Closed micro-physiological systems (MPS) 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 MPS, 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 lesions in closed cell layers. This could be a novel tool to study cellular mechanisms of different cell types after injury. The present project aimed to establish well-defined lesion in cellular layers without removing the dead cells and the molecular signaling that is caused by apoptosis. Considering that, we constructed a MPS that was produced by layer laminate manufacturing. According to the experimental needs, the MPS contains two fluidic circuits which include reservoirs, channels, and an integrated micro pump. To establish the method, blood endothelial outgrowth cells (BOEC) were seeded into the MPS previously coated with collagen (5μg/cm2) at a density of approximately 7,5×104 cells/cm2. After 3 hours of attachment, a pulsatile flow was applied to the channels. When the whole channel was covered with a BOEC monolayer, laser ablation took place between day 3 and 6 after seeding. To induce the selective cell injury we used a JenLas D2.mini laser that was optically integrated into an inverted microscope. The irradiation took several seconds with a wavelength of 532 nm. The damage and the following regeneration processes were observed by fluorescence microscopy using LIVE/DEAD Viability/Cytotoxicity Kit and Time Lapse recording
Florian Schmieder, Deborah Förster, Mechthild Rösler, Jan Sradnick, Udo Klotzbach, Bernd Hohenstein, and Frank Sonntag, "Laser-induced cell injury in closed micro physiological systems: a novel method to study regeneration processes in vitro," Proc. SPIE 11268, Laser-based Micro- and Nanoprocessing XIV, 1126804 (Presented at SPIE LASE: February 03, 2020; Published: 2 March 2020); https://doi.org/10.1117/12.2566162.
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