Development of cell sheet with anisotropic patterns using thermo-sensitive and micropatterned hydrogel

Abstract

Anisotropic tissue structure, in which striated patterns assembled with extracellular matrix (ECM) and cells play an important role in their function. Previously, there were many challenges to mimic these structures using combination of substrates grafted with temperature-responsive polymers, transplantation tools and micropatterning procedure has been used to obtain cell sheets with anisotropic patterns. However, these approaches are found to have limitations such as longer transfer time, deformation of cell sheet by contraction, and complicated processing condition. In this study, I developed micropatterned thermosensitive hydrogels to achieve cell sheet with striated structure of ECM and cells using customized PDMS molds. The hydrogels were fabricated to have various sizes of topologic patterns (25 and 80 μm) using customized PDMS molds. Using this patterned hydrogel, over 90 % of anisotropic cell sheets could be transferred to target substrate by reducing temperature from 37 ℃ to 4 ℃ (size of patterns on hydrogels increased about 1.2 times) within 10 minutes. I demonstrated the effect of different pattern size on alignment of ECM/cell assembly by fast Fourier transform (FFT) analysis of fluorescently stained stress fibers and ECM proteins. The aligned cell sheet maintained their alignments after being transferred to the substrate. In addition, I demonstrated topologic patterns also promoted alignment of myotubes within 20 ° (p<0.05 vs. flat) and induced myotube differentiation about three times (p<0.05 vs. flat) in 3 days of myotube culture. Finally, I could stack cell layers controlled each direction using simple steps. My results showed that the mechanical cues given from patterns on hydrogel in a short time (1 day) could affect the differentiation of cell sheets potentially. Moreover, my platform will be applicable to proof-of-concept in fabrication of anisotropic organized structure.