Li Weizhen, McLeod David, Antonevich Sarah, Pozo Maria R, Li Zhenyu, Entcheva Emilia
Department of Biomedical Engineering, The George Washington University, Washington, DC 20052, USA.
J Mol Cell Cardiol Plus. 2025 Jun 11;13:100464. doi: 10.1016/j.jmccpl.2025.100464. eCollection 2025 Sep.
Adequate oxygen supply is crucial for proper cellular function. The emergence of high-throughput (HT) expansion of human stem-cell-derived cells and HT in vitro cellular assays for drug testing necessitate monitoring and understanding of the oxygenation conditions, yet virtually no data exists for such settings. We used HT label-free optical measurements and computational modeling to gain insights about oxygen availability (pericellular 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 pericellular oxygen dynamics. The developed computational model, trained on the obtained comprehensive data set, revealed that time-variant maximum oxygen consumption rate, V, is needed to faithfully capture the complex pericellular oxygen dynamics in the excitable hiPSC-CMs, but not in the cFBs. Interestingly, hypoxia (<2 % pericellular oxygen) developed within hours in the dense iPSC-CM cultures when the solution volume was sufficiently large. Conversely, hiPSC-CMs grown at low cell density or in smaller solution volume, as well as cFB under all studied conditions, were found to operate in hyperoxic (>7 %) conditions. Pericellular oxygen dynamics of the differentiated hiPSC-CMs evolved over days in culture, with the best improvement in respiration seen in samples operating close to normoxia. Our results and the developed computational model can be used directly to optimize cardiac cell growth in HT plates and achieve desired physiological conditions, which is 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体外细胞检测技术的出现，使得监测和了解氧合条件成为必要，但实际上针对此类情况的数据几乎不存在。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于玻璃底96孔板中生长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。基于所获得的综合数据集进行训练的已开发计算模型表明，需要时变的最大耗氧率V才能准确捕捉可兴奋的hiPSC-CM中复杂的细胞周围氧动力学，而cFB则不需要。有趣的是，当溶液体积足够大时，在密集的iPSC-CM培养物中数小时内就会出现缺氧（细胞周围氧含量<2%）情况。相反，发现以低细胞密度或在较小溶液体积中生长的hiPSC-CM，以及在所有研究条件下的cFB，都处于高氧（>7%）条件下运行。分化后的hiPSC-CM的细胞周围氧动力学在培养过程中会在数天内发生变化，在接近正常氧水平运行的样本中呼吸改善最为明显。我们的结果和已开发的计算模型可直接用于优化HT板中心脏细胞的生长并实现所需的生理条件，这在心脏毒性细胞检测、药物开发、个性化医疗和心脏再生应用中具有重要意义。
