Li Weizhen, McLeod David, Antonevich Sarah, Li Zhenyu, Entcheva Emilia
Department of Biomedical Engineering, The George Washington University, Washington DC 20052, USA.
bioRxiv. 2025 Feb 24:2025.02.19.639086. doi: 10.1101/2025.02.19.639086.
Adequate oxygen supply is crucial for proper cellular function. The emergence of high-throughput (HT) expansion of human stem-cell-derived cells and HT cellular assays for drug testing necessitate monitoring and understanding of the oxygenation conditions, yet virtually no data exists for such settings. For metabolically active cells like cardiomyocytes, variations in oxygenation may significantly impact their maturation and function; conversely, electromechanical activity can drive oxygen demands. We used HT label-free optical measurements and computational modeling to gain insights about oxygen availability (peri-cellular oxygen dynamics) in syncytia of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) and human cardiac fibroblasts (cFB) grown in glass-bottom 96-well plates under static conditions. Our experimental results highlight the critical role of cell density and solution height (oxygen delivery path) in peri-cellular oxygen dynamics. The developed 3D reaction-diffusion model with Michaelis-Menten kinetics, trained on the obtained comprehensive data set, revealed that time-variant maximum oxygen consumption rate, Vmax, is needed to faithfully capture the complex peri-cellular oxygen dynamics in the excitable hiPSC-CMs, but not in the cFB. For the latter, accounting for cell proliferation was needed. Interestingly, we found both hypoxic (< 2%) and hyperoxic (> 7%) conditions can easily emerge in these standard HT plates in static culture and that peri-cellular oxygen dynamics evolves with days in culture. Our results and the developed computational model can directly be used to optimize cardiac cell growth in HT plates to achieve desired physiological conditions, important in cellular assays for cardiotoxicity, drug development, personalized medicine and heart regeneration applications.
充足的氧气供应对于细胞的正常功能至关重要。人类干细胞衍生细胞的高通量（HT）扩增以及用于药物测试的HT细胞分析方法的出现，使得监测和了解氧合条件成为必要，但实际上几乎没有关于此类情况的数据。对于像心肌细胞这样代谢活跃的细胞，氧合的变化可能会显著影响其成熟和功能；相反，机电活动会驱动氧气需求。我们使用HT无标记光学测量和计算模型，以深入了解在静态条件下生长于玻璃底96孔板中的人诱导多能干细胞衍生心肌细胞（hiPSC-CM）和人心脏成纤维细胞（cFB）的合体细胞中的氧可用性（细胞周围氧动力学）。我们的实验结果突出了细胞密度和溶液高度（氧气输送路径）在细胞周围氧动力学中的关键作用。在获得的综合数据集上训练的具有米氏动力学的三维反应扩散模型表明，对于兴奋性hiPSC-CM，需要随时间变化的最大氧消耗率Vmax才能如实地捕捉复杂的细胞周围氧动力学，而对于cFB则不需要。对于后者，需要考虑细胞增殖。有趣的是，我们发现在这些静态培养的标准HT板中，低氧（<2%）和高氧（>7%）条件都很容易出现，并且细胞周围氧动力学随培养天数而变化。我们的结果和开发的计算模型可直接用于优化HT板中心脏细胞的生长，以实现所需的生理条件，这在心脏毒性细胞分析、药物开发、个性化医疗和心脏再生应用中很重要。
