Fabrication of Functional High Density Cell Sheets for Tissue Engineering Using Spin-Coated poly(N-isopropylacrylamide)Thin Films

Abstract

The current goal of cell sheet engineering technology is to provide an improved approach for more predictive and effective generation of viable tissue-like constructs. Success requires validation and characterization of cell sheet growth and detachment. In this study, we perform qualitative and quantitative assessments of cell sheets lifted from thin spin-coated poly(N-isopropylacrylamide) films.Cell morphology, detachment time, metabolic activity and viability of mouse stromal and human corneal epithelial cell sheets were examined before and after lifting and after reattachment. The histological analysis of cell sheets and the immunofluorescence analysis of acting cytoskeleton, paxillin and cadherins were performed. The structure of extracellular matrix and the content of type I collagen in stromal and epithelial cell sheets were assessed by scanning electron microscopy and SDS-PAGE analysis, respectively.Results demonstrate that lifted cell sheets remain viable and maintain tissue-like integrity that strongly depends on reinforcement of acting cytoskeleton and cadherins at cell-cell junctions. Strong cell-cell adhesions and a high density of confluent cell sheets promote collective cell sheet detachment. Preserved extracellular matrix and focal adhesion complexes support cell sheet reattachment.This study demonstrates the potential of spin-coated poly(N-isopropylacrylamide) films in producing viable cell sheets that might be considered as a building block to create large biological tissues. Our results also describe the central cellular mechanisms involved in temperature-controlled collective cell migration. This study should promote further research in engineering three-dimensional tissues with complex organizational architecture.