Abstract 15277: High-Throughput Optical Oxygen Measurements for Human IPSC-Cardiomyocytes and Ventricular Fibroblasts in 96-Well Plates

Abstract

For metabolically active cells like cardiomyocytes, a balanced state of oxygen supply and demand is crucial for basic cell function. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a valuable experimental model due of their scalability and relevance to human physiology. Oxygen consumption and availability have not been characterized in high-throughput format (HT), used in pre-clinical studies. Here, we quantified pericellular oxygen in syncytia of hiPSC-CM and human cardiac fibroblasts, grown in glass-bottom 96-well plates ( Fig 1A-C ). We deployed Ruthenium-based oxygen sensors and a ratiometric optical oxygen measurement system ( Fig 1B ) to track pericellular oxygen over time across all wells. Measured ratios were calibrated to % oxygen using two-point calibration with 5% Na 2 SO 3 and upon saturation with ambient air. Solution height and cell density were varied (4 solution heights, 3 cell densities) to understand their role in oxygen diffusion and oxygen consumption rate (OCR) by the cells. Michaelis-Menten kinetics and the Thiele modulus were used to computationally simulate oxygen availability in matching conditions. Without active perfusion or mass transport, for all tested conditions in the hiPSC-CMs, oxygen was depleted to <5% within approximately an hour. The drop of oxygen occurred in two phases - a zero-order decrease phase, followed by Michaelis-Menten kinetic phase ( Fig 1D ). Increase in culture medium volume/ height was found to be a powerful determinate of oxygen availability in simulations and in experiments ( Fig 1E n = 5 per group). Higher cell density correlated with steeper slope in the first phase of decline in oxygen availability, n = 4 per group. Cardiac fibroblasts followed a similar pattern (n = 4 per group) with noticeably slower OCR. Overall, our results indicate that pericellular oxygen in HT glass-bottom plates may reach hypoxic levels quickly; solutions for active mass transport need to be considered.