Li Weizhen, McLeod David, Ketzenberger John T, Kowalik Grant, Russo Rebekah, Li Zhenyu, Kay Matthew W, Entcheva Emilia
Department of Biomedical Engineering, School of Engineering and Applied Science, The George Washington University, Washington, DC, United States.
Front Bioeng Biotechnol. 2023 Jun 16;11:1214493. doi: 10.3389/fbioe.2023.1214493. eCollection 2023.
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a scalable experimental model relevant to human physiology. Oxygen consumption of hiPSC-CMs has not been studied in high-throughput (HT) format plates used in pre-clinical studies. Here, we provide comprehensive characterization and validation of a system for HT long-term optical measurements of peri-cellular oxygen in cardiac syncytia (human iPSC-CM and human cardiac fibroblasts), grown in glass-bottom 96-well plates. Laser-cut oxygen sensors having a ruthenium dye and an oxygen-insensitive reference dye were used. Ratiometric measurements (409 nm excitation) reflected dynamic changes in oxygen, as validated with simultaneous Clark electrode measurements. Emission ratios (653 nm vs. 510 nm) were calibrated for percent oxygen using two-point calibration. Time-dependent changes in the Stern-Volmer parameter, , were observed during the initial 40-90 min of incubation, likely temperature-related. Effects of pH on oxygen measurements were negligible in the pH range of 4-8, with a small ratio reduction for pH > 10. Time-dependent calibration was implemented, and light exposure time was optimized (0.6-0.8 s) for oxygen measurements inside an incubator. Peri-cellular oxygen dropped to levels <5% within 3-10 h for densely-plated hiPSC-CMs in glass-bottom 96-well plates. After the initial oxygen decrease, samples either settled to low steady-state or exhibited intermittent peri-cellular oxygen dynamics. Cardiac fibroblasts showed slower oxygen depletion and higher steady-state levels without oscillations, compared to hiPSC-CMs. Overall, the system has great utility for long-term HT monitoring of peri-cellular oxygen dynamics for tracking cellular oxygen consumption, metabolic perturbations, and characterization of the maturation of hiPSC-CMs.
人诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）代表了一种与人类生理学相关的可扩展实验模型。在临床前研究中使用的高通量（HT）格式培养板中，尚未对hiPSC-CMs的耗氧量进行研究。在此，我们提供了一个系统的全面表征和验证，该系统用于对生长在玻璃底96孔板中的心脏合胞体（人iPSC-CM和人心脏成纤维细胞）进行细胞周围氧气的HT长期光学测量。使用了具有钌染料和氧不敏感参比染料的激光切割氧传感器。比率测量（409nm激发）反映了氧气的动态变化，同时进行的Clark电极测量验证了这一点。发射比率（653nm对510nm）使用两点校准法针对氧百分比进行校准。在孵育的最初40-90分钟内观察到Stern-Volmer参数的时间依赖性变化，这可能与温度有关。在pH值4-8范围内，pH对氧测量的影响可忽略不计，pH>10时比率略有降低。实施了时间依赖性校准，并优化了在培养箱内进行氧测量的光照时间（0.6-0.8秒）。对于玻璃底96孔板中密集接种的hiPSC-CMs，细胞周围氧气在3-10小时内降至<5%的水平。在最初的氧气减少之后，样品要么稳定在低稳态，要么表现出间歇性的细胞周围氧气动态变化。与hiPSC-CMs相比，心脏成纤维细胞显示出较慢的氧气消耗和较高的稳态水平，且无振荡。总体而言，该系统对于长期HT监测细胞周围氧气动态变化以追踪细胞耗氧量、代谢扰动以及hiPSC-CMs成熟的表征具有很大的实用价值。
