Poly(N-isopropylacrylamide)/Grapheme Oxide Hybrid Hydrogels For Cell Sheet Engineering

Cell sheet engineering,which fabricates 3D tissues without scaffolds,has been proposed as a novel approach for tissue engineering.Thermoresponsive polymer poly(Nisopropylacrylamide)(pNIPAAm)has the ability to switch between a hydrophobic and hydrophilic surface at different physiologically acceptable temperatures,and has been widely used in cell sheet harvesting systems.Hydrogels have been developed to mimic native tissue microenvironments in various applications.Because of its structural and physiological similarity to native extracellular matrices.Unfortunately,native pNIPAAm hydrogels have been reported not suitable for cell culturing and cell sheet harvesting for the low attachment of anchorage dependent cells.To improve the cell attachment behavior of pNIPAAm hydrogels,graphene oxide(GO)was selected as a nano strengthening agent to prepare nanocomposite hydrogels.The work of this thesis is divided into the following parts:The first part is the preparation of pNIPAAm/GO hydrogel and its application in cell sheet collection.In the present study,pNIPAAm/GO composite hydrogels with polyethylene glycol dimethacrylate(PEGDA)as crosslinker were fabricated by in-situ free radical polymerization.The morphology,composition and mechanical properties of the hydrogels were characterized by scanning electron microscopy(SEM),fourier transform infrared spectroscopy(FTIR)and electronic universal testing machine.The swelling properties and temperature-sensitive properties of the hydrogels at different concentrations of GO were also studied.At the same time,the effects of biocompatible GO nanomaterials on the biocompativity of pNIPAAm hydrogels were discussed,and cell detachment behavior from the pNIPAAm/GO composite hydrogels by temperature stimuli was investigated.The second part is the fabrication of 3D cell spheroids from free standing cell sheets.The single-line cell sheet or tumor cell-fibroblast co-cultured cell sheet was collected using the previously prepared pNIPAAm/GO composite hydrogel.The collected cell sheet was transferred to an anti-cell adhesion agar surface culture plate to obtain fibroblast cell spheroids,tumor multicellular spheroids and co-culture cell spheroids.Further,the thermosensitive hydrogel with circular patterns was used to prepare cell sheets of the same area,and then equal size of cell spheroids was obtained.The uniform 3D spheroid could be used as models for screening anticancer drugs.The third part is the preparation of patterned co-cultured cell sheets.Patterned pNIPAAm/GO hydrogel was prepared using PDMS as template to obtain a hydrogel with circular groove patterns on the surface.Because of the existence of the groove,the cells that firstly seeded tended to grow in the areas of the groove by shaking.After the first cells have covered the entire groove,a second cell line was then seeded is inoculated to achieve a patterned co-cultured cell sheet.In this section,due to the weak binding force between HeLa cells,a completed patterned co-cultured cell sheet was not obtained.