Heinson Yuli W, Han Julie L, Entcheva Emilia
Department of Biomedical Engineering, The George Washington University, Washington, DC 20037.
bioRxiv. 2023 Aug 31:2023.08.29.555447. doi: 10.1101/2023.08.29.555447.
We present a simple low-cost system for comprehensive functional characterization of cardiac function under spontaneous and paced conditions, in standard 96 and 384-well plates. This full-plate actuator/imager, OptoDyCE-plate, uses optogenetic stimulation and optical readouts of voltage and calcium from all wells in parallel. The system is validated with syncytia of human induced pluripotent stem cell derived cardiomyocytes, iPSC-CMs, grown as monolayers, or in quasi-3D isotropic and anisotropic constructs using electrospun matrices, in 96 and 394-well format. Genetic modifications, e.g. interference CRISPR (CRISPRi), and nine compounds of acute and chronic action were tested, including five histone deacetylase inhibitors (HDACis). Their effects on voltage and calcium were compared across growth conditions and pacing rates. We also demonstrated deployment of optogenetic cell spheroids for point pacing to study conduction in 96-well format, and the use of temporal multiplexing to register voltage and calcium simultaneously on a single camera in this stand-alone platform. Opto-DyCE-plate showed excellent performance even in the small samples in 384-well plates, in the various configurations. Anisotropic structured constructs may provide some benefits in drug testing, although drug responses were consistent across tested configurations. Differential voltage vs. calcium responses were seen for some drugs, especially for non-traditional modulators of cardiac function, e.g. HDACi, and pacing rate was a powerful modulator of drug response, highlighting the need for comprehensive multiparametric assessment, as offered by OptoDyCE-plate. Increasing throughput and speed and reducing cost of screening can help stratify potential compounds early in the drug development process and accelerate the development of safer drugs.
我们展示了一种简单的低成本系统，用于在标准96孔和384孔板中，在自发和起搏条件下对心脏功能进行全面的功能表征。这种全板致动器/成像仪OptoDyCE-plate，利用光遗传学刺激以及对所有孔中电压和钙的光学读数，并行进行操作。该系统已通过人诱导多能干细胞衍生的心肌细胞（iPSC-CMs）的合体细胞进行验证，这些细胞以单层形式生长，或使用电纺基质在96孔和384孔板中形成准三维各向同性和各向异性结构。测试了基因修饰，如干扰性CRISPR（CRISPRi），以及九种具有急性和慢性作用的化合物，包括五种组蛋白脱乙酰酶抑制剂（HDACi）。比较了它们在不同生长条件和起搏速率下对电压和钙的影响。我们还展示了光遗传学细胞球体在96孔板中用于点起搏以研究传导，以及在这个独立平台上使用时间复用在单个相机上同时记录电压和钙。即使在384孔板中的小样本以及各种配置中，Opto-DyCE-plate也表现出优异的性能。各向异性结构构建体在药物测试中可能具有一些优势，尽管在所测试的配置中药物反应是一致的。对于一些药物，特别是对于心脏功能的非传统调节剂，如HDACi，观察到了电压与钙反应的差异，并且起搏速率是药物反应的有力调节剂，这突出了OptoDyCE-plate所提供的全面多参数评估的必要性。提高通量和速度以及降低筛选成本有助于在药物开发过程的早期对潜在化合物进行分层，并加速更安全药物的开发。
