A serum-free culture medium for MSC cells and its preparation method

Abstract

This disclosure belongs to the field of cell culture technology, and specifically relates to a serum-free culture medium for MSCs and its preparation method. The culture medium contains a fixed ratio of basic nutrients and six key regulatory factors, namely transferrin, ascorbic acid, human serum albumin, human fibroblast growth factor rhFGF, platelet-derived growth factor PDGF-BB, and transforming growth factor TGF-β1. By optimizing the combination and concentration of these six key regulatory factors, the proliferation rate and activity of MSCs are significantly improved. It also provides four suitable formulations: high proliferation, high activity, balanced, and economical. The culture effect is stable and reproducible, with high safety and a simple process, making it suitable for laboratory research and large-scale clinical-grade MSC culture.

Description

Technical Field

[0001] This disclosure belongs to the field of cell culture technology, and specifically relates to a serum-free culture medium for MSC cells and its preparation method. Background Technology

[0002] Mesenchymal stem cells (MSCs) are widely used in biomedicine, especially in cell therapy. Their multipotent differentiation, paracrine signaling, and homing properties give them great potential in combating diseases. MSCs can differentiate into myocytes, osteoblasts, and chondrocytes, making them a potential therapeutic tool in regenerative medicine. However, during in vitro expansion and culture, MSCs often face challenges such as slow passage rates, decreased cell viability with each passage, and easy loss of differentiation potential. These problems significantly limit the development and application of MSCs in the biomedical field.

[0003] To address these issues, a common method is to add fetal bovine serum (FBS) during MSC cell culture. This mixed supplementation aims to improve MSC cell survival and maintain their differentiation potential. However, FBS itself has limitations; it is susceptible to contamination by exogenous pathogens, which can negatively impact MSC cell culture. Furthermore, traditional FBS-containing media result in poor cell morphology, cessation of growth after several passages, and sensitivity to trypsin digestion. Therefore, developing and optimizing a serum-free culture medium suitable for the growth characteristics of MSC cells and capable of maintaining their viability is crucial. This will lay the foundation for the application and development of MSC cells in the biopharmaceutical industry. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides a serum-free culture medium for MSC cells based on experimental design optimization and its preparation method.

[0005] The objective of this disclosure is achieved through the following technical solution: In a first aspect of this disclosure, a serum-free culture medium for MSC cells is provided, the culture medium comprising a fixed ratio of basic nutrients and six key regulatory factors; The basic nutritional components include: L-arginine hydrochloride 0.126 g / L, L-cysteine ​​dihydrochloride 0.0313 g / L, L-glutamine 0.292 g / L, L-histidine hydrochloride monohydrate 0.042 g / L, L-isoleucine 0.052 g / L, L-leucine 0.052 g / L, L-lysine hydrochloride 0.0725 g / L, L-methionine 0.015 g / L, L-phenylalanine 0.032 g / L, L-threonine 0.048 g / L, L-tryptophan 0.01 g / L, L-tyrosine disodium dihydrate 0.0519 g / L, L-valine 0.046 g / L, choline chloride 0.001 g / L, folic acid 0.001 g / L, inositol 0.002 g / L, and nicotinamide 0.001 g / L. The formula contains: 0.001 g / L D-calcium pantothenate, 0.001 g / L pyridoxine hydrochloride, 0.001 g / L riboflavin, 0.001 g / L thiamine hydrochloride, 1.0 g / L glucose, 0.011 g / L phenol red, 0.265 g / L calcium chloride, 0.09767 g / L anhydrous magnesium sulfate, 0.4 g / L potassium chloride, 6.8 g / L sodium chloride, 0.122 g / L anhydrous disodium hydrogen phosphate, and 2.2 g / L sodium bicarbonate; it also contains: 0.00001 g / L insulin, 2 μM biotin, and 1.5 × 10⁻⁶ sodium selenite. -8 g / L, L-glutamine 2 mM, 2-mercaptoethanol 0.1 μM; The six key regulatory factors and their concentration ranges are as follows: transferrin 5~15 μg / mL, ascorbic acid 20~60 μg / mL, human serum albumin 1~10 μg / mL, human fibroblast growth factor rhFGF 1~100 ng / mL, platelet-derived growth factor PDGF-BB 1~100 ng / mL, and transforming growth factor TGF-β1 0.5~500 ng / mL.

[0006] In some specific embodiments of this disclosure, the concentration of human serum albumin is 10 μg / mL, at which concentration human serum albumin significantly enhances the proliferation rate and cell activity of MSC cells.

[0007] In some specific embodiments of this disclosure, the concentration of transferrin is 15 μg / mL, and the concentration of platelet-derived growth factor PDGF-BB is 100 ng / mL. Transferrin and platelet-derived growth factor PDGF-BB synergistically enhance the proliferation rate of MSC cells.

[0008] In some specific embodiments of this disclosure, the culture medium is a high-proliferation formulation, wherein the concentrations of the six key regulatory factors are as follows: transferrin 15 μg / mL, ascorbic acid 60 μg / mL, human serum albumin 10 μg / mL, rhFGF 1 ng / mL, PDGF-BB 100 ng / mL, and TGF-β 1 500 ng / mL.

[0009] In some specific embodiments of this disclosure, the culture medium is a highly active formulation, wherein the concentrations of the six key regulatory factors are as follows: transferrin 10 μg / mL, ascorbic acid 40 μg / mL, human serum albumin 10 μg / mL, rhFGF 50 ng / mL, PDGF-BB 50 ng / mL, and TGF-β1 250 ng / mL.

[0010] In some specific embodiments of this disclosure, the culture medium is a balanced formulation, wherein the concentrations of the six key regulatory factors are as follows: transferrin 8 μg / mL, ascorbic acid 30 μg / mL, human serum albumin 5 μg / mL, rhFGF 10 ng / mL, PDGF-BB 30 ng / mL, and TGF-β1 100 ng / mL.

[0011] In some specific embodiments of this disclosure, the culture medium is an economical formulation, wherein the concentrations of the six key regulatory factors are as follows: transferrin 5 μg / mL, ascorbic acid 20 μg / mL, human serum albumin 3 μg / mL, rhFGF 1 ng / mL, PDGF-BB 1 ng / mL, and TGF-β1 0.5 ng / mL.

[0012] In some specific embodiments of this disclosure, the osmotic pressure of the culture medium is 280~300 mOsm / kg H2O, and the pH is 7.2~7.4; furthermore, the culture medium is used in a static culture environment of 37°C, 5% CO2, and 95% humidity.

[0013] In a second aspect of this disclosure, a method for preparing the aforementioned serum-free culture medium for MSC cells is provided, comprising the following steps: (1) stock solution preparation: transferrin and ascorbic acid are prepared into stock solutions using PBS buffer; rhFGF, PDGF-BB, and TGF-β1 are prepared into stock solutions using PBS buffer containing 0.1% human serum albumin; all stock solutions are filtered and sterilized and then stored at -80°C; (2) basal culture medium preparation: each basic nutrient component is weighed according to the content described in claim 1, dissolved and mixed, and then filtered and sterilized; (3) final culture medium preparation: stock solutions of the six key regulatory factors at any concentration described in claims 1-8 are added to the basal culture medium, mixed, and then filtered and sterilized to obtain the final culture medium.

[0014] In some embodiments of this disclosure, the sterilization by filtration after mixing involves mixing at room temperature for 10-15 minutes after addition, followed by sterilization by filtration using a 0.22 μm filter membrane.

[0015] The technical solution provided in this disclosure has the following technical contributions: (1) This disclosure provides a serum-free culture medium for MSC cells composed of a fixed ratio of basic nutrients and 6 key regulatory factors. The 6 key regulatory factors are transferrin, ascorbic acid, human serum albumin, human fibroblast growth factor rhFGF, platelet-derived growth factor PDGF-BB, and transforming growth factor TGF-β1, and the effective concentration range is determined by optimization.

[0016] (2) This disclosure clearly states that human serum albumin is the core driving factor for enhancing MSC proliferation and activity, confirms that transferrin and PDGF-BB have a significant synergistic proliferative effect, and provides four optimized formulations for industrial application: high-proliferative, high-activity, balanced, and economical formulations.

[0017] (3) The culture medium of this invention can achieve stable culture of MSCs without allogeneic and serum-free sources, with cell viability up to 93.5%, significantly improved proliferation rate, uniform cell morphology, stable passage, high safety, good batch-to-batch consistency, simple preparation process, suitable for laboratory research and development and large-scale expansion and production of clinical-grade MSCs, providing a stable and efficient culture solution for the biomedical application of mesenchymal stem cells. Attached Figure Description

[0018] Figure 1 The response indicator Y1: cell proliferation rate (OD) 450 Pareto plot of the standardization effect of (value).

[0019] Figure 2 This is a Pareto plot of the standardized effect of the response index Y2: cell viability (%).

[0020] Figure 3 These are MSC cells cultured using the high-proliferation culture medium formulation in Experiment Example 1.

[0021] Figure 4 These are MSC cells cultured using the highly active culture medium formulation in Experiment Example 2.

[0022] Figure 5 These are MSC cells cultured using the balanced culture medium formulation in Experiment Example 3.

[0023] Figure 6 These are MSC cells cultured using the economical culture medium formula in Experiment Example 4.

[0024] Figure 7 These are MSC cells cultured using a traditional bovine serum-containing culture medium formula. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it. It should be noted that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, all reagents used in the embodiments are commercially available analytical grade, and all experimental methods used are conventional methods.

[0026] Terminology Explanation Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and not intended to be limiting.

[0027] In this disclosure, the terms “comprising” or “including” are open-ended expressions used to refer to the phrase “including but not limited to” and are used interchangeably with it, meaning that they include the contents specified in this disclosure but do not exclude other contents.

[0028] The present disclosure is further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present disclosure in any way.

[0029] 1. Preparation before the experiment 1.1 Cell resuscitation and passage Frozen MSC cells were revived and seeded into T25 culture flasks. Basic culture medium containing only basic nutrients (excluding 6 key influencing factors) was added and the flasks were placed in a CO2 incubator for 48 hours. When the cell confluence reached 70%-80%, the cells were digested and passaged with 0.025% low-concentration trypsin. Cells from passages 3-5 with viability ≥95% were selected for experiments. Table 1. Serum-free culture medium formulation for MSCs 2. Identification of key influencing factors The formulation of serum-free culture medium for MSCs is a core factor determining cell growth status, activity, and proliferation efficiency. The selection of its components must be based on the physiological metabolic needs, nutritional supply characteristics, and functional regulation mechanisms of MSCs, while combining existing research results with preliminary experimental verification to ensure that the selected components have clear functional relevance and experimental necessity. Transferrin, as a key carrier of cellular iron metabolism, can maintain the iron supply required for the normal proliferation of MSCs and avoid cellular metabolic disorders caused by iron deficiency. Ascorbic acid can scavenge reactive oxygen species generated during cell culture, protect cells from oxidative damage, and promote MSC cell proliferation and phenotypic stability. Human serum albumin can maintain the osmotic pressure balance of the culture medium, bind and transport nutrients, provide a stable growth microenvironment for MSCs, and reduce cell apoptosis. Human fibroblast growth factor (rhFGF), platelet-derived growth factor (PDGF-BB), and transforming growth factor (TGF-β1) are all key regulators of MSC cell proliferation and differentiation. Among them, rhFGF can significantly promote MSC cell adhesion and proliferation, PDGF-BB can enhance intercellular adhesion and synergistically promote proliferation, and TGF-β1 can regulate the balance between cell proliferation and differentiation. The three work together to ensure the normal growth and functional integrity of MSCs.

[0030] 3. Preliminary experiments with single-component absence Based on the clear functional correlation of the above components, a preliminary experiment was designed for the absence of a single component. The normal group with all factors was used as a positive control, with three replicates in each group. Cells were cultured for 72 hours according to the experimental procedure described below, and the cell proliferation rate (OD) was detected. 450 The core indicators of cell viability (%) were used to verify the impact on MSC cell growth by using a relatively decreasing amount.

[0031] 3.1 Reagent Preparation Based on the levels of each key influencing factor, the stock solutions of each target factor were precisely prepared (transferrin and ascorbic acid were prepared with PBS buffer, and growth factors were prepared with PBS buffer containing 0.1% human serum albumin to avoid loss of activity). After the stock solutions were filtered and sterilized, they were stored at -80℃ for later use. 3.2 Instrument Calibration Before the experiment, calibrate instruments such as electronic balances, pipettes, and microplate readers to ensure that the accuracy of the instruments meets the experimental requirements; adjust the CO2 incubator to the set temperature, humidity, and CO2 concentration in advance and stabilize it for more than 24 hours. 3.3 Preparation of single-component missing culture medium Based on the experimental protocol for the absence of a single component among the six key influencing factors, culture media for each target factor with a single component absence were prepared, the concentration of each component was precisely controlled, and after thorough mixing, the media were filtered to remove bacteria and dispensed into 96-well cell culture plates (100 μL per well), and the groups were labeled accordingly.

[0032] 3.4 Cell Seeding and Culture Cell counting and dilution: After passage, MSC cells were digested, washed twice with PBS buffer, resuspended in an appropriate amount of basal culture medium, counted by trypan blue staining, and the cell concentration was adjusted to 1×10⁶. 4 cells / mL; Inoculation: Add 100 μL of cell suspension to each well of a 96-well plate containing culture media for each experimental group, ensuring that the cell inoculation amount per well is 1 × 10³ cells (uniform inoculation density). Gently shake the 96-well plate to distribute the cells evenly.

[0033] Culture: Place the inoculated 96-well plates in a CO2 incubator and statically culture for 72 hours at 37°C, 5% CO2, and 95% humidity. Avoid shaking the culture plates during the culture period, observe cell morphology regularly, and record any contamination, abnormal cell adhesion, or other issues.

[0034] 3.5 Indicator Testing After 72 hours of incubation, the following three core indicators were tested on parallel samples from each experimental group. All testing procedures were strictly performed in accordance with the kit instructions.

[0035] Cell viability assay (trypan blue staining): Take 10 μL of cell suspension from each well, add 10 μL of trypan blue staining solution, mix well and let stand for 5 min, count under a microscope and calculate cell viability (cell viability = number of live cells / total number of cells × 100%). Cell proliferation rate assay (CCK-8 assay): Add 10 μL of CCK-8 reagent to each well, mix gently, incubate in a CO2 incubator for 2 h, and then measure the absorbance (OD value) of each well at a wavelength of 450 nm using an ELISA reader. The higher the OD value, the faster the cell proliferation rate.

[0036] 3.6 Results Analysis According to the detection indicators in 3.5, calculate the decrease rate of each experimental group relative to the positive control group (decrease rate = (CK group indicator value - experimental group indicator value) / CK group indicator value × 100%). Table 2. Experimental results of single component missingness in 6 key influencing factors Based on the verification results of the preliminary experiment that "the absence or abnormal concentration of a single component will significantly affect the growth of MSC cells", the above six components were confirmed as key influencing factors.

[0037] 4. Factor-based design screening Partial factorial design is a highly efficient screening design method in Design of Experiments (DOE). By rationally selecting a subset of typical experimental sites, it replaces full factorial experiments (a 6-factor, two-level full factorial experiment requires 64 experimental sites). While ensuring experimental accuracy and identifying main effects and key interactions, it significantly reduces the number of experiments and improves experimental efficiency. This experiment selected six target factors, each with two levels (low and high). A partial factorial design was used based on Minitab software. By detecting growth-related indicators of MSCs in each experimental group, combined with analysis of variance (ANOVA) and effect analysis, the significance of the effects of each factor and their interactions on MSC growth was determined, core influencing factors were screened, and the optimal culture medium formulation was obtained by adjusting the concentration of these core influencing factors.

[0038] 4.16 Level Settings for Key Influencing Factors This experiment selected six key influencing factors: transferrin (A), ascorbic acid (B), human serum albumin (C), human fibroblast growth factor (D), platelet-derived growth factor (E), and transforming growth factor (F). Two levels were set for each factor (low level: -1, high level: +1). The level settings were based on experience in developing basal culture media to ensure a reasonable range (covering the effective concentration range of the factor while avoiding excessively high / low concentrations that could lead to abnormal cell growth or factor inactivation). The specific factors and level settings are shown in Table 3 below. Table 3. Level settings for 6 key influencing factors 4.2 Culture medium preparation According to the experimental protocol generated by Minitab software, each target factor was added to the basal culture medium, the concentration of each factor was precisely controlled, the mixture was thoroughly mixed, filtered to remove bacteria, and dispensed into 96-well cell culture plates (100 μL per well), and the plates were labeled accordingly.

[0039] 4.3 Cell Seeding and Culture Cell counting and dilution: After passage, MSC cells were digested, washed twice with PBS buffer, resuspended in an appropriate amount of basal culture medium, counted by trypan blue staining, and the cell concentration was adjusted to 1×10⁶. 4 cells / mL; Inoculation: Add 100 μL of cell suspension to each well of a 96-well plate containing culture media for each experimental group, ensuring that the cell inoculation amount per well is 1 × 10³ cells (uniform inoculation density). Gently shake the 96-well plate to distribute the cells evenly.

[0040] Culture: Place the inoculated 96-well plates in a CO2 incubator and statically culture for 72 hours at 37°C, 5% CO2, and 95% humidity. Avoid shaking the culture plates during the culture period, observe cell morphology regularly, and record any contamination, abnormal cell adhesion, or other issues.

[0041] 4.4 Indicator Testing After 72 hours of incubation, the following three core indicators were tested on parallel samples from each experimental group. All testing procedures were strictly performed in accordance with the kit instructions.

[0042] Cell viability assay (trypan blue staining): Take 10 μL of cell suspension from each well, add 10 μL of trypan blue staining solution, mix well and let stand for 5 min, count under a microscope and calculate cell viability (cell viability = number of live cells / total number of cells × 100%). Cell proliferation rate assay (CCK-8 assay): Add 10 μL of CCK-8 reagent to each well, mix gently, incubate in a CO2 incubator for 2 h, and then measure the absorbance (OD value) of each well at a wavelength of 450 nm using an ELISA reader. The higher the OD value, the faster the cell proliferation rate.

[0043] 4.5 Data Processing and Analysis The compiled experimental data (average cell viability and average CCK-8 OD value of each experimental group) were entered into Minitab 19 software and correlated with the corresponding experimental factor levels to establish a data model.

[0044] This experiment used Minitab 19 to design a factorial experiment, establishing Y1 as the cell proliferation rate (OD). 450 Two response indicators were used: Y1 and Y2: cell viability (%). To ensure statistical significance, three biological parallel samples were set up for each group of experiments. The experimental results are presented as mean ± standard deviation (SD), and the coefficient of variation (CV, %) was calculated. The experimental results are shown in Table 4 below: Table 4. Results of Partial Factor Analysis Test Y1: The results of regression analysis of cell proliferation rate factors are shown in Table 5-7 below: Table 5. Encoded Coefficients of Y1 Table 6. Model Summary Table 7. Analysis of Variance Y2: The regression analysis results of cell viability (%) factors are shown in Table 8-10 below: Table 8. Y2 Coefficients Table 9. Model Summary Table 10. Analysis of Variance Analysis of variance showed that the constructed DOE model was highly significant overall (P<0.001), effectively explaining the changes in cell activity. In the model, the linear main effect term dominated the contribution to the response (F = 2384.96, P<0.001), while the interaction and curvature terms did not reach significant levels, suggesting that cell activity is mainly regulated by the independent effects of each added factor, rather than by synergistic or antagonistic effects between factors.

[0045] The standardized Pareto plot results were consistent with those of ANOVA. Human serum albumin, platelet-derived growth factor (PDGF-BB), transforming growth factor (TGF-β1), and human fibroblast growth factor (rhFGF) all had highly significant effects on cell viability (P<0.001). Human serum albumin showed the most prominent positive promoting effect and was the core driving factor for enhancing cell viability. Transferrin and ascorbic acid also exhibited significant positive regulatory effects (P<0.05). Based on the results, the concentrations of key components in the formulation were determined, and four optimal experimental examples were summarized. This targeted improvement of mesenchymal stem cell culture efficiency, combined with actual raw material prices, enhanced the culture effect and stability of the serum-free culture medium formulation. 5. Validation of the optimized formulation through experimental examples Based on the results of participial factorial design, analysis of variance, and effect analysis using Minitab software, combined with cell proliferation rate (OD) 450 The optimization targets were determined by two indicators: cell activity (%) and cell viability (%). While taking into account the cost of raw materials and the feasibility of practical application, four optimal formulations of serum-free culture medium for MSC cells were selected. Under the premise of fixed basic nutrient components, only the concentrations of six key regulatory factors were adjusted. The specific formulation parameters and verification results are as follows. All verification experiments followed the experimental procedures in 4.2 to 4.4 above. Three parallel samples were set up for each group, and the average value of the results was taken.

[0046] Experimental Example 1: Serum-free culture medium for highly proliferating MSC cells This formulation aims to enhance the in vitro proliferation rate of MSCs and is suitable for applications requiring rapid expansion of large numbers of MSCs. The concentrations of the six key regulatory factors are as follows: transferrin: 15 μg / mL, ascorbic acid: 60 μg / mL, human serum albumin: 10 μg / mL, human fibroblast growth factor (rhFGF): 1 ng / mL, platelet-derived growth factor (PDGF-BB): 100 ng / mL, and transforming growth factor (TGF-β1): 500 ng / mL.

[0047] Validation results: After 72 hours of culture, the cell proliferation rate OD 450 The cell viability was 91.8% with a cell adhesion rate of >95% and a spindle cell ratio of over 98%. There was no abnormal differentiation. Compared with the traditional culture medium (Shanghai Yamei Biomedical Technology Co., Ltd., catalog number: CB015) containing fetal bovine serum (Suzhou Shuangru Biotechnology Co., Ltd., catalog number: S711-001s), the proliferation rate was increased by 31.7%, meeting the application requirements for rapid expansion.

[0048] Experimental Example 2: Serum-free culture medium for highly active MSC cells This formulation aims to maintain the high activity of MSC cells and preserve their differentiation potential. It is suitable for applications with stringent requirements for cell activity, such as regenerative medicine and cell therapy. The concentrations of the six key regulatory factors are as follows: transferrin: 10 μg / mL, ascorbic acid: 40 μg / mL, human serum albumin: 10 μg / mL, human fibroblast growth factor (rhFGF): 50 ng / mL, platelet-derived growth factor (PDGF-BB): 50 ng / mL, and transforming growth factor (TGF-β1): 250 ng / mL.

[0049] Verification results: After 72 hours of culture, cell viability reached 93.5%, and the cell proliferation rate OD... 450 The value was 0.802; the cell morphology was uniform, and the cell viability remained above 90% after 5 passages, with no significant loss of differentiation potential. Compared with the traditional culture medium (Shanghai Yamei Biomedical Technology Co., Ltd., catalog number: CB015) containing fetal bovine serum (Suzhou Shuangru Biotechnology Co., Ltd., catalog number: S711-001s), the cell viability was increased by 8.2%, and there was no risk of exogenous pathogen contamination.

[0050] Experimental Example 3: Serum-free culture medium for balanced MSC cells This formulation balances cell proliferation rate and cell activity, making it suitable for general scenarios such as routine laboratory research and basic MSC cell culture, while controlling raw material costs. The concentrations of the six key regulatory factors are as follows: transferrin: 8 μg / mL, ascorbic acid: 30 μg / mL, human serum albumin: 5 μg / mL, human fibroblast growth factor (rhFGF): 10 ng / mL, platelet-derived growth factor (PDGF-BB): 30 ng / mL, and transforming growth factor (TGF-β1): 100 ng / mL.

[0051] Validation results: After 72 hours of culture, the cell proliferation rate OD 450 The value was 0.786, and the cell viability was 90.2%; the cells adhered normally, the spindle-shaped proportion was 97%, and there was no abnormal apoptosis. Compared with the high proliferation and high activity formula, the raw material cost was reduced, meeting the cost-effectiveness requirements of conventional culture.

[0052] Experiment Example 4: Serum-free culture medium for economical MSC cells This formulation aims to reduce the concentration of growth factors and control the overall cost of culture medium preparation. It is suitable for low-cost applications such as large-scale industrial culture and the construction of MSC cell seed banks. The concentrations of the six key regulatory factors are as follows: transferrin: 5 μg / mL, ascorbic acid: 20 μg / mL, human serum albumin: 3 μg / mL, human fibroblast growth factor (rhFGF): 1 ng / mL, platelet-derived growth factor (PDGF-BB): 1 ng / mL, and transforming growth factor (TGF-β1): 0.5 ng / mL.

[0053] Validation results: After 72 hours of culture, the cell proliferation rate OD 450 The value was 0.723, and the cell viability was 88.5%. The cells were in a stable growth state with an adhesion rate of over 90% and no obvious morphological abnormalities. Compared with the balanced formulation, it meets the cost control requirements for large-scale culture.

[0054] 6. Experimental Conclusions The above four optimized formulations were all obtained through DOE experimental design optimization. They can achieve serum-free and efficient culture of MSCs under static culture conditions of 37 ℃, 5% CO2, and 95% humidity. Compared with traditional culture media containing fetal bovine serum, they effectively solve problems such as exogenous pathogen contamination, cell viability reduction with passage, and loss of differentiation potential. At the same time, different formulations can be flexibly selected according to actual application scenarios such as proliferation requirements, activity requirements, and cost control, adapting to the MSC cell culture needs of multiple fields such as biomedical research, cell therapy, and regenerative medicine.

[0055] Furthermore, the DOE experimental design method adopted in this invention significantly reduces the number of experiments through partial factorial design (only 19 experiments replace 64 full factorial experiments), and accurately screens core influencing factors by combining variance analysis and effect analysis. This provides a scientific and efficient methodological reference for the formulation optimization of serum-free cell culture media, and can be extended to the research and optimization of other types of cell culture media.

[0056] The above specific embodiments are merely illustrative of the content of this disclosure and do not represent a limitation thereof. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and substance of this disclosure, and these modifications and improvements are also considered to be within the scope of protection of this disclosure.

Claims

1. A serum-free culture medium for MSC cells, characterized in that, The culture medium contains a fixed ratio of basic nutrients and six key regulatory factors. The basic nutritional components include: L-arginine hydrochloride 0.126 g / L, L-cysteine ​​dihydrochloride 0.0313 g / L, L-glutamine 0.292 g / L, L-histidine hydrochloride monohydrate 0.042 g / L, L-isoleucine 0.052 g / L, L-leucine 0.052 g / L, L-lysine hydrochloride 0.0725 g / L, L-methionine 0.015 g / L, L-phenylalanine 0.032 g / L, L-threonine 0.048 g / L, L-tryptophan 0.01 g / L, L-tyrosine disodium dihydrate 0.0519 g / L, L-valine 0.046 g / L, choline chloride 0.001 g / L, folic acid 0.001 g / L, inositol 0.002 g / L, and nicotinamide 0.001 g / L. The formula contains: 0.001 g / L D-calcium pantothenate, 0.001 g / L pyridoxine hydrochloride, 0.001 g / L riboflavin, 0.001 g / L thiamine hydrochloride, 1.0 g / L glucose, 0.011 g / L phenol red, 0.265 g / L calcium chloride, 0.09767 g / L anhydrous magnesium sulfate, 0.4 g / L potassium chloride, 6.8 g / L sodium chloride, 0.122 g / L anhydrous disodium hydrogen phosphate, and 2.2 g / L sodium bicarbonate; it also contains: 0.00001 g / L insulin, 2 μM biotin, and 1.5 × 10⁻⁶ sodium selenite. -8 g / L, L-glutamine 2 mM, 2-mercaptoethanol 0.1 μM; The six key regulatory factors and their concentration ranges are as follows: transferrin 5~15 μg / mL, ascorbic acid 20~60 μg / mL, human serum albumin 1~10 μg / mL, human fibroblast growth factor rhFGF 1~100 ng / mL, platelet-derived growth factor PDGF-BB 1~100 ng / mL, and transforming growth factor TGF-β1 0.5~500 ng / mL.

2. The serum-free culture medium for MSC cells according to claim 1, characterized in that, The concentration of human serum albumin was 10 μg / mL.

3. The serum-free culture medium for MSC cells according to claim 1, characterized in that, The concentration of transferrin was 15 μg / mL, and the concentration of platelet-derived growth factor PDGF-BB was 100 ng / mL.

4. The serum-free culture medium for MSC cells according to claim 1, characterized in that, The culture medium is a high-proliferation formulation, wherein the concentrations of the six key regulatory factors are as follows: transferrin 15 μg / mL, ascorbic acid 60 μg / mL, human serum albumin 10 μg / mL, rhFGF 1 ng / mL, PDGF-BB 100 ng / mL, and TGF-β 1 500 ng / mL.

5. The serum-free culture medium for MSC cells according to claim 1, characterized in that, The culture medium is a highly active formulation, wherein the concentrations of the six key regulatory factors are as follows: transferrin 10 μg / mL, ascorbic acid 40 μg / mL, human serum albumin 10 μg / mL, rhFGF 50 ng / mL, PDGF-BB 50 ng / mL, and TGF-β1 250 ng / mL.

6. The serum-free culture medium for MSC cells according to claim 1, characterized in that, The culture medium is a balanced formulation, wherein the concentrations of the six key regulatory factors are as follows: transferrin 8 μg / mL, ascorbic acid 30 μg / mL, human serum albumin 5 μg / mL, rhFGF 10 ng / mL, PDGF-BB 30 ng / mL, and TGF-β1 100 ng / mL.

7. The serum-free culture medium for MSC cells according to claim 1, characterized in that, The culture medium is an economical formula, wherein the concentrations of the six key regulatory factors are as follows: transferrin 5 μg / mL, ascorbic acid 20 μg / mL, human serum albumin 3 μg / mL, rhFGF 1 ng / mL, PDGF-BB 1 ng / mL, and TGF-β1 0.5 ng / mL.

8. The serum-free culture medium for MSC cells according to any one of claims 1 to 7, characterized in that, The osmotic pressure of the culture medium is 280~300 mOsm / kg H2O, and the pH is 7.2~7.

4. Furthermore, the culture medium is used in a static culture environment of 37°C, 5% CO2, and 95% humidity.

9. A method for preparing serum-free culture medium for MSC cells according to any one of claims 1 to 8, characterized in that, Includes the following steps: (1) Preparation of stock solutions: Transferrin and ascorbic acid were prepared into stock solutions using PBS buffer; rhFGF, PDGF-BB, and TGF-β1 were prepared into stock solutions using PBS buffer containing 0.1% human serum albumin; all stock solutions were filtered and sterilized and then stored at -80°C. (2) Preparation of basic culture medium: Weigh each basic nutrient component according to the content described in claim 1, dissolve and mix well, and then filter to remove bacteria; (3) Preparation of final culture medium: Add stock solutions of the six key regulatory factors at any concentration described in claims 1-8 to the basal culture medium, mix well, filter and sterilize to obtain the final culture medium.

10. The preparation method according to claim 9, characterized in that, The process of adding the product and then filtering it for sterilization involves mixing it at room temperature for 10-15 minutes and then filtering it through a 0.22 μm filter membrane.

Images