Laser-induced cell injury in closed microphysiological systems: A novel method to study regeneration processes

Closed microphysiological systems are miniaturized, chip-sized platforms that can be used as cellularized organoid systems to study cellular processes like migration, regeneration or proliferation in-vitro. Due to the limited accessibility of the cells inside of closed microphysiological systems, the establishment of a well-defined mechanism to induce specific cell damage is difficult. Here we present a novel laser based method to induce well-defined leasions in closed cell layers. This could be a novel tool to study cellular mechanisms of different cell lines after injury. The present project aimed to establish well-defined lesion in cellular layers without removing the dead cells and their signals. Considering that we constructed a microphysiological system that was produced by layer laminate manufacturing at Fraunhofer IWS. According to the experimental needs the microphysiological system contains two fluidic circuits wich includes reservoirs, channels, and an integrated micropump. To establish the method, blood endothelial outgrowth cells (BEOC) were seeded into the microfluidic system previously coated with collagen (5ìg/cm²) at a density of approximately 7,5x104 cells/cm². After 3 hours of attachment, a pulsatile flow was applied to the channels. When the whole channel was covered with an BEOC monolayer, laser ablation took place between day 3 and 6 after seeding. To induce the selective cell injury we used an JenLas® D2.mini laser that was optically integrated into an inverted microscope. The irradiation took several seconds at a power of 3W with a wave length of 532 nm. The damage and the following regeneration processes were observed by fluorescence microscopy using LIVE/DEAD® Viability/Cytotoxicity Kit. Time Lapse recording was used to visualize the regeneration of the injured cell layers and to study the interacton between different cell types within the system.