Sumaru, K. | Search bioRxiv

bioRxiv2025

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

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 ContentA 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 O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/663334v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@22844aorg.highwire.dtl.DTLVardef@1b3938org.highwire.dtl.DTLVardef@149a748org.highwire.dtl.DTLVardef@85834c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2018

Automated Adherent Cell Elimination by a High-Speed Laser Mediated by a Light-Responsive Polymer

Conventional cell handling and sorting methods require manual dissociation, which decreases cell quality and quantity and is not well suited for monitoring applications. To purify adherent cultured cells, in situ cell purification technologies that are high throughput and can be utilized in an on-demand manner are expected. Previous demonstrations using direct laser-mediated cell elimination revealed only limited success in terms of their usability and throughput. Here, we developed a Laser-Induced, Light-responsive-polymer-Activated, Cell Killing (LILACK) system that enables high-speed and on-demand adherent cell sectioning and purification. This system employs a visible laser beam, which does not kill cells directly, but induces local heat production through the trans-cis-trans photo-isomerization of azobenzene moieties in only the irradiated area of a light-responsive thin layer. Using this system in each passage for sectioning, human induced pluripotent stem cells (hiPSCs) were maintained their pluripotency and self-renewal during long-term culture. Furthermore, combined with deep machine-learning analysis on fluorescent and phase contrast images, a label-free and automatic cell processing system was developed by eliminating unwanted spontaneously differentiated cells in undifferentiated hiPSC culture conditions.

bioengineering↗