Micropatterned neural induction with heat-inactivated extracellular matrix protein by on-demand high-speed laser.

Introduction: 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. Methods: 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. Results: 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. Conclusions: Laser-induced heat inactivation of ECM on culture substrates enables lithography-, hydrogel-, and PDMS-free micropatterning, facilitating on-demand regulation of cell-autonomous tissue formation, the effect of teratogenic substances, and precise tissue engineering in regenerative medicine.