Preparation Of Thermo-sensitive Culture Dish And The Study About Cell Sheet From This Special Dish

Objective (1) To explore the procedure for the synthesis of poly N-isopropylacry-lamide (PNIPAAm) and the thermo-sensitive culture dish grafted with PNIPAAm hydrogels by the radiation polymerization, which could be used for follow-up cell culture. (2) To research the growth conditions and the biological characteristics when the cells are seeded and then proliferate on this thermo-sensitve culure dish. The cells detache as single cell sheets from the thermo-sesitive surface. (3) The suitable procedure of the culture of rabbit oral mucosal epithelial cells (OMECs) is explored and the biological characteristics of OMECs are studied.Methods (1) N-isopropylacrylamide(NIPAAm) monomer was dissolved in 2-propanol at concentrations of 55% with 0.5% N,N'-Methylene bisacrylamide (MBA). Solution (70ul) was added and spread uniformly over tissue culture polystyrene (TCP) surfaces. These dishes were immediately subjected to irradiation with 250 kgy electron beam. A suitable synthetic method for thermo-sensitive PNIPAAm formation which could be used for follow-up cell culture was explored. The physical and chemical properties were determined by Fourier transform infrared (FTIR), scanning electron microscopy (SEM), and atomic force microsocoy (AFM). (2) PIPAAm-grafted dishes were sterilized and washed extensively with cold distilled water to remove unreacted NIPAAm monomer and ungrafted PIPAAm. The rabbit corneal stroma cells were cultured on the thermo-sensitive PNIPAAm. Phase contrast microscope eximination was performed daily. SEM eximination was carried out to observe the growth, arrangement and adhesion of cultivated cells. H&E staining were carried to observe the cell sheet without any scafford. (3) The rabbit OMECs with the digestion method were isolated and cultured. In order to explore the growing rule and influence factors of OMECs and make them more convenient and available application, phase contrast microscope and SEM examination were performed to observe the cellular morphology, biological characteristics. The expression of pan-keratin was examined by immunofluorescence. The proliferation of keratocytes was assessed by CCK-8 method. According to calcium concentration of K-SFM medium, we divided OMECs into the group A (without calcium) and group B (with 0.09mmol/L calcium).Results (1) Thermo-sensitive culture dish grafted with PNIPAAm hydrogels could be achieved when 55% NIPAAm monomer with 0.5%MBA were irradiated under 250kgy electron beam. FTIR showed that the hydrogels possessed the special spectral characteristics of PNIPAAm. SEM showed that the PNIPAAm hydrogels had multi-microporous structure. AFM displayed that the surfaces of thermo-sensitive materials were slight rough. (2) The phase contrast microscope and SEM showed that corneal stromal cells grew well in the thermo-sensitive surface. When the cell reached confluence, lowered the culture temperature and got the corneal stromal cell sheet. H&E staining showed that this cell sheet was without any scafford. (3) The phase contrast microscope showed that at the seventh day, primary OMECs reached confluence and looked like cobble-stone. And immunofluorescence revealed that the pan-keratin, marker of the epithelium cell, was localized in the cytoplasm of OMECs. SEM revealed that OMECs attached closely to each other with cell junctions. The CCK8 assay displayed that there were no difference between the group A and group B for OMECs proliferation.Conclusions:(1) According to the procedures described in this experiment, we could synthesize thermo-sensitive culture dish grafted with PNIPAAm hydrogels by the radiation polymerization. (2) Our thermo-sensitive culture dish provides a useful way by which we can get the cell sheet without any scafford. (3) This experiment provides an improved OMECs culture method. We can get suitable seeded OMECs for cell sheet and future tissue engineering.