Preparation Of Cell Sheets By Thermosensitive Coating Attachment On The Surface Of Elastic Silica Gel

Objective: In this study,polydimethylsiloxane(PDMS),a silica gel with excellent mechanical properties,was used as the substrate material,on whose surface thermosensitive PNIPAAm polymer film was grafted for culturing mouse osteoblast(MC3T3)sheets.During the experiment,we explored the adherence,growth,proliferation of cells on the material surface and the detachment of cell sheets.This study intends to provide an easy-operation,non-destructive method of constructing cell sheets for bone tissue engineering.The study also expects to carry out the in-vitro mechanical dynamic test during cell cultivation by utilizing the excellent mechanical characteristics of PDMS in the later experiments while harvesting cell sheets with better properties.Experimentation and results: In this experiment,the PDMS mechanically stretched dish was prepared by specially designed tensile mould.The PNIPAAm monomer was graft-copolymerized onto the surface of plasma-treated PDMS stretched dish by UV light,and the surface of the stretched dish bottom was subjected to SEM observation,spectroscopic analysis and FT-IR analysis.The experimental results demonstrated the presence of grafted layer.Afterwards,the surface proteins were modified by adding fibronectin solution into the successfully surface-grafted culture dish,and then MC3T3 cells were seeded onto the PDMS stretched dish of the experimental group.Cells in the PNIPAAm-g-PDMS group were closely stacked to each other on the5 th day of cultivation,and individual cells were morphologically vague,with appearance of macroscopically visible cellular membrane sheets.After the temperature dropped to 4?,the edges of cell membranes began to tilt and extended gradually towards the middle.30 min later,the cell sheets were detached completely.After viable/dead staining,the detached cell sheets were stained green,which exhibited a clear outline.Hence,successful construction and detachment of the cell sheets were proved.Conclusions: The experiment uses plasma and UV light grafting technique to attach thermosensitive PNIPAAm film on the surface of elastic silica to successfully cultivate mouse osteoblasts(MC3T3)sheets,which are then successfully detached by temperature control technology.The experiment confirms the feasibility of such method for preparing novel intelligent material while simultaneously constructing cell sheets,which provides a new idea for the non-destructive preparation of cell sheets and the experimental method of cell mechanics.