Pericellular oxygen dynamics in human cardiac fibroblasts and iPSC-cardiomyocytes in high-throughput plates: insights from experiments and modeling

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_max, 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.