细胞培养肉是在体外模拟动物肉类的生长机理，培养细胞获得可食用肉类的新型蛋白生产方式，是缓解全球性肉类供应和资源环境禀赋不足的潜在方式之一。传统细胞体外培养中使用血清，存在着成本高昂、批次不稳定、病原体传播等问题，因此低成本高效的无血清培养基开发研究是细胞培养肉产业面临的关键问题之一。本论文以猪肌肉干细胞为种子细胞，以建立高效的无血清培养基增殖与分化体系为目标，分析猪肌肉干细胞代谢与转录水平特点，建立高通量培养基筛选平台，成功开发用于猪肌肉干细胞的无血清增殖培养基和无血清分化培养基，为细胞培养肉的低成本高效生产打下研究基础。具体研究内容如下：

1.血清浓度对猪肌肉干细胞增殖影响与代谢特点研究

为研究添加不同浓度胎牛血清的增殖培养基对猪肌肉干细胞的体外增殖能力的影响，取第五代的猪肌肉干细胞为对象，通过细胞表型、增殖倍数、细胞活率和细胞增殖检测试剂盒来评估细胞短期增殖能力；通过细胞连续传代培养的扩增倍数来评估细胞长期增殖情况。另外，通过代谢组学比较含血清培养基中培养细胞前后代谢物的变化，总结培养过程中的细胞代谢特点。研究结果显示，当胎牛血清添加量小于1%，细胞的短期增殖能力会出现显著性差异，无胎牛血清组的细胞将直接死亡。胎牛血清添加量小于5%会显著影响细胞的长期增殖能力。另外，代谢组分析表明细胞对培养基的吸收代谢变化主要集中在脂类及脂肪酸代谢、溶血磷脂类代谢、甘油磷脂类代谢、氨基酸代谢、能量代谢等相关的代谢物中。上述结果为后续无血清培养基的开发确定研究起点与指明方向。

2.猪肌肉干细胞无血清增殖培养基配方研制及其转录特点探究

基于前人研究，本研究采用10 cm培养皿传统培养，胰酶消化传代计数的测试方式，选取并测试胰岛素-转铁蛋白-硒-乙醇胺（ITS-X）、碱性成纤维细胞生长因子（Basic fibroblast growth factor，bFGF）、胰岛素样生长因子（Insulin-like growth factors，IGFs）、聚乙烯醇（Polyvinyl alcohol，PVA）、白血病抑制因子（Leukemia inhibitory factor，LIF）、抗坏血酸、表皮生长因子（Epidermal growth factor，EGF）、4-(2-羟乙基)-1-哌嗪乙烷磺酸（4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid，HEPES）、皮质醇、牛血清白蛋白（Bovine serum albumin，BSA）、SB203580、Y-27632 2HCl、脂质补充剂、L-抗坏血酸-2-磷酸盐（L-ascorbic acid 2-phosphate，2PAA）、精胺、转化生长因子β1（Transforming growth factor β1，TGFβ1）作为初代无血清培养基组分，迭代开发了无血清增殖培养基Allin-4，该无血清增殖培养基配方能够支持猪肌肉干细胞7天扩增7倍，显著提高表征细胞干性的配对盒基因（Paired box gene 7，PAX7）表达量，成肌分化抗原（Myogenic differentiation antigen，MYOD）基因与肌细胞生成素（Myogenin，MYOG）基因的表达量。同时对比含血清培养基与无血清培养基在转录水平的差异，发现在细胞周期、抗氧化衰老、肌动蛋白和细胞外基质合成、营养能量代谢等方面存在差异，并整理出对应调节的关键因子，为进一步的无血清培养基优化确定方向。

3.孔板高通量平台建立与无血清增殖培养基优化

为了高效地进行无血清培养基优化工作，本研究将96孔板的细胞培养技术、Hoechst活细胞荧光染色技术与高内涵细胞成像系统整合建立了孔板高通量培养基筛选平台，并通过含血清培养基的细胞培养验证了该平台数据稳定性，该平台能将培养基测试效率提高10倍。基于此，本研究进一步筛选了数十种功能组分及其浓度，迭代获得了Allin-19配方，该培养基的促细胞增殖能力为Allin-4的222%，能够支持猪肌肉干细胞连续3代每代增殖超过3倍，活率稳定在80%以上，显著提高PAX7表达量，维持细胞干性，MYOD基因表达升高，MYOG基因表达降低，恢复成肌分化潜力。

4.基于转录研究的猪肌肉干细胞无血清分化培养基的研制

本研究测试了猪肌肉干细胞在血清饥饿诱导下成肌分化过程的转录特点，分析发现了INSR、IRS2、IGF1R、IGF1、IGFBP7、IGFBP4、TFRC、LPAR1、LPAR2、ALB、EGFR等关键差异表达的受体蛋白基因。基于转录组结果，以肌管分化百分比、肌管直径、肌球蛋白重链（Myosin heavy chain，MYHC）蛋白表达量为评价指标，分别测试了ITS-X、IGF-1、IGF-2、BSA、脂质补充剂、EGF在无血清成肌分化配方中的作用，确定了ITS-X、IGF-1、IGF-2、BSA和脂质补充剂的组合，并优化了IGF-1与IGF-2的浓度为25 ng/mL，形成了无血清成肌诱导分化培养基（版本3）。该培养基能提高细胞分化效率至73.8±5.1%，分化形成的平均肌管直径为62.9±17.8 μm。并通过转录组测序对比含血清与无血清诱导成肌分化过程，发现无血清诱导分化培养基诱导的细胞分化进程与含血清分化培养基诱导的分化进程类似，主要通过心肌收缩、心肌病、激素合成和分泌、活性氧和氧化磷酸化信号通路进行成肌诱导分化。

5.无血清增殖和分化培养基效果验证及成本分析

本研究将Allin-19用于培养猪成纤维细胞和猪平滑肌细胞，发现其均能实现连续3代传代培养，具有猪肌源性细胞普遍适应性。另外，比较发现了无血清成肌分化培养基在分化效率、肌管形成的质量和产生肌球蛋白含量等方面效果均优于传统的血清饥饿分化方案。不过肌肉干细胞在体外培养中出现干性衰退的问题，因此难以实现长期无血清增殖与分化的联用，后续发现课题组的细胞周期依赖性激酶抑制剂2A （Cyclin-dependent kinase inhibitor 2A，CDKN2A）基因敲除的猪肌肉干细胞可以实现长期培养并保持分化能力。我们将该细胞在无血清培养基中测试，发现针对CDKN2A基因敲除的猪肌肉干细胞，无血清增殖培养基Allin-19的长期传代能力与含血清配方类似，且能维持细胞干性基因的表达，同时促进细胞成肌分化。配合无血清成肌诱导分化培养基，建立了无血清增殖与成肌分化培养基的联合使用体系。同时，出于产业化成本考虑，计算发现无血清增殖培养基的价格仅为含血清培养基的33.6%，无血清分化培养基的价格仅为含血清培养基的10.9%，均明显低于含血清培养基。

综上所述，本研究分别探究了猪肌肉干细胞在增殖过程和成肌分化过程中转录与代谢特点，以此分别开发了猪肌肉干细胞的无血清增殖培养基（Allin-19）和无血清成肌诱导分化培养基（版本3），并在CDKN2A基因敲除细胞中实现了无血清培养基联合使用体系。为低成本、高效的细胞培养肉产业化推进提供理论与技术支持。

Cultured meat is a novel protein production method that simulates the growth mechanisms of animal meat in vitro, using cultured cells to obtain edible meat. It is one of the potential solutions to alleviate global meat supply shortages and resource environmental constraints. Traditional serum-based cell culture methods face challenges such as high costs, batch instability, and the risk of pathogen transmission. Therefore, developing low-cost and efficient serum-free culture media is one of the key issues facing the cultured meat industry. This thesis uses pig muscle stem cells as seed cells, aiming to establish an efficient serum-free proliferation and differentiation system. It analyzes the metabolic and transcriptional characteristics of pig muscle stem cells, establishes a high-throughput culture medium screening platform, and successfully develops serum-free proliferation and differentiation media for pig muscle stem cells, providing a research foundation for the low-cost and efficient production of cultured meat. The specific research content is as follows:

1. Effect of serum concentration on porcine muscle stem cell proliferation and metabolic characteristics.

Using P5 generation porcine muscle satellite cells as the subject, this study investigates the effect of proliferation media with varying concentrations of fetal bovine serum (FBS) on the in vitro proliferation capacity of these cells. Short-term proliferation capacity was assessed through cell phenotype, proliferation fold, cell viability, and EDU assays. Long-term proliferation was evaluated by the expansion fold of cells during continuous passaging. Furthermore, metabolomics analysis was performed to compare metabolite changes in cells cultured in serum-containing media before and after culture, summarizing the cellular metabolic characteristics during the culture process. The results showed that when the FBS addition was less than 1%, there were significant differences in the short-term proliferation capacity of the cells, and the serum-free group experienced direct cell death. FBS addition of less than 5% significantly affected the long-term proliferation capacity of the cells. In addition, metabolomic analysis indicated that the cellular absorption and metabolic changes of the culture medium were mainly concentrated in metabolites related to lipid and fatty acid metabolism, lysophospholipid metabolism, glycerophospholipid metabolism, amino acid metabolism, and energy metabolism. These results establish a starting point and direction for the subsequent development of serum-free culture media.

2. Development of a serum-free proliferation medium formulation for porcine muscle stem cells and research of their transcriptional characteristics.

Based on previous research, this study employed traditional 10 cm dish culture and trypsin digestion for passaging and cell counting. We selected and tested ITS-X, bFGF, IGFs, PVA, LIF, ascorbic acid, EGF, HEPES, Cortisol, BSA, SB203580, Y-27632 2HCl, Lipid Supplement, 2PAA, Spermine, and TGFβ1 as components of the initial serum-free medium. Through iterative development, we created a serum-free proliferation medium, Allin-4, which supports a 7-fold expansion of porcine muscle stem cells over 7 days. It significantly increased the expression of the PAX7 gene, a marker of stemness, while the expression levels of MYOD and MYOG genes gradually approached those of the control group. Concurrently, a comparison of the transcriptional differences between serum-containing and serum-free media revealed variations in cell cycle, antioxidant senescence, actin and extracellular matrix synthesis, and nutrient energy metabolism. Key regulatory factors related to these differences were identified, providing direction for further optimization of the serum-free medium.

3. Establishment of a microplate-based high-throughput platform and optimization of serum-free proliferation medium.

To efficiently optimize serum-free culture media, this study integrated 96-well plate cell culture techniques, Hoechst live-cell fluorescence staining, and a high-content cell imaging system to establish a high-throughput plate-based media screening platform. The data stability of this platform was validated through cell culture using serum-containing media, demonstrating a 10-fold improvement in media testing efficiency. Based on this platform, we further screened dozens of functional components and their concentrations, iteratively obtaining the Allin-19 formulation. This medium exhibited a 222% increase in cell proliferation compared to the Allin-4 formulation, supporting the continuous passage of porcine muscle stem cells for three generations with each generation exhibiting over 3-fold proliferation and a stable viability of over 80%. It significantly enhanced PAX7 expression, maintaining cell stemness, increased MYOD gene expression, decreased MYOG gene expression, and restored myogenic differentiation potential.

4. Development of a serum-free differentiation medium for porcine muscle stem cells based on transcriptomic studies.

This study investigated the transcriptional characteristics of porcine muscle satellite cells during myoblast differentiation induced by serum starvation, and identified key differentially expressed receptor protein genes, including INSR, IRS2, IGF1R, IGF1, IGFBP7, IGFBP4, TFRC, LPAR1, LPAR2, ALB, and EGFR. Based on the transcriptomic results, the effects of ITS-X, IGF-1, IGF-2, BSA, lipid supplement, and EGF in a serum-free myoblast differentiation formula were tested using myotube differentiation percentage, myotube diameter, and MYHC protein expression as evaluation indicators. A combination of ITS-X, IGF-1, IGF-2, BSA, and lipid supplement was determined, and the concentrations of IGF-1 and IGF-2 were optimized to 25 ng/mL, resulting in a serum-free myoblast induction and differentiation medium (version 3). This medium increased cell differentiation efficiency to 73.8±5.1%, and the average myotube diameter of the differentiated cells was 62.9±17.8 μm. Transcriptome sequencing comparison of serum-containing and serum-free induced myoblast differentiation processes revealed that the cell differentiation process induced by the serum-free differentiation medium was similar to that induced by the serum-containing differentiation medium, primarily through pathways involving myocardial contraction, cardiomyopathy, hormone synthesis and secretion, reactive oxygen species, and oxidative phosphorylation.

5. Serum-free proliferation and differentiation medium efficacy validation and cost analysis.

This study used Allin-19 to culture porcine fibroblasts and porcine smooth muscle cells, finding that it could achieve continuous passage for three generations in both, demonstrating universal adaptability for porcine muscle-derived cells. In addition, the serum-free myogenic differentiation medium was compared for differentiation efficiency, quality of myotube formation, and myosin protein content, and the results were superior to the traditional serum starvation differentiation protocol. However, muscle stem cells exhibit stemness decline during in vitro culture, making long-term serum-free proliferation and differentiation difficult to combine. Subsequently, it was found that the research group's CDKN2A gene knockout porcine muscle stem cells could be cultured long-term and maintain differentiation capacity. We tested our own serum-free medium and found that for CDKN2A gene knockout porcine muscle stem cells, the long-term passage ability of the serum-free proliferation medium Allin-19 was similar to that of serum-containing formulations, and it could maintain the expression of cell stemness genes while promoting myogenic differentiation. In combination with the serum-free myogenic induction differentiation medium, a combined usage system of serum-free proliferation and myogenic differentiation media was established. At the same time, considering industrialization costs, calculations show that the price of serum-free proliferation medium is only 33.6% of that of serum-containing medium, and the price of serum-free differentiation medium is only 10.9% of that of serum-containing medium, both significantly lower than the serum-containing medium.

In summary, this study investigated the transcriptional and metabolic characteristics of pig muscle stem cells during the processes of proliferation and myogenic differentiation. Consequently, a serum-free proliferation medium (Allin-19) and a serum-free myogenic induction differentiation medium (Version 3) were developed specifically for pig muscle stem cells. Additionally, a combined system of serum-free media was implemented in CDKN2A gene knockout cells. This research provides theoretical and technical support for the cost-effective and efficient industrialization of cell-cultured meat.