The Preparation, Characterization And Cell Culture On The Surface Of Thermo-sensitive Cell Culture Dish

Tissue engineering techniques rely largely on scaffold-based approaches, often using biodegradable and biologically derived polymersto provide a porous, space-filling, temporary extracellularmatrix (ECM) of desired macroscopic form within which cells grow and integrate. However, such scaffold architectures are primitive compared to those they intend to duplicate in vivo. Additionally, insufficient cell migration into and retention within scaffolds, different rates of cell proliferation compared to scaffold degradation,and host inflammatory reactions remain problems.Further improvements in scaffold design may overcome some of these problems.In contrast to conventional methods, a novel cell sheet-based technique has been proposed .This technique intend to achieve high cell density and function by layering confluent viable cell sheets without the use of acellular scaffolds that limit mass transport. Using thermo-sensitive cell culture surfaces, confluently cultured cells spontaneously detach as intact sheets simply by reducing the culture temperature. Covalently grafted poly (N- isopropylacrylamide) (PNIPAm),which exhibits reversible temperature- dependent soluble/insoluble changes (i.e., lower critical solution temperature of 32℃) in aqueous solution, facilitates both cell adhesion and cell detachment, eliminating use of cell-harvesting enzymes that dissociate cell cultures into dispersed cells.This paper was mainly about the surface modification of cell culture dish by conbining plasma pretreatment and UV-incued PNIPAm graft polymerization, in orer to aquire the thermo-sensitive cell culture dish. Moreover, the surface was characterized and analysed by FT-IR,XPS,AFM and SEM. Cytotoxicity of the dish was also evaluated.In the end ,two types of cells (OLC and HPLC)were cultured and cell sheets detachment can be observed by reducing the culture tempareture.1. The preparation of thermo-sensitive cell culcure dishThe normal cell culture dishes (Polystyrene) were subjected to plasma pretreatment and UV-incued PNIPAm graft polymerization,without photo-initiator. The changement of contactact angle was used as the indication of the surface graft modifaction and orthogonal experimental design was used to arrange the experiment groups. Four different levels of four factors were analysed,intending to find an optimized combinations.The optimized combinations were W/FM 0.08GJ/Kg,plasma pretreatment time 2min,NIPAm monomo concentration 30%,UV irradiation time 15min,under which conditions the changement of contactact angle were maximal.2. The analysis and characterization of thermo-sensitive cell culcure dish surfaceThe surface of prepared cell culture dish was characterized and analysed by FT-IR,XPS,AFM and SEM. The IR spectrum showed the function group-amide I peak appeared on the grafting surface,which was characteristic function group of PNIAPm.The XPS analysis showed the presence of atom C ,N ,O on the grafting surface ,which was the chemical components of NIAPm.The AFM and SEM showed the similar appearance of grating surface ,apparently different to normal suiface.Obvious difference were confirmed between the grafting and normal surface of cell culture dish,which proved the results that PNIAPm had been successfully grafted onto the surface of cell culture dish.3. The cytotoxic evaluation and observation of cell culcure on the thermo-sensitive cell culcure dishThe cytotoxicity of thermo-sensitive cell culcure dish was evaluated by direct contact with MTT-based method.The relative cell groth rate of L929 cell represented the proliferation of cells on materials with respect to control over time. The result showed that cytotoxicity of thermo-sensitive cell culcure dish was none or little,which can be used for lab use.Additional cell culture was performed with two types of cell (OLC and HPLC)and cell sheets detachment were observed by reducing the culture tempareture after the confluently growth of the two types of cells.