Skrivergaard Stig, Rasmussen Martin Krøyer, Therkildsen Margrethe, Young Jette Feveile
Department of Food Science, Aarhus University, Aarhus, Denmark.
Stem Cell Rev Rep. 2025 Jul 3. doi: 10.1007/s12015-025-10915-7.
Quantifying muscle satellite cell proliferation and differentiation is crucial for applications in muscle regeneration, disease modeling, and cultivated meat research. Traditional fluorescence-based assays, while sensitive, are labor-intensive, endpoint-restricted, and disruptive to myotube integrity.
In this study, we present a novel high-contrast brightfield (HCBF) imaging technique for high-throughput, label-free assessment of both satellite cell proliferation and myogenic differentiation. Using the BioTek Cytation 5 automated imager and Gen5 software (Agilent Technologies), we optimized imaging parameters to achieve continuous, highly time-resolved quantification in standard 96- and 384-well formats without any additional reagents or cell manipulation needed.
Our approach enabled detailed kinetic profiling of satellite cell behavior, revealing myotube formation dynamics, species-specific media responses, optimal seeding conditions and the influence of mechanical factors on differentiation. We also demonstrated that serum-free media formulations could support efficient myotube formation in both bovine and porcine satellite cells, while having very different myotube kinetics and morphology than serum-containing samples. Furthermore, we highlighted the high degree of well-to-well variation and the sporadic formation and detachment of myotubes in culture, and the interesting phenomena of a second wave of myotubes being formed following detachment in serum-containing samples. Additionally, the 384-well format enabled a label-free screening method to assess clonal myogenicity of isolated satellite cells.
By eliminating the need for genetic labeling, invasive staining or specialized consumables, our high-throughput HCBF methodology advances myogenic research, offering new opportunities for efficient screening and highly detailed kinetic data acquisition for serum-free media development, drug discovery and pathophysiological testing for both cultivated meat and musculoskeletal research.
量化肌肉卫星细胞的增殖和分化对于肌肉再生、疾病建模及人造肉研究的应用至关重要。传统的基于荧光的检测方法虽然灵敏，但劳动强度大、受终点限制，且会破坏肌管完整性。
在本研究中，我们提出了一种新型的高对比度明场（HCBF）成像技术，用于对卫星细胞增殖和肌源性分化进行高通量、无标记评估。使用BioTek Cytation 5自动成像仪和Gen5软件（安捷伦科技公司），我们优化了成像参数，以在标准的96孔和384孔板中实现连续、高度时间分辨的定量分析，无需任何额外试剂或细胞操作。
我们的方法能够对卫星细胞行为进行详细的动力学分析，揭示肌管形成动态、物种特异性培养基反应、最佳接种条件以及机械因素对分化的影响。我们还证明，无血清培养基配方能够支持牛和猪卫星细胞高效形成肌管，同时其肌管动力学和形态与含血清样本有很大不同。此外，我们强调了培养中孔间差异程度高以及肌管的零星形成和脱离现象，以及在含血清样本中脱离后形成第二波肌管的有趣现象。此外，384孔板形式实现了一种无标记筛选方法，用于评估分离的卫星细胞的克隆肌源性。
通过无需基因标记、侵入性染色或特殊耗材，我们的高通量HCBF方法推动了肌源性研究，为无血清培养基开发、药物发现以及人造肉和肌肉骨骼研究的病理生理测试提供了高效筛选和高度详细的动力学数据采集的新机会。
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