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<p>The advancement of cultured fat toward viable industrial production is currently hindered by economic and technical constraints, including the formulation of cost-effective, chemically defined culture media. This study addresses serum dependency in bovine mesenchymal stem cells (bMSCs), a common cell source for cellular agriculture, and particularly for cultured fat given their well-characterized adipogenic potential. We show that while supplementing reduced-serum media with insulin-transferrin-selenium-BSA (ITS+) and fibroblast growth factor 2 (FGF2) mitigates short-term viability loss, these factors fail to sustain prolonged proliferation in serum-free media. To uncover the molecular barriers to serum-free adaptation, we performed a targeted CRISPR-Cas9 screen, identifying TP53 as the primary checkpoint restricting survival. Targeted TP53 disruption conferred a robust survival advantage, enabling long-term expansion in serum-free conditions. By contrast, <i>PTEN</i> knockout displayed an intermediate phenotype, improving viability relative to wild-type cells but falling short of the proliferative capacity achieved by TP53 ablation. Furthermore, we demonstrated that mitigating proteolytic stress using soybean trypsin inhibitor is essential for effective serum-free subculturing. These findings establish that p53 pathway disruption decouples adipose-derived bMSCs (Ad-bMSCs) expansion from serum dependency, offering a scalable strategy for chemically defined fat production.</p>

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Eliminating serum dependency of bovine mesenchymal stem cell expansion via media composition and genetic screen

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年8月5日

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  • Amit Zirman,
  • Dar Yaron,
  • Yuval Peled,
  • Shahar Amichai,
  • Roni Rak

摘要

The advancement of cultured fat toward viable industrial production is currently hindered by economic and technical constraints, including the formulation of cost-effective, chemically defined culture media. This study addresses serum dependency in bovine mesenchymal stem cells (bMSCs), a common cell source for cellular agriculture, and particularly for cultured fat given their well-characterized adipogenic potential. We show that while supplementing reduced-serum media with insulin-transferrin-selenium-BSA (ITS+) and fibroblast growth factor 2 (FGF2) mitigates short-term viability loss, these factors fail to sustain prolonged proliferation in serum-free media. To uncover the molecular barriers to serum-free adaptation, we performed a targeted CRISPR-Cas9 screen, identifying TP53 as the primary checkpoint restricting survival. Targeted TP53 disruption conferred a robust survival advantage, enabling long-term expansion in serum-free conditions. By contrast, PTEN knockout displayed an intermediate phenotype, improving viability relative to wild-type cells but falling short of the proliferative capacity achieved by TP53 ablation. Furthermore, we demonstrated that mitigating proteolytic stress using soybean trypsin inhibitor is essential for effective serum-free subculturing. These findings establish that p53 pathway disruption decouples adipose-derived bMSCs (Ad-bMSCs) expansion from serum dependency, offering a scalable strategy for chemically defined fat production.