Probing the Timeline of Integration between Three-Dimensional “Spark-Cell” Spheroids and Human Cardiac Tissue

Abstract

Prior work by our group in cardiac optogenetics has established the tandem-cell-unit strategy for pacing, i.e. light-sensitive (channelrhodopsin-2) non-myocytes, such as human embryonic kidney cells (ChR2-HEKs), when put in contact with human stem-cell-derived cardiomyocytes (hiPS-CMs), can be used for optical stimulation. We have successfully engineered 3D spheroids of ChR2-HEK “spark cells” and now set out probe the timeline of emergence of optical pacing after these structures are deposited onto hiPS-CM syncytia.ChR2-HEKs were seeded at 2 x 104cells/well onto 96-well microplates chemically designed to promote cellular self-assembly. Cells were cultured over 24 hours. Each resulting “spark-cell” spheroid was then transferred onto an iPS-CM monolayer and monitored in 2-hour increments. Blue light (470 nm) pulsed at selected frequencies in the range of 0.5 Hz to 2.0 Hz at <1mW/mm2was used to probe for responsiveness, while optical measurements with genetically-encoded calcium indicator R-GECO was used for capturing the response. We have observed a 1:1 relationship between blue light pulses and calcium transients paced at the selected frequencies as early as 2 hours after deposition of the cellular conduit on the hiPS-CM tissue.Our results demonstrate a simpler, scalable, and cost-effective method to optically stimulate engineered cardiac tissue compared to traditional stimulation approaches. In particular, we show that 3D “spark cell” spheroids can confer cardiac optical pacing within a much shorter time frame compared to direct optogenetic transformation of cardiomyocytes by plasmid transfection or viral infection, which typically requires more than 48 hours. Our future work involves extension of the method to other “spark cell” types such as cardiac progenitor cells, and robotic automation of the spheroid handling. We envision our work to improve high-throughput technology for pharmacological testing in cardiomyocytes.