A high-efficiency serum-free proliferation culture medium for culturing porcine muscle stem cells

Abstract

This invention discloses a serum-free proliferation medium for efficiently culturing porcine muscle stem cells, belonging to the field of cell culture technology. This serum-free proliferation medium is based on DMEM / F12 and contains ITS, L-ascorbic acid, BSA, Fetuin, FGF-2, and other components. This invention optimized the concentrations of key factors such as BSA, Fetuin, and FGF-2 through single-factor analysis, Plackett-Burman assays, and steepest ascent and Box-Behnken response surface methodology, successfully constructing a low-cost, green serum-free culture medium suitable for porcine muscle stem cells. The short-term culture effect of this serum-free medium on porcine muscle stem cells is comparable to that of complete culture media, with a significant cost advantage, laying the foundation for further long-term cell culture and functional maintenance under serum-free conditions.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to a serum-free proliferation culture medium for efficiently culturing porcine muscle stem cells and its preparation method. Background Technology

[0002] With population growth and economic development, the depletion of limited resources and environmental pollution are becoming increasingly severe, making traditional meat production methods unable to meet future demands. Cell-cultured meat, through the in vitro culture and directed differentiation of animal cells, achieves sustainable meat production, demonstrating advantages in terms of greenness, environmental friendliness, and health. In vitro culture of animal cells typically relies on nutrients containing 10-20% of the necessary nutrients to support and maintain normal cellular life activities, such as commonly used fetal bovine serum (FBS), which serves as an important source of growth factors, hormones, and some nutrients in cell culture. However, the use of serum involves numerous scientific, ethical, and safety issues, such as high cost, unclear composition, significant batch-to-batch variations in different seasons and regions, the presence of endotoxins and foreign antigens, frequent instances of commercially fraudulent mixed serums, and acquisition methods that violate humanitarian and animal welfare principles. Serum-free medium (SFM) is inexpensive, has clearly defined composition, effectively avoids blood-borne contamination problems associated with serum, and its preparation process is simple, improving experimental stability, accuracy, and efficiency, thus providing possibilities for large-scale, low-cost cell culture.

[0003] Serum-free culture media, which do not contain animal serum, consist of a basal medium and various supplements. However, the interactions between these components can affect cell behavior, and the effects of component concentrations are often non-linear. Therefore, optimizing serum-free culture media is a dynamic process that must be continuously adjusted based on specific cell characteristics and experimental needs. Selecting effective methods to determine the optimal formulation is crucial. Optimized serum-free culture media can maintain their original or even more efficient culture effects without significantly impacting the biological functions of animal cells.

[0004] Porcine muscle stem cells (pMuSCs) are one of the most abundant adult stem cell populations in tissue-resident organisms. Despite their significant advantages in culture medium development, their large-scale application still faces a key bottleneck: after eight in vitro passages, the proliferation rate drops sharply and stem cell markers are lost, leading to a loss of terminal differentiation capacity. Therefore, establishing efficient strategies to regulate the in vitro proliferation and differentiation of muscle stem cells has become a pressing technical challenge. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention develops a serum-free proliferation culture medium for culturing porcine muscle stem cells, aiming to reduce the technical problem of the high dependence of existing porcine muscle stem cell in vitro culture on serum, and to provide a reference for promoting the development of serum-free culture media in the field of cell cultured meat.

[0006] The specific technical solution of the present invention is as follows: In a first aspect, the present invention provides a serum-free proliferation medium for efficiently culturing porcine muscle stem cells, which is based on DMEM / F12 and contains insulin-transferrin-selenium complex (ITS), L-ascorbic acid, bovine serum albumin (BSA), Fetuin, and FGF-2.

[0007] Preferably, in the serum-free proliferation medium described above, the content of ITS is 1% (i.e., 100 mg / mL), the content of L-ascorbic acid is 200 μg / mL, the content of BSA is 523.32 μg / mL, the content of Fetuin is 976.99 μg / mL, and the content of FGF-2 is 79.41 μg / mL.

[0008] Secondly, the present invention provides the application of the above-mentioned serum-free proliferation medium in the in vitro culture of porcine muscle stem cells, and the application directions include the preparation of cultured meat using porcine muscle stem cells.

[0009] Thirdly, the present invention provides a method for long-term passage of porcine muscle stem cells, which uses the serum-free proliferation medium provided by the present invention. In some embodiments of the present invention, an indirect passage strategy is employed: each passage is first performed using a serum-containing medium for adhesion, and then transferred to the serum-free proliferation medium for further culture.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention optimized the concentrations of key factors such as BSA, Fetuin, and FGF-2 through univariate analysis, Plackett-Burman experiments, and the steepest ascent and Box-Behnken response surface methodology, ultimately obtaining a serum-free culture medium suitable for the in vitro culture of porcine muscle stem cells. Under these serum-free conditions, short-term cell proliferation was comparable to that in the complete culture medium group. Although proliferative activity decreased and cell stemness could not be effectively maintained after long-term culture, the cost advantage was significant. This invention provides a scientific basis for further achieving long-term cell culture and functional maintenance under serum-free conditions, and contributes to the large-scale acquisition of high-quality and abundant seed cells for cell-cultured meat. Attached Figure Description

[0011] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0012] Figure 1 The figure shows the results of the analysis of the effects of various growth factors on the proliferation of porcine muscle stem cells in Example 1 (48 h).

[0013] Figure 2 The figure shows the results of the significance factor analysis of the Plackett-Burman experiment in Example 1, where A is the half-normal probability effect of the standardized effect (α=0.05) and B is the Pareto plot of the standardized effect (α=0.05).

[0014] Figure 3 This is a graph showing the results of the difference analysis between the experimental and predicted values ​​in the Box-Behnken experiment in Example 1.

[0015] Figure 4 The results of response surface and cubic plot analysis of BSA, Fetuin, and FGF-2 in Example 1 are shown.

[0016] Figure 5 This is a comparison of the morphological changes of porcine muscle stem cells cultured in complete culture medium (pMuSC-CM) and serum-free culture medium (pMuSC-SFM) for 96 h in Example 2.

[0017] Figure 6 The graph shows the proliferation and apoptosis analysis results of porcine muscle stem cells in the pMuSC-SFM culture system in Example 2. A represents the 7-day growth curve, B represents the short-term proliferation detection, and CE represents the apoptosis detection using the Annxin V / PI double staining method. Statistical difference analysis was performed using the t-test (n=3). express p <0.05, express p <0.01, express p <0.001.

[0018] Figure 7 The image shows the morphology of different generations of porcine muscle stem cells in the pMuSC-SFM culture system in Example 3. The scale bar is 100 μm.

[0019] Figure 8The image shows the results of myogenic properties of porcine muscle stem cells in the pMuSC-SFM culture system after long-term passage in Example 3. A shows typical immunofluorescence images of MyHC-positive cells (red) after induction and differentiation at passages 2, 5, 8, and 10 (cell nuclei via DAPI: blue, scale bar: 50 μm). B shows the quantitative analysis of myotube fusion rate (%) at passages 2, 5, 8, and 10. One-way ANOVA was used for comparison, and post-hoc tests were performed. Different lowercase letters indicate significant differences. p <0.05), while the same lowercase letter indicates no significant difference ( p >0.05).

[0020] Figure 9 The figure shows the cost analysis results of pMuSC-SFM and pMuSC-CM culture media in Example 4. Detailed Implementation

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification and claims of this invention are intended to cover non-exclusive inclusion.

[0022] Screening and optimizing alternative serum or other animal-derived components and their concentrations are major challenges in developing serum-free culture media. Through rational combination of media components, optimization of culture conditions, and systematic validation, efficient and stable serum-free media can be developed to meet the needs of cell culture and scientific research. This invention isolated and identified porcine muscle stem cells and, using the Design of Experiments (DOE) method, developed and optimized a serum-free culture medium formulation that supports short-term, efficient cell proliferation through single-factor screening, Plackett-Burman experiments, and Box-Behnken response surface methodology.

[0023] This invention provides a serum-free proliferation medium for porcine muscle stem cells, using DMEM / F12 as the basal medium and ITS, L-ascorbic acid, BSA, Fetuin, and FGF-2 as additives. Previous experiments showed that DMEM / F12 and DMEM medium are significantly superior to RPMI 1640 in terms of cell adaptability for porcine muscle stem cells. Furthermore, based on the more comprehensive nutritional characteristics of DMEM / F12 medium, it was ultimately selected as the basal medium system. In this invention, ITS and L-ascorbic acid are essential additives in the basal medium, and the remaining components are optimized through systematic experiments to finally obtain a serum-free proliferation medium.

[0024] In a preferred embodiment of the present invention, the serum-free proliferation medium is based on DMEM / F12, containing 1% ITS, 200 μg / mL L-ascorbic acid, 523.32 μg / mL BSA, 976.99 μg / mL Fetuin, and 79.41 μg / mL FGF-2. This medium provides comparable initial cell culture performance to complete culture media, but at a lower cost, and is expected to replace serum-based culture media.

[0025] However, experimental data show that under serum-free culture conditions, porcine muscle stem cells experience cell adhesion and morphological stability issues during long-term passage, which affects their proliferation and differentiation capabilities. Those skilled in the art can further optimize the culture system using the serum-free proliferation medium provided in this invention, such as by supplementing key growth factors and adhesion molecules, to maintain cell viability and myogenic differentiation potential, thereby achieving long-term stability of serum-free culture.

[0026] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0027] Example 1 This example demonstrates the development of a serum-free culture medium suitable for porcine muscle stem cells, comprising the following steps: (1) Isolation and purification of porcine muscle stem cells.

[0028] After sterilizing clean tweezers, scissors, trays, etc., separate the leg or back muscles of the pig. Wash the muscle tissue block three times with PBS, remove the fascia (the white, smooth membrane attached to the muscle) and blood vessels, wash again, and then cut into a paste with scissors. Centrifuge at 3000 rpm for 5 min, resuspend the pellet in PBS, and repeat twice on ice. Then add collagenase digestion solution to the cut meat paste and digest at 200 rpm in a shaker at 37°C for 3 h, until it becomes visibly viscous. Add a 1:1 ratio of proliferation medium to the digested tissue to stop digestion. Filter the tissue sequentially using 100 μm, 70 μm, and 40 μm cell filters. After differential centrifugation for 3 h, obtain PSCs cells. Culture in RPMI 1640 medium containing 20% ​​FBS for 2-4 days. When the PSCs cell density reaches 80%-90%, trypsin digest and transfer to 10 cm cell culture dishes. After the cells reach confluence, freeze or passage for use.

[0029] (2) Obtaining key factors in serum-free culture medium.

[0030] DMEM / F12 was used as the basal medium, with 1% ITS and 200 μg / mL L-ascorbic acid as basal supplements, and any one of BSA, Fetuin, HGF, FGF-2, EGF-1, PDGF-BB, IGF-1, TGF-β3 and NRG1 added.

[0031] The concentrations of BSA, Fetuin, HGF, FGF-2, EGF-1, PDGF-BB, IGF-1, TGF-β3, and NRG1 in the culture medium were adjusted, and the effects of each factor on porcine muscle stem cell proliferation were assessed by measuring the absorbance at 450 nm using the CCK-8 assay. Results are as follows: Figure 1 As shown, all tested factors exhibit absorbance peaks within specific concentration ranges.

[0032] according to Figure 1 The distribution pattern of characteristic peaks was shown to determine the concentration gradient range of each factor in the Plackett-Burman experiment. The Plackett-Burman experiment used the CCK-8 assay at a wavelength of 450 nm to detect OD450 as the response index, evaluating the effects of the nine additives on the growth of porcine muscle stem cells. The experimental results showed that the maximum OD450 value was 1.49, and the minimum OD450 value was 0.610 (Table 1).

[0033] Table 1. Plackett-Burman experimental design matrix and response results

[0034] The Plackett-Burman experimental results were analyzed using Design-Expert 13 software using ANOVA. The results are shown in Table 2. A linear regression equation was obtained through linear fitting, as shown in the formula below. OD 450 =1.05+0.2281 A+0.1199 B-0.0214 C-0.0769 D+0.0409 E+0.0439 F+0.0647 G+0.0467 H+0.0216 J.

[0035] Table 2. Significance analysis results of the Plackett-Burman experimental design.

[0036] Note: Determination coefficient (R²) = 0.9946; Adjusted determination coefficient (Adj R²) = 0.8049; Coefficient of variation (CV) = 4.96%; This indicates that p < 0.05.

[0037] The results data analysis showed that the statistical reliability of the PB experimental design model met the research standards: the model p-value was 0.0242 (p < 0.05), confirming that the model had good fit within the regression domain; the F-value was 40.78, indicating that the system noise interference was controllable; the coefficient of determination was 0.9946, meaning that 99.46% of the variables could be explained by the model, indicating good correlation; the adjusted coefficient of determination was 0.8049, which was not significantly different from the coefficient of determination, indicating that the model had good fit; the CV value was 4.96%, indicating that the Plackett-Burman experiment had good reliability and accuracy; the precision of this experiment reached 19.3316, far exceeding the benchmark threshold of 4.0, further confirming the stability of the detection system.

[0038] The Lenth test was used to screen the Plackett-Burman experimental data for significance, and a half-normal distribution probability plot of the standardized effects was constructed. Figure 2 A) and its Pareto distribution map ( Figure 2 B). The results show that, Figure 2 The standardized effect values ​​of the three parameters A (BSA), B (Fetuin), and D (FGF-2) in A significantly deviated from the confidence interval (p < 0.05), and their statistical significance was double-validated in the Pareto plot by the distribution characteristics beyond the critical t-value.

[0039] Based on the correlation coefficients of the obtained first-order regression equations, the influence of each factor on the growth of porcine muscle stem cells was ranked as follows: A (BSA) > B (Fetuin) > D (FGF-2). Among them, BSA and Fetuin showed significant positive regulatory effects (p < 0.05), while FGF-2 showed growth inhibitory properties.

[0040] Based on the Plackett-Burman experimental results, the concentration ranges of BSA, Fetuin, and FGF-2 were further broadened through the steepest ramp experiment. The CCK-8 method was used to detect OD450 at a wavelength of 450 nm as the response index. The experimental results (Table 3) show that the OD450 value first increases and then decreases with increasing concentrations of BSA, Fetuin, and FGF-2. The maximum OD450 value appears at the 0+3Δ gradient, with an OD450 value of 1.21, corresponding to the following concentrations: BSA = 1400 μg / mL, Fetuin = 650 μg / mL, and FGF-2 = 15 ng / mL.

[0041] Table 3 Response results of the steepest climb experiment design

[0042] (3) Optimization of serum-free culture medium components.

[0043] The optimal combination of BSA, Fetuin, and FGF-2 was optimized using Box-Behnken response surface methodology, and the OD450 was measured at 450 nm using the CCK-8 method as the response index. Experimental results are shown in Table 4.

[0044] Table 4 Box-Behnken Experimental Design Matrix and Response Results

[0045] ANOVA analysis of the OD450 values ​​was performed using Design-Expert 13 software. The results showed that the model p-value was 0.0012 (p < 0.05), proving that the model was statistically significant. The coefficient of determination (R² = 0.9085) and the adjusted coefficient of determination (AdjR² = 0.8367) were not significantly different, indicating that the model had good overall fit and could explain 90.85% of the response variance. The quadratic regression equation fitted based on the results of the analysis of variance is shown below: OD 450 =1.19-0.0839 A+0.0478 B-0.0644 C-0.0729 AB-0.0639 AC+0.1184 BC+0.0417 A 2 -0.0899 B 2 +0.1968 C 2 .

[0046] Table 5. Significance Analysis Results of the Box-Behnken Experiment Design

[0047] Table 6 Supplementary Results of Significance Analysis of Box-Behnken Experimental Design

[0048] Note: R2=0.9634; Adj R2=0.9085; CV=3.96%; This indicates that p < 0.05.

[0049] Statistical analysis showed that the main effect of BSA (option A), the main effect and secondary effect of Fetuin and FGF-2 (linear terms B and D, B) were significant. 2 D 2 The nonlinear term and the two-factor interaction effects among the three factors (AB, AD, and BD terms) all reached the statistical significance threshold (p < 0.05). By comparing the predicted values ​​and experimental values ​​of the model, it was found that most of the experimental data points fell on the predicted line, and the two showed good agreement. The differences between the two are shown in […]. Figure 3 As shown.

[0050] To further determine the effects of BSA, Fetuin, and FGF-2 on the growth of porcine muscle stem cells and the optimal concentrations of these three substances, the CCK-8 assay was used to detect OD450 at a wavelength of 450 nm as a response index, and response surface models were constructed for the three substances. Three-dimensional response surface visualization showed that when the third variable was fixed at the encoded value of -1, the BSA-related surface exhibited a typical saddle-shaped configuration. Figure 4 (A, B), while the combination of Fetuin and FGF-2 exhibited multimodal fluctuation characteristics, with each parameter combination showing a clear response peak. Through analysis using a combination of three-dimensional response surface methodology and cube plots, the optimal concentration parameters were finally determined to be BSA 523.32 μg / mL, Fetuin 976.99 μg / mL, and FGF-2 79.41 ng / mL, with a theoretical OD450 prediction value of 1.64.

[0051] The validation experiment showed that under optimal culture conditions, the mean OD450 of porcine muscle stem cells was 1.60 ± 0.03 (n=6), and the measured OD450 value was 1.60, which was 95.0% of the predicted value. The relative deviation from the theoretical value was controlled within 5%, which fully verified the reliability of the Box-Behnken model in the optimization of serum-free culture system.

[0052] The culture medium system was named pMuSC-SFM, and its specific composition was as follows: DMEM / F12 as the basal medium, containing 1% ITS, 200 μg / mL L-ascorbic acid, 523.32 μg / mL BSA, 976.99 μg / mL Fetuin, and 79.41 ng / mL FGF-2.

[0053] Example 2 This study investigated the effects of serum-free culture medium pMuSC-SFM on the physiological characteristics of porcine muscle stem cells. The specific experiment is as follows: The complete culture medium (pMuSC-CM) was used as a control. The pMuSC-CM contained 10% FBS, 1% penicillin-streptomycin, 0.5% CEE, 1% NAEE, Gln, and 2.5 ng / mL FGF-2, and was supplemented with RPMI 1640 medium.

[0054] Porcine muscle stem cells were cultured using pMuSC-SFM and pMuSC-CM, respectively. Short-term culture observations were conducted on porcine muscle stem cells under different culture conditions, and growth curves, CCK-8 assays, and apoptosis assays were used to evaluate cell proliferation and apoptosis.

[0055] The results of cell morphology detection are as follows: Figure 5 As shown: In the initial stage (0 h), both groups of cells exhibited typical spindle shapes and were relatively dispersed. After 24 h of culture, cells in both the pMuSC-CM and pMuSC-SFM groups adhered well to the culture wall, demonstrating initial growth activity. By 48 h, the cell proliferation rate in the pMuSC-SFM group was slightly faster, but the cell arrangement was looser, and individual morphological differences were greater, suggesting that cells may exhibit irregular growth due to a lack of sufficient adhesion factors under serum-free conditions. Further observation showed that at 72 h and 96 h, cells in the CM group gradually tended to fuse, exhibiting a consistent spindle or fusiform shape, with plump cell morphology and continuously increasing density, indicating that serum components contribute to stable cell growth and continuous proliferation. These results demonstrate the important role of serum growth factors in maintaining cytoskeleton integrity and enhancing intercellular adhesion. In contrast, although the pMuSC-SFM group maintained normal growth within 72 h, between 72 and 96 h, cells gradually detached from the culture wall, and some cells began to become elongated and irregular in shape, accompanied by signs of apoptosis.

[0056] The results of the cell proliferation capacity test are as follows: Figure 6As shown in Figure A: Within the initial 48 h of culture, the proliferation capacity of the pMuSC-SFM group was comparable to that of the pMuSC-CM group; however, after 72 h, the pMuSC-CM group cells rapidly entered the exponential growth phase, while the proliferation of the pMuSC-SFM group cells slowed significantly and was accompanied by abnormal growth. CCK-8 assay results showed that the relative viability of the pMuSC-SFM group cells was 82.64 ± 1.48% (p < 0.05) within 24 h, and there was no significant difference between the pMuSC-SFM group and the pMuSC-CM group at 48 h. However, thereafter, the pMuSC-SFM group cells began to partially detach from the cell wall and undergo apoptosis, resulting in significantly lower proliferation at 72 h and 96 h compared to the pMuSC-CM group (p < 0.01). Figure 6 B).

[0057] Further apoptosis assays (at 48, 60, 72, and 96 h) yielded the following results: Figure 6 As shown in CE, the total apoptosis rates of the pMuSC-SFM group were 8.46±2.16%, 17.04±4.63% (p<0.001), 10.62±3.87% (p<0.05), and 37.92±6.34% (p<0.01), respectively, all significantly higher than those of the pMuSC-CM group. Among them, early apoptosis was lower than that of the pMuSC-CM group at 48, 60, and 72 h, but significantly higher than that of the pMuSC-CM group at 96 h. Late apoptosis was consistently higher than that of the pMuSC-CM group after 48 h, reaching 31.20±3.57% (p<0.01) at 96 h.

[0058] The above results demonstrate that the serum-free culture medium pMuSC-SFM constructed in this invention can effectively maintain the adherence, basic morphology, and proliferative activity of porcine muscle stem cells in the early stages of culture, exhibiting culture effects similar to those of complete culture medium within 48 hours, indicating that it can provide necessary short-term growth support for porcine muscle stem cells. Furthermore, this culture medium is serum-independent, possessing relatively well-defined components and high system controllability, providing an experimental basis for the subsequent establishment of a standardized serum-free culture system.

[0059] Example 3 The effect of serum-free culture medium pMuSC-SFM on the myogenic properties of porcine muscle stem cells was originally tested. The specific experiment is as follows: To evaluate the performance of serum-free culture medium and achieve long-term passage, an indirect passage strategy was adopted in this study: each passage was first performed using complete culture medium (pMuSC-CM) to allow cells to adhere within approximately 6 hours, and then immediately replaced with serum-free culture medium (pMuSC-SFM). Under these conditions, porcine muscle stem cells from different passages (P2, P5, P8, P10, P12, P13) were morphologically observed under pMuSC-SFM conditions. Cells from different passage numbers (P2, P5, P8, P10) were selected and induced to differentiate for 3 days. Their myogenic differentiation efficiency was then assessed by MyHC immunofluorescence staining.

[0060] Morphological observation results of different generations of porcine muscle stem cells as follows: Figure 7 As shown, the serum-free culture medium pMuSC-SFM constructed in this invention can maintain the basic morphological characteristics of porcine muscle stem cells and a certain level of myogenic differentiation potential during low passage stages.

[0061] The results of long-term passage of porcine muscle stem cells under pMuSC-SFM culture conditions are as follows: Figure 8 As shown, P2 generation cells still exhibited a high myogenic differentiation rate under this system, reaching 59.46±3.81%, suggesting that this culture medium has the feasibility of further optimizing as a basic formulation for a serum-free long-term culture system of porcine muscle stem cells.

[0062] Example 4 This example provides a simple cost analysis of the serum-free culture medium developed in this invention. Taking the optimal combination of pMuSC-SFM culture medium as an example, it is a serum-free culture system composed of DMEM / F12 as the base medium, with the addition of 1% ITS, 200 μg / mL L-ascorbic acid, and supplemented with 523.32 μg / mL BSA, 976.99 μg / mL Fetuin, and 79.41 ng / mL FGF-2.

[0063] Overall price comparison Figure 9 As shown, the cost of pMuSC-SFM medium is 2308.90 yuan / liter, significantly lower than the price of pMuSC-CM medium at 4460.78 yuan / liter, reducing culture costs by 48.24%. While ITS, Fetun, and FGF-2 are relatively expensive, purchasing these supplements in large quantities would further improve the cost-effectiveness. These findings indicate that the pMuSC-CM developed in this invention is an economically viable serum-free culture medium, laying the foundation for further development of serum-free culture media.

[0064] In summary, under the serum-free culture conditions of this invention, cell proliferation in the early stages of short-term culture is similar to that of complete culture medium, but the cost of serum-free culture medium is significantly lower than that of complete culture medium (reducing culture costs by 48.24%). Therefore, under certain conditions, the serum-free culture medium provided by this invention can completely replace serum culture medium. Furthermore, in long-term passaging, directly using serum-free culture conditions faces problems such as low cell adhesion and morphological changes, which can be solved through an indirect passaging strategy (pMuSC-CM adheres and then is converted to pMuSC-SFM). This invention utilizes response surface methodology to perform gradient optimization of key substances in serum-free culture medium, successfully constructing a low-cost, green pMuSC-SFM medium. This provides a scientific basis for further achieving long-term cell culture and functional maintenance under serum-free conditions. In the future, long-term cell activity and myogenic differentiation potential can be further improved by supplementing key growth factors or optimizing passaging strategies, thereby providing technical support for the commercial application of cultured meat.

[0065] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A serum-free proliferation culture medium for efficiently culturing porcine muscle stem cells, characterized in that, The basal medium was DMEM / F12, containing ITS, L-ascorbic acid, BSA, Fetuin, and FGF-2.

2. The serum-free proliferation culture medium according to claim 2, characterized in that, The ITS content is 100 mg / mL, the L-ascorbic acid content is 200 μg / mL, the BSA content is 523.32 μg / mL, the Fetuin content is 976.99 μg / mL, and the FGF-2 content is 79.41 μg / mL.

3. The application of the serum-free proliferation medium as described in claim 1 or 2 in the in vitro culture of porcine muscle stem cells.

4. The application according to claim 3, characterized in that, The application is the preparation of cultured meat using porcine muscle stem cells.

5. A method for long-term passage of porcine muscle stem cells, characterized in that, Using the serum-free proliferation medium as described in claim 1 or 2.

6. The subculture method according to claim 5, characterized in that, Each passage is first cultured on a serum-containing medium for adhesion, and then transferred to the serum-free proliferation medium for further culture.

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