Preparation Of PVDF-g-PNIPAAm Thermo-sensitive Surface And Cell Cultivation And Harvest On It

Trypsin digestion was usually used to harvest anchorage-dependent cells cultured in vitro. The principle of trypsin digestion was using trypsin to degradate the extracellular matrix proteins to disconnect the cell-cell and cell-surface adhesions, then cells could divide. However, these proteins played an important role in cells adhering and growing. This approach undermined both the integrity of cell structure and function. Studies have shown that temperature-sensitive materials with Poly (N-isopropyl acrylamide) can be used to culture and harvest cells. When the cultured temperature was at37℃, which was higher than the LCST of PNIPAAm, the surface presents hydrophobicity which was suitable for cells adhesion and proliferation. When the temperature lowered to below LCST, the surface transferred to become hydrophilic, cells could detach from the surface automatically. Cell harvested with this new method could retain the complete membrane receptor proteins and the connections between cells were not destroyed, illustrating advantages to traditional cell harvest methods.In this paper, the preparation of a new kind of temperature-sensitive PVDF-g-PNIPAAm cell culture surface was explored. First, PVDF were treated with KOH at a concentration of2.5mol/L and then AIBN was used as an initiator to graft PNIPAAm on PVDF. Fourier transform infrared spectroscopy and1HNMR were applied to detect the structure of PVDF-g-PNIPAAm copolymer, the results confirmed the success grafting of PNIPAAm. The PVDF-g-PNIPAAm copolymer were then transformed to be temperature-sensitive films with the method of immersion precipitation phase transfer. X-ray photoelectron spectroscopy was used to detect the chemical composition on membrane surfaces, and PNIPAAm grafting rate was calculated according to the formula. The membrane surface morphology was detected by environmental scanning electron microscope. The surface contact angle analyzer was used to detect the static contact angle of different films at25℃and37℃. The results showed that the contents of N, O element of PVDF-g-PNIPAAm membranes were significantly improved compared with the PVDF membrane, and the membrane surface was porous honeycomb structure; with the increasing of PNIPAAm grafting rate, the surface contact angle decreased at25℃while increased at37℃, and the contact angle at25℃was lowered than that at37℃.Finally, Hela cells were seeded on the PVDF-g-PNIPAAm membranes, and the adhesion and cooling detaching behaviors of Hela cells on different membranes were investigated. The live/dead situation of Hela cells on membranes cultured for48h were stained by fluorescent dyes. The morphology of Hela cells cultured for24h and72h on membranes were observed. The viability of cells grown at different membrane surface was examined by MTT assay. Cells harvested by trypsin digestion method and temperature reduction method were stained by fluorescent dyes. The results showed the cell compatibility of PVDF-g-PNIPAAm membranes was good; cells could proliferate normally on the thermo-sensitive membranes. The results of MTT showed that cells can best proliferate on M32and M11. Cell detachment experiments showed that cells can detach from the thermal-sensitive membranes spontaneously, and the harvest rate was:M11> M32> M21> M23. Moreover, the activity of cells harvested by temperature reduction method was higher than that by trypsin digestion method, which proved the temperature reduction method had more advantages than the trypsin digestion method.