multi-directional differentiation potential, stem cells have demonstrated their tissue repair advantages in clinical research on spinal cord injury repair, functional reconstruction after myocardial infarction, healing of diabetic foot ulcers, and intervention in degenerative diseases (such as Parkinson's disease). Some stem cell therapies have entered phase III clinical trials, creating an urgent need for standardized, high-dose clinical-grade stem cell preparations. In the construction of disease models, organoids induced from patient-derived stem cells can simulate the pathological processes of Alzheimer's disease, non-alcoholic liver disease, and other diseases, providing a key carrier for research on personalized diagnosis and treatment mechanisms. This requires a large number of highly homogeneous stem cells.

[0003] Traditional stem cell culture methods often use single-material microcarriers such as pure gelatin and pure chitosan, which lack cell-specific binding sites for RGD peptides. This results in low stem cell adhesion rates and easy detachment during stirring, making it difficult to provide cells with a suitable three-dimensional growth space. In terms of nutrient supply, they generally rely on timed feeding modes, which are not easy to match the dynamic metabolic needs of cells in real time. During the high proliferation phase, glucose shortage and excessive lactate accumulation are prone to occur, resulting in low stem cell viability. On the other hand, excessive feeding during the low proliferation phase will waste glucose and glutamine. Therefore, a large-scale stem cell culture method is needed. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies, which often use single-material microcarriers such as pure gelatin or pure chitosan, lacking cell-specific binding sites for RGD peptides. This results in low stem cell adhesion rates and easy detachment during stirring, making it difficult to provide suitable three-dimensional growth space for cells. The technology also relies heavily on timed feeding, which is not easy to match the dynamic metabolic needs of cells in real time. During the high proliferation phase, glucose shortage and excessive lactate accumulation are common, leading to low stem cell viability. On the other hand, excessive feeding during the low proliferation phase can waste glucose and glutamine. Therefore, this invention proposes a large-scale stem cell culture method.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for large-scale stem cell culture includes the following steps: S1. Stem cell pretreatment: P3-P5 generation mesenchymal or embryonic stem cells were digested with trypsin containing 0.01%-0.05% EDTA at 37℃±0.5℃ for 2-3 min to obtain a single-cell suspension. The suspension was then resuspended in DMEM / F12 containing 10%-15% fetal bovine serum and the cell density was adjusted to 1×10⁶ cells / mL. 5 -5×10 5 cells / mL; S2. Construction of composite culture system: Pretreated cells were seeded into a bioreactor containing modified microcarriers. The microcarriers were a composite material of chitosan and gelatin with a pore size of 50-100 μm, a particle size of 200-300 μm, and grafted with RGD peptides. A mixed gas of 5% CO2 + 3% O2 ​​was introduced to maintain pH 7.2-7.4 and temperature 37℃±0.2℃. S3. Dynamic fed-batch culture: The concentrations of glucose and lactate are monitored in real time using an intelligent feedback system. The glucose concentration is maintained at 1.5-3.0 g / L and the lactate concentration is maintained at ≤2.0 g / L. When the glucose concentration is lower than 1.5 g / L, a nutrient supplement containing glucose, glutamine and recombinant human insulin is automatically fed at a flow rate of 5-10 mL / h for 7-10 days. S4. Harvesting and purification: After culture, add 0.02%-0.04% collagenase, incubate at 37℃ for 15-20 min, collect the suspension, filter through a 200-mesh filter, and centrifuge at 800xg for 20 min with 1.073g / mL Percoll solution to obtain stem cells with a viability ≥95% and homogeneity ≥90%.

[0006] The above technical solution further includes: Specifically, the modified microcarrier described in S2 is prepared by dissolving chitosan and gelatin in a 1% acetic acid solution at a mass ratio of 3:2, adding 0.5% RGD peptide, preparing microspheres by emulsification-crosslinking method, crosslinking with 0.1% glutaraldehyde for 2 hours, and then freeze-drying under vacuum.

[0007] Specifically, the nutritional supplement solution described in S3 consists of: glucose 20-30 g / L, glutamine 2-4 g / L, recombinant human insulin 5-10 μg / mL, vitamin C 0.1-0.2 g / L, and EGF 10-20 ng / mL.

[0008] Specifically, the bioreactor in S2 is a stirred bioreactor with a stirring rate of 20-30 rpm, and the inner wall of the reactor is coated with an anti-adsorption coating, which is a phosphate buffer containing 2% bovine serum albumin.

[0009] Specifically, the intelligent feedback feed system in S3 includes: an online glucose sensor, an online lactate sensor, a PLC control unit, and a peristaltic pump. The sensor detection frequency is once every 2 hours, and the PLC control unit automatically adjusts the flow acceleration rate of the peristaltic pump based on the detection data.

[0010] The present invention has the following beneficial effects: This invention breaks through the limitations of traditional single-material microcarriers by using a chitosan and gelatin composite carrier grafted with RGD peptides. This not only improves cell adhesion rate but also provides a three-dimensional growth space for cells through specific pore / particle size design, solving the problem of poor cell uniformity in large-scale culture.

[0011] In this invention, the traditional timed feeding mode is abandoned. Instead, the nutrient supply is dynamically adjusted by real-time detection of glucose and lactic acid concentrations to maintain the cell metabolic environment, improve stem cell viability, reduce nutrient waste, and lower culture medium costs. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the overall process of a large-scale stem cell culture method proposed in this invention; Detailed Implementation

[0013] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0014] Example

[0015] like Figure 1 As shown, the present invention proposes a large-scale stem cell culture method, which includes the following steps: S1. Stem cell pretreatment: P3-P5 generation mesenchymal or embryonic stem cells were digested with trypsin containing 0.01%-0.05% EDTA at 37℃±0.5℃ for 2-3 min to obtain a single-cell suspension. The suspension was then resuspended in DMEM / F12 containing 10%-15% fetal bovine serum and the cell density was adjusted to 1×10⁶ cells / mL. 5 -5×10 5 cells / mL; S2. Construction of composite culture system: Pretreated cells were seeded into a bioreactor containing modified microcarriers. The microcarriers were a composite material of chitosan and gelatin with a pore size of 50-100 μm, a particle size of 200-300 μm, and grafted with RGD peptides. A mixed gas of 5% CO2 + 3% O2 ​​was introduced to maintain pH 7.2-7.4 and temperature 37℃±0.2℃. S3. Dynamic fed-batch culture: The concentrations of glucose and lactate are monitored in real time using an intelligent feedback system. The glucose concentration is maintained at 1.5-3.0 g / L and the lactate concentration is maintained at ≤2.0 g / L. When the glucose concentration is lower than 1.5 g / L, a nutrient supplement containing glucose, glutamine and recombinant human insulin is automatically fed at a flow rate of 5-10 mL / h for 7-10 days. S4. Harvesting and purification: After culture, add 0.02%-0.04% collagenase, incubate at 37℃ for 15-20 min, collect the suspension, filter through a 200-mesh filter, and centrifuge at 800xg for 20 min with 1.073g / mL Percoll solution to obtain stem cells with a viability ≥95% and homogeneity ≥90%.

[0016] Furthermore, P4 generation mesenchymal stem cells were digested with trypsin containing 0.03% EDTA at 37°C for 2.5 min to obtain a single-cell suspension. The suspension was then resuspended in DMEM / F12 medium containing 12% fetal bovine serum, and the cell density was adjusted to 3 × 10⁶ cells / year. 5 Pretreatment was completed using cells / mL; The single-cell suspension was then inoculated into a stirred bioreactor containing modified microcarriers. The modified microcarriers were prepared by emulsification-crosslinking microspheres (3:2 mass ratio of chitosan and gelatin dissolved in 1% acetic acid solution, 0.5% RGD peptide added, followed by crosslinking with 0.1% glutaraldehyde for 2 hours and then freeze-drying under vacuum). The microspheres had a pore size of 80 μm and a particle size of 250 μm. The inner wall of the reactor was coated with a phosphate buffer solution containing 2% bovine serum albumin as an anti-adsorption coating. A mixed gas containing 5% CO2 and 3% O2 ​​was introduced into the reactor to maintain the culture environment at pH 7.3 and temperature 37℃±0.2℃. The stirring rate was controlled at 25 rpm to construct a composite culture system. During the culture process, an intelligent feedback feed system consisting of an online glucose sensor, an online lactate sensor, a PLC control unit, and a peristaltic pump was used. The sensors detected the glucose level in the culture medium every 2 hours. For glucose and lactate concentrations, when the glucose concentration is below 1.5 g / L, the peristaltic pump is adjusted by the PLC control unit to automatically add a nutrient supplement containing 25 g / L glucose, 3 g / L glutamine, 8 μg / mL recombinant human insulin, 0.15 g / L vitamin C, and 15 ng / mL EGF at a rate of 8 mL / h, maintaining the glucose concentration at 1.5-3.0 g / L and the lactate concentration ≤2.0 g / L, and continuing the culture for 8 days. After the culture is completed, 0.03% collagenase is added to the bioreactor, and the cells are incubated at 37°C for 18 min. The cell suspension is collected, filtered through a 200-mesh filter, and purified by density gradient centrifugation at 1.073 g / mL Percoll solution at 800 x g for 20 min, finally obtaining large-scale mesenchymal stem cells with a viability of ≥95% and homogeneity of ≥90%.

[0017] The preparation method of the modified microcarrier in S2 is as follows: chitosan and gelatin are dissolved in 1% acetic acid solution at a mass ratio of 3:2, 0.5% RGD peptide is added, microspheres are prepared by emulsification-crosslinking method, and after crosslinking with 0.1% glutaraldehyde for 2 hours, they are freeze-dried under vacuum.

[0018] Further, take low molecular weight chitosan (molecular weight 50-100kDa) and pharmaceutical grade gelatin and mix them at a mass ratio of 3:2. Add the mixture to a 1% acetic acid solution prepared in advance with deionized water (solid-liquid ratio 1:20). Place the mixture in a 37°C constant temperature water bath and stir magnetically at 300 rpm until completely dissolved to form a homogeneous and transparent aqueous solution. Subsequently, 0.5% of recombinant human RGD peptide (sequence Arg-Gly-Asp) was added, and stirring was continued at 200 rpm for 30 min until the peptide was completely dispersed. When preparing microspheres using the emulsion-crosslinking method, the above aqueous solution was first slowly injected into liquid paraffin containing 5% Span 80 (oil-water volume ratio 1:5), and mechanically stirred at 800 rpm for 30 min at room temperature to form a stable W / O emulsion. Then, 0.1% glutaraldehyde solution (volume ratio to aqueous phase 1:10) was added dropwise to the emulsion, and crosslinking was carried out at room temperature for 2 h while maintaining a stirring rate of 200 rpm. During this period, samples were taken every 30 min to observe the morphology of the microspheres. After crosslinking, the microspheres were allowed to settle, the upper oil phase was discarded, and the microspheres were washed three times with anhydrous ethanol to remove residual liquid paraffin and Span 80. Then, the microspheres were washed five times with deionized water until the pH was neutral (to remove unreacted glutaraldehyde). Finally, the washed microspheres were transferred to a freeze-drying tray, pre-frozen at -50℃ for 4 hours, and then freeze-dried in a vacuum freeze dryer (vacuum degree 0.1mbar, cold trap temperature -80℃) for 24 hours to obtain white porous chitosan and gelatin composite microcarriers with pore size of 50-100μm, particle size of 200-300μm, and surface RGD peptide grafting rate ≥85%.

[0019] The nutritional supplement solution in S3 consists of: glucose 20-30 g / L, glutamine 2-4 g / L, recombinant human insulin 5-10 μg / mL, vitamin C 0.1-0.2 g / L, and EGF 10-20 ng / mL.

[0020] Furthermore, when preparing the S3 nutrient supplement solution, first use sterile, pyrogen-free DMEM / F12 basal culture medium (equilibrated at 25°C for 30 min beforehand) as a solvent. Accurately weigh pharmaceutical-grade anhydrous glucose (purity ≥99.5%) using a 0.01% analytical balance, and add it to the culture medium at a final concentration of 20-30 g / L. Place the solution in a 37°C constant temperature water bath and magnetically stir at 150 rpm for 15 min until the glucose is completely dissolved. Next, add L-glutamine (purity ≥98%, sterile lyophilized powder) at a final concentration of 2-4 g / L, maintaining a temperature of 37°C and stirring at 120 rpm for 10 min. During this time, monitor the pH value of the solution in real time using a pH meter. If pH fluctuations occur due to glutamine dissolution, adjust the pH using 0.1% of the solution. Adjust the sodium hydroxide solution to 7.2-7.4 mol / L; then add recombinant human insulin (purity ≥99%, activity 27.5 IU / mg). First, dissolve the insulin in a small amount of 0.1 mol / L hydrochloric acid solution (sterile) to a stock solution concentration of 1 mg / mL, then slowly add it dropwise at a final concentration of 5-10 μg / mL while stirring at 80 rpm for 5 min to prevent insulin aggregation; then add L-ascorbic acid (vitamin C, purity ≥99%) at a final concentration of 0.1-0.2 g / L, protecting the solution from light throughout (using a brown stirring flask), and stir for 8 min until completely dissolved; finally, add recombinant human epidermal growth factor (EGF, purity ≥98%, activity ≥5×10⁻⁶). 5 First, dilute EGF with sterile enzyme-free PBS buffer to a stock solution of 10 μg / mL, then add it to a final concentration of 10-20 ng / mL, and gently stir at 50 rpm for 3 min. After all components are dissolved, bring the volume to the required level with sterile DMEM / F12 medium, filter through a 0.22 μm polyethersulfone sterile filter membrane (pre-washed 3 times with sterile water) for sterilization, collect the filtrate in a sterile storage bottle, and store at 4°C protected from light. Before use, it needs to be restored to 37°C. The glucose concentration is verified to be 22-28 g / L and the glutamine concentration to be 2.5-3.5 g / L by high performance liquid chromatography, and the insulin concentration is verified to be 6-9 μg / mL and the EGF concentration to be 12-18 ng / mL by enzyme-linked immunosorbent assay (ELISA) to ensure that the content of each component meets the design requirements.

[0021] The bioreactor in S2 is a stirred bioreactor with a stirring rate of 20-30 rpm, and the inner wall of the reactor is coated with an anti-adsorption coating, which is a phosphate buffer containing 2% bovine serum albumin.

[0022] Furthermore, in preparing the S2 stirred bioreactor, a 5L stainless steel stirred bioreactor (equipped with a paddle stirrer, temperature sensor, pH electrode, and gas inlet port) was first selected. The inner wall of the reactor was first flushed with 0.1mol / L hydrochloric acid solution for 30 minutes (to remove metal residue), and then repeatedly flushed with sterile deionized water 5 times until the pH of the effluent was neutral. Subsequently, it was sterilized by autoclaving at 121℃ for 30 minutes, cooled to room temperature, and then transferred to a sterile operating room. Next, the anti-adsorption coating solution was prepared: sterile enzyme-free phosphate buffer (pH 7.2-7.4) was taken, and bovine serum albumin with a purity ≥98% was added. The solution was stirred to dissolve and the final concentration of bovine serum albumin was adjusted to 2%. The solution was then filtered through a 0.22μm polyethersulfone filter membrane for sterilization. The above-mentioned 2% bovine serum albumin phosphate buffer solution was injected into the reactor at a ratio of 20 mL per liter of reactor volume. After closing the reactor port, the reactor was slowly rotated (5 rpm) to make the coating solution evenly cover all areas of the inner wall of the reactor. Then, the reactor was kept tilted at 30° and incubated in a constant temperature incubator at 37°C for 2 hours (to ensure that bovine serum albumin is fully adsorbed onto the inner wall). After incubation, open the reactor exhaust valve and slowly pour out the residual coating solution. Then, introduce sterile compressed air (flow rate 1L / min) into the reactor and continue to dry it for 30 minutes (to avoid residual liquid affecting the culture system). Subsequently, install a paddle stirrer that has been autoclaved at 121℃ for 30 minutes, connect the temperature and pH monitoring system, and add sterile DMEM / F12 culture medium to the reactor for pre-equilibration for 30 minutes. During this period, adjust the stirring speed to 20-30 rpm (determined according to the suspension state of the microcarriers, preferably 25 rpm, to ensure uniform suspension of the microcarriers and no significant shear force damage to the cells). At the same time, introduce a mixed gas containing 5% CO2 and 3% O2 ​​to maintain the pH of the culture medium at 7.2-7.4 and the temperature at 37℃±0.2℃. After the parameters stabilize, it can be used for the inoculation of pretreated mesenchymal stem cells.

[0023] The intelligent feedback feeding system in S3 includes an online glucose sensor, an online lactate sensor, a PLC control unit, and a peristaltic pump. The sensor detects data once every 2 hours, and the PLC control unit automatically adjusts the flow rate of the peristaltic pump based on the detection data.

[0024] Furthermore, when setting up the S3 intelligent feedback feed system, the core components are first pre-treated: the online glucose sensor (using an enzyme electrode type, detection range 0.1-10 g / L) and the online lactate sensor (using an optical type, detection range 0.1-5 g / L) are rinsed three times with sterile PBS buffer, and then immersed in 1 g / L glucose standard solution and 1 g / L lactate standard solution respectively for 30 min to ensure that the detection error is ≤2%; then the two sensors are inserted into the culture system through the sterile sampling interface on the side wall of the reactor (insertion depth 1 / 3 from the bottom of the reactor to avoid touching the agitator and microcarrier), and connected to the signal acquisition module of the PLC control unit (using an industrial grade, supporting analog input / output) through signal cables. Meanwhile, a bio-corrosion-resistant silicone tube (0.8 mm inner diameter) was selected as the delivery pipeline. One end was connected to the sterile storage tank of nutrient supplement (sterilized at 121℃ for 30 min in advance), and the other end was connected to the sterile feeding port of the reactor through a peristaltic pump (a stepper motor driven type with a flow rate range of 0.1-50 mL / h). The control cable of the peristaltic pump was connected to the execution module of the PLC control unit. During the system debugging phase, the following parameters were preset in the PLC control unit: the sensor detection frequency was set to 1 time / 2h, the glucose concentration maintenance threshold was 1.5-3.0g / L, and the lactic acid concentration control threshold was ≤2.0g / L. A linkage logic between glucose concentration and peristaltic pump flow rate was also set: when the detected glucose concentration was below 1.5g / L, the PLC automatically triggered the peristaltic pump to start, with an initial flow rate of 5mL / h. For every 0.5g / L increase in glucose concentration, the flow rate decreased by 1mL / h until the glucose concentration stabilized at 2.0-2.5g / L, maintaining a flow rate of 8mL / h. If the detected lactic acid concentration exceeded 2.0g / L, the PLC immediately reduced the peristaltic pump flow rate by 2mL / h and simultaneously triggered an alarm module. During the cultivation process, the system automatically completes a test every 2 hours: the sensor converts the real-time concentration signal into an electrical signal and transmits it to the PLC. After analyzing the data, the PLC sends an adjustment command to the peristaltic pump to realize the dynamic delivery of the nutrient supplement solution. No manual intervention is required throughout the process, and all test data and adjustment commands are automatically stored in the PLC's database and can be exported and traced at any time.

[0025] In this embodiment, the specific implementation method is as follows: I. Preliminary Preparations Preparation of modified microcarriers: Low molecular weight chitosan (molecular weight 50-100kDa) and pharmaceutical grade gelatin were mixed at a mass ratio of 3:2 and added to a 1% acetic acid solution prepared with deionized water (solid-liquid ratio 1:20). The mixture was placed in a 37°C constant temperature water bath and magnetically stirred at 300 rpm until completely dissolved, forming a homogeneous and transparent aqueous solution. Then, 0.5% of recombinant human RGD peptide (sequence Arg-Gly-Asp) was added, and stirring was continued at 200 rpm for 30 min until the peptide fragments were completely dispersed. When preparing microspheres using the emulsification-crosslinking method, the above aqueous solution was first slowly injected into liquid paraffin containing 5% Span 80 (oil-water volume ratio 1:5), and mechanically stirred at 800 rpm for 30 min at room temperature to form a stable W / O emulsion. Then, the mixture was further... 0.1% glutaraldehyde solution (volume ratio of 1:10 to aqueous phase) was added dropwise to the emulsion, and crosslinking was carried out at room temperature for 2 hours while maintaining a stirring rate of 200 rpm. During this period, samples were taken every 30 minutes to observe the morphology of the microspheres. After crosslinking, the microspheres were allowed to settle, the upper oil phase was discarded, and the microspheres were washed three times with anhydrous ethanol to remove residual liquid paraffin and Span 80. Then, they were washed five times with deionized water until the pH value was neutral (to remove unreacted glutaraldehyde). Finally, the washed microspheres were transferred to a freeze-drying tray, pre-frozen in an ultra-low temperature freezer at -50℃ for 4 hours, and then freeze-dried in a vacuum freeze dryer (vacuum degree 0.1 mbar, cold trap temperature -80℃) for 24 hours to obtain white porous chitosan-gelatin composite microcarriers (pore size 80 μm, particle size 250 μm, surface RGD peptide grafting rate ≥85%).

[0026] Bioreactor Preparation: A 5L stainless steel stirred bioreactor (equipped with a paddle stirrer, temperature sensor, pH electrode, and gas inlet) was selected. The reactor inner wall was first flushed with 0.1mol / L hydrochloric acid solution for 30 minutes (to remove metal residue), then rinsed repeatedly with sterile deionized water 5 times until the effluent pH was neutral. Subsequently, it was autoclaved at 121℃ for 30 minutes and cooled to room temperature before being transferred to a sterile operating room. Preparation of the anti-adsorption coating solution: Sterile enzyme-free phosphate buffer (pH 7.2-7.4) was taken, and bovine serum albumin (BSA) with a purity ≥98% was added. The mixture was stirred to dissolve and the final BSA concentration was adjusted to 2%. The solution was then filtered through a 0.22μm polyethersulfone membrane for sterilization. The 2% BSA phosphate buffer was added to 20m³ of water per liter of reactor volume. Inject L into the reactor, close the reactor port, and slowly rotate the reactor (5 rpm) to evenly cover all areas of the reactor's inner wall with the coating solution. Then, keep the reactor tilted at 30° and incubate at 37°C for 2 hours (to ensure full adsorption of bovine serum albumin). After incubation, pour out the residual coating solution and blow it dry with sterile compressed air (1 L / min) for 30 minutes. Install a paddle stirrer that has been autoclaved at 121°C for 30 minutes, connect the temperature and pH monitoring system, add sterile DMEM / F12 medium for pre-equilibration for 30 minutes, adjust the stirring speed to 25 rpm, and introduce a mixed gas containing 5% CO2 and 3% O2 ​​to maintain the medium pH at 7.3 and the temperature at 37°C ± 0.2°C. After the parameters stabilize, it is ready for use.

[0027] Preparation of nutrient supplement solution: Use sterile, pyrogen-free DMEM / F12 basal medium (equilibrated at 25℃ for 30 min) as the solvent. Weigh pharmaceutical-grade anhydrous glucose (purity ≥99.5%) using a balance and add it to a final concentration of 25 g / L. Stir magnetically at 150 rpm for 15 min in a 37℃ constant temperature water bath until completely dissolved. Add L-glutamine (purity ≥98%, sterile lyophilized powder) to a final concentration of 3 g / L. Maintain stirring at 120 rpm for 10 min at 37℃. Finely adjust the pH with 0.1 mol / L sodium hydroxide solution. 7.3; Add recombinant human insulin (purity ≥99%, activity ≥27.5 IU / mg): First, dissolve it in a small amount of sterile 0.1 mol / L hydrochloric acid solution to a stock solution of 1 mg / mL, then slowly add it dropwise at a final concentration of 8 μg / mL while stirring at 80 rpm for 5 min; Add L-ascorbic acid (vitamin C, purity ≥99%) at a final concentration of 0.15 g / L, protecting from light throughout (using a brown stirring flask), and stir for 8 min until dissolved; Add recombinant human epidermal growth factor (EGF, purity ≥98%, activity ≥5 × 10⁻⁶). 5IU / mg): First, dilute with sterile enzyme-free PBS buffer to a stock solution of 10 μg / mL, then add to a final concentration of 15 ng / mL, and gently stir at 50 rpm for 3 min; bring to volume with sterile DMEM / F12 medium, filter through a 0.22 μm polyethersulfone filter membrane (pre-washed 3 times with sterile water) for sterilization, collect in a sterile storage bottle, and store at 4°C protected from light (before use, restore to 37°C, and test results show glucose 25 g / L, glutamine 3 g / L, insulin 8 μg / mL, and EGF 15 ng / mL).

[0028] Intelligent feedback fed-batch system setup: Pretreatment of the online glucose sensor (enzyme electrode type, detection range 0.1-10 g / L) and online lactate sensor (optical type, detection range 0.1-5 g / L): Rinse three times sequentially with sterile PBS buffer, then calibrate for 30 min by immersing in 1 g / L glucose standard solution and 1 g / L lactate standard solution respectively (ensuring detection error ≤2%); insert both sensors into the culture system through the sterile sampling interface on the reactor side wall (depth from 1 / 3 of the bottom, avoiding the stir bar and microcarrier), and connect them to the signal acquisition module of the industrial-grade PLC control unit via signal cables; select bio-corrosion resistant silicone tubing (inner diameter 0.8 mm), one end connected to a 121℃ autoclaved... The sterile storage tank for nutrient supplementation solution for bacteria (30 min) is connected to the reactor's sterile feeding port via a stepper motor-driven peristaltic pump (flow rate range 0.1-50 mL / h). The peristaltic pump control cable is connected to the PLC execution module. The PLC is preset with the following parameters: sensor detection frequency once every 2 hours, glucose maintenance threshold of 1.5-3.0 g / L, and lactate control threshold ≤2.0 g / L. The linkage logic is as follows: when glucose <1.5 g / L, the peristaltic pump is started (initial flow rate 5 mL / h), and the flow rate is reduced by 1 mL / h for every 0.5 g / L increase, maintaining 8 mL / h when it is stable at 2.0-2.5 g / L; when lactate >2.0 g / L, the flow rate is reduced by 2 mL / h and an alarm is triggered. All data is automatically stored and traced.

[0029] II. Stem Cell Culture Process Stem cell pretreatment: P4 generation mesenchymal stem cells (identified with CD44 and CD90 positivity ≥95%) were selected and digested with trypsin containing 0.03% EDTA at 37°C for 2.5 min (until microscopic observation showed increased intercellular spaces and rounded cell shapes). Digestion was immediately terminated by adding DMEM / F12 medium containing 12% fetal bovine serum (verified for proliferation compatibility). Cells were gently pipetted into a single-cell suspension, and the cell density was adjusted to 3 × 10⁶ cells / cells using a cell counting chamber. 5 cells / mL.

[0030] Construction of the composite culture system: The pretreated single-cell suspension was inoculated into the prepared stirred bioreactor (containing the prepared modified microcarrier) at a cell to microcarrier ratio of 1:5. A mixed gas containing 5% CO2 and 3% O2 ​​was introduced, and the pH value was maintained at 7.3, the temperature at 37℃±0.2℃, and the stirring rate at 25 rpm.

[0031] Dynamic fed-batch culture: The intelligent feedback fed-batch system is activated, and the concentrations of glucose and lactate in the culture medium are detected every 2 hours. When glucose < 1.5 g / L, the peristaltic pump is adjusted by PLC to automatically feed nutrient supplement at a rate of 8 mL / h to maintain glucose at 1.5-3.0 g / L and lactate ≤ 2.0 g / L for 8 days.

[0032] Stem cell harvesting and purification: After culture, 0.03% collagenase was added to the reactor, incubated at 37℃ for 18 min with intermittent gentle stirring, and the cell suspension was collected and filtered through a 200-mesh filter (to remove residual microcarrier fragments). The suspension was centrifuged at 800xg for 20 min using 1.073 g / mL Percoll solution, and the middle milky white cell layer was aspirated and washed twice with physiological saline. Finally, large-scale mesenchymal stem cells with a viability ≥95% and homogeneity ≥90% were obtained (flow cytometry showed CD44 and CD90 positive rates ≥95%, CD34 / CD45 negative rates ≥99%, and osteogenic / adipogenic induction verified multi-lineage differentiation potential).

[0033] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their likenesses.

Claims

1. A method for large-scale stem cell culture, characterized in that, Includes the following steps: S1. Stem cell pretreatment: P3-P5 generation mesenchymal or embryonic stem cells were digested with trypsin containing 0.01%-0.05% EDTA at 37℃±0.5℃ for 2-3 min to obtain a single-cell suspension. The suspension was then resuspended in DMEM / F12 containing 10%-15% fetal bovine serum and the cell density was adjusted to 1×10⁶ cells / mL. 5 -5×10 5 cells / mL; S2. Construction of composite culture system: Pretreated cells were seeded into a bioreactor containing modified microcarriers. The microcarriers were a composite material of chitosan and gelatin with a pore size of 50-100 μm, a particle size of 200-300 μm, and grafted with RGD peptides. A mixed gas of 5% CO2 + 3% O2 ​​was introduced to maintain pH 7.2-7.4 and temperature 37℃±0.2℃. S3. Dynamic fed-batch culture: The concentrations of glucose and lactate are monitored in real time using an intelligent feedback system. The glucose concentration is maintained at 1.5-3.0 g / L and the lactate concentration is maintained at ≤2.0 g / L. When the glucose concentration is lower than 1.5 g / L, a nutrient supplement containing glucose, glutamine and recombinant human insulin is automatically fed at a flow rate of 5-10 mL / h for 7-10 days. S4. Harvesting and purification: After culture, add 0.02%-0.04% collagenase, incubate at 37℃ for 15-20 min, collect the suspension, filter through a 200-mesh filter, and centrifuge at 800xg for 20 min with 1.073g / mL Percoll solution to obtain stem cells with a viability ≥95% and homogeneity ≥90%.

2. The large-scale stem cell culture method according to claim 1, characterized in that, The modified microcarrier described in S2 is prepared by dissolving chitosan and gelatin in a 1% acetic acid solution at a mass ratio of 3:2, adding 0.5% RGD peptide, preparing microspheres by emulsification-crosslinking method, crosslinking with 0.1% glutaraldehyde for 2 hours, and then freeze-drying under vacuum.

3. The large-scale stem cell culture method according to claim 1, characterized in that, The nutritional supplement solution described in S3 consists of: glucose 20-30 g / L, glutamine 2-4 g / L, recombinant human insulin 5-10 μg / mL, vitamin C 0.1-0.2 g / L, and EGF 10-20 ng / mL.

4. The large-scale stem cell culture method according to claim 1, characterized in that, The bioreactor in S2 is a stirred bioreactor with a stirring rate of 20-30 rpm, and the inner wall of the reactor is coated with an anti-adsorption coating, which is a phosphate buffer containing 2% bovine serum albumin.

5. The large-scale stem cell culture method according to claim 1, characterized in that, The intelligent feedback feeding system in S3 includes an online glucose sensor, an online lactate sensor, a PLC control unit, and a peristaltic pump. The sensor detects data once every 2 hours, and the PLC control unit automatically adjusts the flow rate of the peristaltic pump based on the detection data.

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