MSC culture medium composition and culture method

Abstract

The invention discloses an MSC culture medium composition and a culture method, and belongs to the field of biological medicine. Aiming at the problems of low MSC amplification efficiency and poor function maintenance in the prior art, the invention provides the MSC culture medium combination, which comprises a basic culture medium and functional factors, and the functional factors comprise an activation functional factor, an amplification functional factor and a steady-state functional factor which are sequentially added according to a preset time interval; wherein the activating functional factors comprise b-FGF, EGF and TGF-beta 1; the amplification functional factors comprise b-FGF (Fibroblast Growth Factor), PDGF-BB (Platelet Derived Growth Factor The steady-state functional factors comprise a b-FGF (Fibroblast Growth Factor) and IGF-1. The cell cycle is quickly started by activating the functional factors, differentiation is inhibited, and the functional factors are amplified to promote large-scale cell amplification; steady-state functional factor equilibrium amplification and dryness maintenance are carried out; and different functional factors are sequentially added according to a preset time interval, and the concentration of the factors is dynamically adjusted, so that differentiation tendency or aging acceleration caused by long-time high-concentration stimulation of a single factor is avoided, long-term efficient amplification of the MSC is realized, and the multifunctionality of the MSC is kept.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to an MSC culture medium combination and culture method. Background Technology

[0002] Mesenchymal stem cells (MSCs) have broad application prospects in regenerative medicine, immune regulation, and tissue engineering. Currently, common MSC expansion media are mostly based on DMEM / F12 or α-MEM basal media, supplemented with animal-derived or heterologous components such as fetal bovine serum (FBS) or human platelet lysate (HPL). These methods suffer from problems such as large batch-to-batch variability, high immunogenicity risk, and high cost. While serum-free media have been explored in existing technologies, they still suffer from low expansion efficiency (typically senescence within <10 passages), cell functional degradation (such as decreased osteogenic / adipogenic differentiation capacity), and the inability to simultaneously accommodate high proliferation and multi-lineage differentiation potential.

[0003] Corresponding improvements have been made to address the aforementioned issues. For example, Chinese patent application CN201910201406.9, published on June 28, 2019, discloses a serum-free, animal-derived, and clearly defined culture medium and its application, relating to the field of cell culture technology. The serum-free, animal-derived, and clearly defined culture medium provided by this invention includes a basal culture medium and cytokines, hormones, transferrin, albumin, fetal globulin, putrescine, and serotonin added to the basal culture medium. The shortcomings of this patent are: the lack of antioxidants, resulting in poor culture effects; and the fact that fetal globulin is usually extracted from bovine fetal serum. Even after purification, its source is still animal, introducing the risk of unknown pathogens and batch-to-batch variability.

[0004] For example, Chinese patent application number CN201710295010.6, published on June 30, 2017, discloses a serum-free culture medium for mesenchymal stem cells, comprising a basal culture medium and additives added to the basal culture medium, wherein the additives include L... Glutamine, non-essential amino acids, L Ascorbic acid, sodium selenite, fibronectin, ethanolamine, hydrocortisone, trypsin inhibitor, human transferrin, human insulin, bFGF, TGF β1 and PDGF BB, this culture medium overcomes the problems of poor cell adhesion, relatively complex composition, and lack of support for primary cell culture in existing serum-free media. The shortcomings of this patent are: although it uses a combination of strong growth factors (bFGF, TGF-β1, PDGF-BB) to strongly stimulate cell proliferation, long-term culture under high concentrations of growth factors may lead to cells entering a replicative senescence state more quickly; and the strong proliferative pressure may increase the risk of genomic instability, screening out cells with abnormal karyotypes. Summary of the Invention

[0005] 1. The problem to be solved To address the problems of low MSC amplification efficiency and poor functional maintenance in existing MSCs, this invention provides an MSC culture medium combination and method. The culture medium combination of this invention rapidly initiates the cell cycle and inhibits differentiation by activating functional factors, while amplifying functional factors promote large-scale cell expansion; homeostatic functional factors balance amplification and stemness maintenance; and different functional factors are added sequentially at predetermined time intervals to dynamically adjust factor concentrations, avoiding differentiation tendency or accelerated senescence caused by prolonged high-concentration stimulation of a single factor, thus achieving long-term, efficient MSC amplification while maintaining its multifunctionality. The culture method of this invention achieves a 20-fold amplification within 72 hours, far exceeding that of FBS culture (<2); and stemness is maintained: post-amplification MSCs express CD73. + / CD90 + / CD105 + >95%, maintaining trilineage differentiation potential; at the same time, the proportion of SA-β-gal positive cells is less than 5% within 72 hours, exhibiting a low senescence rate.

[0006] 2. Technical Solution To solve the above problems, the present invention adopts the following technical solution.

[0007] An MSC culture medium composition includes a basal medium and functional factors, wherein the functional factors include an activating functional factor, an amplifying functional factor, and a homeostatic functional factor added sequentially at predetermined time intervals; wherein: The activating functional factors include b-FGF, EGF, and TGF-β1; The amplification functional factors include b-FGF, PDGF-BB, and IL-6; The steady-state functional factors include b-FGF and IGF-1.

[0008] Furthermore, the concentration of b-FGF in the steady-state functional factor is lower than the concentration of b-FGF in the amplification functional factor and the concentration of b-FGF in the activation functional factor.

[0009] Furthermore, the concentration of β-FGF in the activating functional factors is 1-2 ng / mL; the concentration of EGF is 2-5 ng / mL; and the concentration of TGF-β1 is 0.2-0.5 ng / mL. The concentrations of the amplification functional factors are as follows: b-FGF: 0.5-5 ng / mL; PDGF-BB: 0.5-5 ng / mL; IL-6: 0.2-2 ng / mL. The concentration of b-FGF in the homeostatic functional factors is 0.5-1 ng / mL; the concentration of IGF-1 is 5-10 ng / mL.

[0010] Furthermore, the basal culture medium includes a basal solution and nutrient supplements added to the basal solution; the nutrient supplements include rHSA, transferrin, insulin, ascorbic acid-2-phosphate, ethanolamine, ethanolamine phosphate, and Nicotinamide and alpha-lipoic acid.

[0011] Furthermore, the base solution is a mixture of DMEM / F12 and α-MEM, with a volume ratio of DMEM / F12 to α-MEM of 1:(1~1.5). The concentrations of the nutritional supplements are as follows: rHSA: 100-200 μg / mL; transferrin: 0.5-5 μg / mL; insulin: 0.5-5 μg / mL; ascorbic acid-2-phosphate: 20-50 μM; ethanolamine: 2 μM; ethanolamine phosphate: 0.2-2 μM; Nicotinamide: 0.2-2 mM; α-lipoic acid: 0.5-5 μM.

[0012] A method for culturing MSCs using the MSC culture medium combination described in any of the above technical solutions includes the following steps: S1: Obtain sample cells; S2: Prepare a cell suspension from the sample cells and inoculate the cell suspension into a culture dish, which includes the cells and the basic culture medium. S3: Add activation factor to the culture dish within 0-24h; add amplification factor within 24-48h; add homeostasis factor within 48h-72h.

[0013] Furthermore, the inoculation density in step S2 is: 3 × 10⁻⁶ 4 cells / cm ² The culture conditions were 37℃ and 5% CO2.

[0014] Furthermore, the addition of the steady-state functional factor in step S3 specifically includes: preparing fresh solution: mixing DMEM / F12 and α-MEM at a volume ratio of 1:(1~1.5), then adding b-FGF and IGF-1; and then adding fresh solution to the culture vessel after 48 hours.

[0015] 3. Beneficial effects (1) In the culture medium combination of the present invention, on the one hand, the activating functional factor promotes the cells to enter the proliferation cycle through b-FGF and EGF, and TGF-β1 stabilizes stemness; the amplification functional factor rapidly promotes cell division through b-FGF and PDGF-BB; and the homeostatic functional factor balances amplification and stemness through b-FGF and IGF-1. On the other hand, by adding the activating functional factor, amplification functional factor and homeostatic functional factor in sequence according to a predetermined time interval, the concentration of factors is dynamically adjusted, avoiding the differentiation tendency or accelerated aging caused by long-term high-concentration stimulation of a single factor, and ensuring that the cells maintain a low SA-β-gal positive rate while having a high amplification rate. The entire culture medium combination realizes long-term high-efficiency amplification of MSCs and maintains their multifunctionality, solving the problems of low amplification efficiency, poor function maintenance and high cost in the prior art. (2) The concentration settings of b-FGF in the activating functional factor, amplification functional factor and homeostatic functional factor of this invention are different. b-FGF in the activating functional factor mainly promotes cell cycle entry during the activation phase, b-FGF in the amplification functional factor mainly strongly drives division during the amplification phase, and b-FGF in the homeostatic functional factor mainly reduces stimulation but maintains survival signal during the homeostatic phase. The different concentration stages make the entire amplification process more controllable and reliable, and provide a smooth state transition for cells, which is particularly suitable for large-scale amplification processes that require continuous passage. It achieves effective control of production costs while ensuring amplification efficiency. (3) This invention adds different kinds of nutrient supplements to the basal culture medium, which reflects the mechanism of low senescence rate; Nicotinamide can increase cellular NAD. + Levels of activity activate Sirt1 to delay aging; α-lipoic acid, as a mitochondrial antioxidant, can reduce the accumulation of reactive oxygen species (ROS) and maintain mitochondrial function. Together, they reduce oxidative stress and DNA damage during the expansion process, thereby reducing premature aging of MSCs. (4) The MSC culture method of the present invention, by adding corresponding functional factors at different stages, achieves a 20-fold expansion within 72 h based on the time-sequential management of cell cycle regulation, signal pathway activation and stemness maintenance, which is much higher than FBS culture (<2); and stemness is maintained: after expansion, MSCs express CD73 + / CD90 + / CD105 +>95%, maintaining trilineage differentiation potential; at the same time, the proportion of SA-β-gal positive cells is less than 5% within 72 hours, exhibiting a low senescence rate. Attached Figure Description

[0016] Figure 1 This is a microscopic image of MSCs amplified in Example 1; Figure 2 This is a microscopic image of MSCs amplified in Comparative Example 3. Figure 3 This is a flow cytometry result diagram of this application; Figure 4 This diagram illustrates the amplification results after three consecutive passages using the culture method described in this application. Detailed Implementation

[0017] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0018] An MSC culture medium composition includes a basal medium and functional factors, wherein the functional factors include an activating functional factor, an amplifying functional factor, and a homeostatic functional factor added sequentially at predetermined time intervals; wherein: The activating functional factors include b-FGF, EGF, and TGF-β1; The amplification functional factors include b-FGF, PDGF-BB, and IL-6; The steady-state functional factors include b-FGF and IGF-1.

[0019] Specifically, a core aspect of this embodiment is the composition of the functional factors, wherein: Among the activating functional factors, b-FGF and EGF jointly activate the MAPK / ERK pathway, rapidly driving resting MSCs into the G1 / S transition of the cell cycle, thereby shortening the time required for cell proliferation. Among the amplification functional factors, DNA synthesis and mitosis rates are enhanced through the PI3K / AKT and JAK / STAT3 pathways. Simultaneously, IL-6 can enhance cellular metabolism and ensure amplification efficiency by activating STAT3. Among the homeostasis functional factors, IGF-1 synergizes with IL-6 in the amplification functional factors: IL-6 enhances the secretion of immunomodulatory molecules (such as IDO and PGE2) through STAT3 signaling, while IGF-1 improves cellular tolerance to inflammatory environments. Together, they give amplified MSCs stronger immunosuppressive and immunoregulatory functions. In other words, by rapidly initiating the cell cycle and inhibiting differentiation through activating functional factors, promoting large-scale cell expansion through amplification functional factors, and balancing amplification and stemness maintenance through homeostasis functional factors, long-term, efficient amplification of MSCs while maintaining their multifunctionality is achieved, solving the problems of low amplification efficiency, poor functional maintenance, and high cost in existing technologies. Another key aspect is the timing of the addition of activating, amplifying, and homeostatic functional factors. Specifically, these factors are added at specific intervals. These intervals can be set according to the specific circumstances; in this example, they are set every 24 hours. That is, the activating factor is added to the basal culture medium, followed by the amplifying factor after 24 hours, and then the homeostatic factor after another 24 hours. This design aims to avoid the differentiation tendency or accelerated senescence caused by prolonged high-concentration stimulation of a single factor, ensuring that cells maintain a low SA-β-gal positivity rate while achieving a high amplification rate.

[0020] In one specific embodiment, the concentration of b-FGF in the homeostatic functional factor is lower than the concentration of b-FGF in the amplification functional factor and the concentration of b-FGF in the activation functional factor. It should be noted that since b-FGF is present in all three functional factors, the role of b-FGF in each functional factor is different: the activation phase mainly initiates the cell to enter the proliferation cycle, preparing for subsequent explosive proliferation; the amplification phase maximizes cell output; and in the homeostatic functional phase, its main role shifts from "strongly driving proliferation" to "maintaining cell survival and basic metabolic activity." By designing the concentration of b-FGF in the homeostatic functional factor to be minimized, and with a stepwise variation in the concentration of b-FGF in the three functional factors, the different concentration stages make the entire amplification process more controllable and reliable, providing a smooth transition of cell states, which is particularly suitable for large-scale amplification processes requiring continuous passage; thus, production costs are effectively controlled while ensuring amplification efficiency.

[0021] More specifically, the concentrations of the activating factors are as follows: b-FGF is 1-2 ng / mL; EGF is 2-5 ng / mL; and TGF-β1 is 0.2-0.5 ng / mL. It should be noted that the concentrations of these three activating factors have a significant impact on MSC proliferation rate, stemness maintenance, and senescence status. If the b-FGF concentration is below 1 ng / mL, the FGFR / ERK signaling pathway cannot be fully activated, delaying the MSC proliferation cycle and prolonging adhesion time. If the b-FGF concentration is above 5 ng / mL, it easily leads to FGFR hyperphosphorylation, triggering metabolic stress, causing cell morphology to flatten and exhibiting early signs of senescence.

[0022] When the concentration of EGF is below 2 ng / mL, the activation effect on EGFR is insufficient, MSCs are in a resting state, and the proliferation rate is significantly reduced; while when the concentration is above 20 ng / mL, it will cause continuous activation of EGFR, induce oxidative stress accumulation and epithelial-like transformation, manifested as increased E-cadherin expression and decreased immunomodulatory function.

[0023] When TGF-β1 is below 0.1 ng / mL, its role in maintaining stemness is insufficient, and although MSCs proliferate rapidly, their immunomodulatory function is weakened; while when it is above 1 ng / mL, it will overactivate the Smad2 / 3 pathway, induce cells to differentiate into myofibroblast-like structures (α-SMA and Collagen I are significantly upregulated), inhibit MSC expansion, and accelerate senescence.

[0024] Therefore, the concentration range selected in this embodiment can achieve a dynamic balance between initiating the cell cycle, maintaining stemness, and preventing excessive differentiation, thereby ensuring rapid expansion of MSCs and stable stemness.

[0025] Furthermore, the concentration of b-FGF in the amplification functional factors is 0.5-5 ng / mL; the concentration of PDGF-BB is 0.5-5 ng / mL; the concentration of IL-6 is 0.2-2 ng / mL; the concentration of b-FGF in the homeostatic functional factors is 0.5-1 ng / mL; and the concentration of IGF-1 is 5-10 ng / mL.

[0026] Excessive β-FGF concentrations can induce excessive cell proliferation, even leading to genetic instability and premature aging, while insufficient concentrations directly result in low proliferation efficiency, potentially causing spontaneous cell differentiation and accelerated aging. For PDGF-BB, excessively high concentrations may abnormally activate downstream signaling, inducing fibrosis-like phenotypes; conversely, excessively low concentrations weaken MSCs' crucial migration ability and their synergistic effect with other factors in promoting proliferation. IL-6, as a pleiotropic factor, has a particularly sensitive concentration window. Excessively high concentrations can transform it from a beneficial regulator into a pro-inflammatory signal, impairing the core immune regulatory function of MSCs and promoting aging; while excessively low concentrations deprive cells of important autocrine survival signals, making them more vulnerable to stress and disrupting the homeostasis of the cytokine network.

[0027] Finally, IGF-1 is a key survival factor. Excessive concentrations can lead to oxidative stress and metabolic disorders, causing a shift in cell differentiation potential and posing potential safety risks; however, excessively low concentrations have a more devastating effect, significantly weakening cell viability, increasing apoptosis rates, and causing overall proliferation retardation and functional decline due to insufficient energy metabolism.

[0028] In one specific embodiment, the basal culture medium comprises a basal solution and nutrient supplements added to the basal solution; the nutrient supplements include rHSA, transferrin, insulin, ascorbic acid-2-phosphate, ethanolamine, ethanolamine phosphate, and Nicotinamide and α-lipoic acid.

[0029] Specifically, the core ingredients of the nutritional supplement in this embodiment are as follows: Nicotinamide: Its stable application in existing serum-free systems is rarely observed. This embodiment clearly defines its key role as an anti-aging metabolic factor. Stable application of nicotinamide is achieved primarily through its combination with α-lipoic acid to reduce its degradation and auto-oxidation rates during culture. α-Lipoic acid acts as a free radical scavenger in the culture medium, maintaining redox balance and thus stabilizing the structural activity of nicotinamide, preventing its excessive oxidation to nicotinamide N-oxide. The synergistic effect of both maintains intracellular NAD+. + Metabolic homeostasis ensures continuous activation of the Sirt1 pathway, maintaining a low senescence rate and high activity of MSCs over the long term. Mechanism of action: Increases NAD+. + At the level of MSCs, it activates the Sirt1 pathway, delays MSC senescence, improves DNA repair capacity, and maintains genome stability; it is a key factor in maintaining a low senescence rate and stemness stability of MSCs. Alpha-lipoic acid (ALA): ALA is rarely added to traditional MSC culture systems, mainly because this molecule is unstable in serum-containing environments and is easily inactivated by metal ions or proteins. Simultaneously, its antioxidant capacity is suppressed in high-glucose culture systems, making it difficult to maintain mitochondrial homeostasis. This invention uses a serum-free system and optimizes the ratio of reducing cofactors (such as ascorbic acid-2-phosphate and ethanolamine) in the culture medium, significantly improving the stability of ALA. Its synergistic effect with Nicotinamide, by reducing ROS accumulation and maintaining mitochondrial membrane potential and ATP homeostasis, solves the problem of early MSC senescence caused by energy metabolism disorders in traditional systems. This embodiment innovatively introduces ALA to enhance mitochondrial function; its mechanism of action is as a mitochondrial antioxidant, reducing ROS accumulation; improving energy metabolism, enabling cells to maintain normal ATP levels under high proliferation conditions; synergistically maintaining cellular metabolic homeostasis with Nicotinamide; and it is a key metabolic regulator for achieving long-term MSC expansion and high activity. In one specific embodiment, the base fluid is a mixture of DMEM / F12 and α-MEM, and the volume ratio of DMEM / F12 to α-MEM is 1:(1~1.5). The concentrations of the nutritional supplements are as follows: rHSA: 100-200 μg / mL; transferrin: 0.5-5 μg / mL; insulin: 0.5-5 μg / mL; ascorbic acid-2-phosphate: 20-50 μM; ethanolamine: 2 μM; ethanolamine phosphate: 0.2-2 μM; Nicotinamide: 0.2-2 mM; α-lipoic acid: 0.5-5 μM.

[0030] A culture method using the MSC culture medium combination as described in any of the above embodiments includes the following steps: S1: Obtain sample cells; S2: Prepare a cell suspension from the sample cells and inoculate the cell suspension into a culture dish, which includes the cells and the basic culture medium. S3: Add activation factor to the culture dish within 0-24h; add amplification factor within 24-48h; add homeostasis factor within 48h-72h.

[0031] Specifically, the inoculation density in step S2 is 3 × 10⁻⁶. 4 cells / cm ² The culture conditions were 37℃, 5% CO2, and saturated humidity.

[0032] The MSC culture method in this embodiment adds corresponding functional factors at different stages, based on the temporal management of cell cycle regulation, signaling pathway activation, and stemness maintenance, such as... Figure 3 and Figure 4 As shown, a 20-fold amplification was achieved within 72 hours, far exceeding that of FBS culture (<2); and stemness was maintained: post-amplification MSCs expressed CD73. + / CD90 + / CD105 + >95%, maintaining trilineage differentiation potential; at the same time, the proportion of SA-β-gal positive cells is less than 5% within 72 hours, exhibiting a low senescence rate.

[0033] In one specific embodiment, the addition of steady-state functional factors in step S3 specifically includes: preparing fresh medium: mixing DMEM / F12 and α-MEM at a volume ratio of 1:(1~1.5), then adding b-FGF and IGF-1; then adding fresh medium to the culture vessel after 48 hours; passage method: after amplification within 72 hours, MSCs reach 20-fold. That is, in this embodiment, the medium replacement strategy is: not to completely replace the medium within 72 hours, but only to add 50% of the original culture medium volume of fresh culture medium at 48 hours (DMEM / F12 and α-MEM at a volume ratio of 1:1-1:1.5, b-FGF reduced to 0.5-1 ng / mL, and IGF-1 added at 5-10 ng / mL); while the activation functional factors and amplification functional factors are added in the form of factor concentrates, which only contain the corresponding factors and do not contain other components.

[0034] This medium replacement strategy avoids the stress on cells caused by a full medium change; replenishment is only performed once every 48 hours within a 72-hour period, making the operation simpler and the environment more stable; it also ensures that the exact same density, culture medium, and intervention timing are used in each passage cycle, ensuring the controllability of the amplification process and the reproducibility of the results.

[0035] To further understand this application, the following examples are provided: Example 1 A method for culturing MSCs includes the following steps: Sample source: Umbilical cord-derived MSCs (P2, used after cryopreservation and thawing, viability >90%). Culture medium composition (100 mL): DMEM / F12:α-MEM=1:1, rHSA 200 μg / mL, transferrin 5 μg / mL, insulin 5 μg / mL, ascorbic acid-2-phosphate 50 μM, ethanolamine / phosphoethanolamine each 2 μM, Nicotinamide 2 mM, α-lipoic acid 5 μM; Factor addition: 0–24h b-FGF 2 ng / mL+EGF 5 ng / mL+TGF-β1 0.5 ng / mL; 24–48h b-FGF 5 ng / mL+PDGF-BB 5 ng / mL+IL-6 2 ng / mL; 48–72h b-FGF 1 ng / mL+IGF-1 10 ng / mL; Operating procedure: Inoculation density 3×10 4 cells / cm²; cultured at 37℃ with 5% CO2, adding 50% fresh culture medium after 48 hours, and subcultured 3 times consecutively; Detection: such as Figure 1 As shown, the amplification fold, flow cytometry phenotype, trilineage differentiation, SA-β-gal staining, and immune function were observed under a microscope (MSC+PBMC co-culture). Results: After 72 hours, the cells were in good condition, uniform under the microscope, and the expansion factor was 23.2 ± 0.15. Phenotype: CD73 + >99%, CD90 + >99%, CD105 + >99%, CD34 - <1%, CD45 - <1%; Differentiation potential: 93% for osteogenic synthesis, 91% for adipogenesis, and 88% for chondrogenesis; Senescence rate: SA-β-gal positive cells 4.5% (FBS control 11.2%); T cell inhibition rate: 72% (FBS control 49%); where FBS control refers to cells cultured in a medium containing fetal bovine serum as the baseline group in the experiment.

[0036] Comparative Example 1: Control group without TGF-β1 supplementation Experimental conditions: This comparative example is basically the same as Example 1, except that TGF-β1 is not added in the functional factor activation stage (0–24 h), while the other components and operating procedures remain the same.

[0037] Experimental results: After 72 h, the MSC amplification fold was 17 ± 0.3 (significantly lower than 23.2 ± 0.15 in Example 1, p < 0.01); cell morphology was relatively dispersed, some cells increased in size, and the nucleus-cytoplasm ratio decreased; flow cytometry results showed that CD73 + 95%, CD90 + 93%, CD105 + The percentage of positive SA-β-gal cells decreased to 88%; the percentage of positive SA-β-gal cells increased to 9.5%; and in the immunosuppression experiment, the T cell inhibition rate was 56% (compared to 72% in Example 1).

[0038] Comparative Example 2: Control experiment without α-lipoic acid: This comparative example is basically the same as Example 1, except that α-lipoic acid in the nutritional supplement is removed, and the other components and culture conditions remain unchanged.

[0039] Experimental results: Within 72 h, the MSC expansion rate was 16 ± 0.2, with uneven cell adhesion and some cells showing granular cytoplasm. Mitochondrial staining showed a decrease in membrane potential of approximately 25%, and a 1.8-fold increase in ROS levels compared to Example 1. The proportion of SA-β-gal positive cells was 12%. CD105... + Expression decreased to 90%, and T cell inhibition rate decreased to 60%.

[0040] Comparative Example 3: Control without Nicotinamide Experimental conditions: This comparative example is basically the same as Example 1, except that the culture medium does not contain Nicotinamide.

[0041] Result: As Figure 2 As shown, the cells under the microscope after 72 hours showed a fold increase of 15 ± 0.25 vacuoles. Percentage of SA-β-gal positive cells: 13.6%; Phenotype: CD105 + Reduced to 85%; T cell inhibition rate: 50%.

[0042] Example 2 The basic process is the same as in Example 1, except that the sample source is fat-derived MSC (P2).

[0043] Results: 72h amplification fold: 21±0.2; phenotype: CD73 +96%, CD90 + 95%, CD105 + 94%; Differentiation potential: success rate of trilineage differentiation >85%; Cell inhibition rate: 68% (FBS control 46%).

[0044] Example 3 The process was basically the same as in Example 1, except that when harvesting MSCs at 72h, the ROCK inhibitor Y-27632 (5 μM) was added before passage and cultured for 24h.

[0045] Result: Adhesion efficiency increased by 28%; Survival rate 24 hours after passage: 96% (control 84%). Phenotypic and trilineal differentiation potential were well maintained.

[0046] The examples described herein are merely preferred embodiments of the invention and are not intended to limit the concept and scope of the invention. Any modifications and improvements made by those skilled in the art to the technical solutions of the invention without departing from the design concept of the invention should fall within the protection scope of the invention.

Claims

1. An MSC culture medium composition, characterized in that: It includes a basal culture medium and functional factors, wherein the functional factors include an activating functional factor, an amplifying functional factor, and a homeostatic functional factor added sequentially at predetermined time intervals; wherein: The activating functional factors include b-FGF, EGF, and TGF-β1; The amplification functional factors include b-FGF, PDGF-BB, and IL-6; The steady-state functional factors include b-FGF and IGF-1.

2. The MSC culture medium composition according to claim 1, characterized in that: The concentration of b-FGF in the steady-state functional factor is less than the concentration of b-FGF in the amplification functional factor and the concentration of b-FGF in the activation functional factor.

3. The MSC culture medium composition according to claim 2, characterized in that: The concentration of β-FGF in the activating functional factors is 1-2 ng / mL; the concentration of EGF is 2-5 ng / mL; and the concentration of TGF-β1 is 0.2-0.5 ng / mL. The concentrations of the amplification functional factors are as follows: b-FGF: 0.5-5 ng / mL; PDGF-BB: 0.5-5 ng / mL; IL-6: 0.2-2 ng / mL. The concentration of b-FGF in the homeostatic functional factors is 0.5-1 ng / mL; the concentration of IGF-1 is 5-10 ng / mL.

4. The MSC culture medium composition according to claim 1, characterized in that: The basal culture medium includes a basal solution and nutrient supplements added to the basal solution; the nutrient supplements include rHSA, transferrin, insulin, ascorbic acid-2-phosphate, ethanolamine, ethanolamine phosphate, and Nicotinamide and α-lipoic acid.

5. The MSC culture medium composition according to claim 4, characterized in that: The base solution is a mixture of DMEM / F12 and α-MEM, with a volume ratio of DMEM / F12 to α-MEM of 1:(1~1.5). The concentrations of the nutritional supplements are as follows: rHSA: 100-200 μg / mL; transferrin: 0.5-5 μg / mL; Insulin: 0.5-5 μg / mL; Ascorbic acid-2-phosphate: 20-50 μM; Ethanolamine: 2 μM; Phosphoethanolamine: 0.2-2 μM; Nicotinamide: 0.2-2 mM; Alpha-lipoic acid: 0.5-5 μM.

6. A method for culturing MSCs using the MSC culture medium combination as described in any one of claims 1-5, characterized in that: Includes the following steps: S1: Obtain sample cells; S2: Prepare a cell suspension from the sample cells and inoculate the cell suspension into a culture dish, which includes the cells and the basic culture medium. S3: Add activation factor to the culture dish within 0-24h; add amplification factor within 24-48h; add homeostasis factor within 48h-72h.

7. The MSC culture method according to claim 6, characterized in that: Therefore, the inoculation density in step S2 is: 3 × 10⁻⁶ 4 cells / cm ² The culture conditions were 37℃ and 5% CO2.

8. The MSC culture method according to claim 6, characterized in that: The addition of the steady-state functional factor in step S3 specifically includes: preparing fresh solution: mixing DMEM / F12 and α-MEM at a volume ratio of 1:(1~1.5), then adding b-FGF and IGF-1; and then adding fresh solution to the culture vessel after 48 hours.

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