MICROPATTERNED NEURAL INDUCTION WITH HEAT-INACTIVATED EXTRACELLULAR MATRIX PROTEIN BY ON-DEMAND HIGH-SPEED LASER

Abstract Microenvironmental heterogeneity in cultured cells can compromise cell quality, reduce experimental reproducibility, and weaken the confidence of cell therapeutic efficacy. Although micropatterned cell cultures are more homogeneous, conventional micropatterning methods lack flexibility. In this study, we develop a micropatterning technology by denaturing extracellular matrix (ECM) proteins in specific areas through heat inactivation using a high-speed laser via a light-responsive polymer layer. We have successfully seeded human induced pluripotent stem cells (hiPSCs) in flexible patterns and examine their neural induction in circular geometries of varying diameters. Size-dependent and cell-autonomous neural structures are formed on this substrate when hiPSCs differentiate into neural lineages in circles of different diameters. This self-organized pattern results from the mitotic orientation and localization of differentiating cells. Furthermore, teratogenic substances can modulate these patterns. Laser-induced heat inactivation of the ECM on culture substrates enables on-demand patterning, facilitating studies of cell-autonomous tissue formation, the effect of teratogenic substances, and precise tissue engineering in regenerative medicine. Table of Content A lithography-, hydrogel-, and PDMS-free micropatterning technology is developed to enable flexible and rapid cell patterning directly onto culture substrates via high-speed laser irradiation. Using this technology, size-dependent, cell-autonomous tissue formation from human induced pluripotent stem cells were driven by mitotic orientation and spatial localization of differentiating cells. These self-organized structures can be applied for safety tests of teratogenic substances. ToC figure