Preparation Of PVDF-g-PNIPAAm Thermo-sensitive Fiber Membranes By Electrostatic Spinning And Application In Cultivation And Harvest Of Cells

Trypsin digestion has long been the main way to harvest anchorage-dependent cells. But trypsin will damage the proteins of extracellular matrix (ECM), leading to the degradation of the structure and function of cell integrity. Cultivating cells on thermos-sensitive material, then harvesting cells by lowering the temperature, can make the ECM of cells maintain integrity, and have more advantages. Poly(N-isopropyl acrylamide)(PNIPAAm), as a thermos-sensitive polymer with widespread use, has the lower critical solution temperature (LCST) close to the body temperature. Since the normal temperature for cell culture is higher than LCST, the PNIPAAm presents hydrophobicity, suitable for cell adhesion and proliferation. When the temperature is blow the LCST, the PNIPAAm presents hydrophilic, which is not suitable for cells to adhere, so that the cells will peal off by themselves. Polyvinylidene fluoride (PVDF) is a good material, and the PNIPAAm grafted on its molecular chain can effectively improve the mechanical and spinnability of PNIPAAm. Nanofiber can simulate the cell EMCS structure, conductive to the adhesion and growth of cells. It is possible to use the thermos-sensitive polymer to prepare nanofiber structure by electrostatic spinning technology, then to cultivate cells on the surface. In this way, the cells can adhere on the surface well, and can be harvested efficiently with temperature reduction.In this study, we used the method of alkali treatment to modify the PVDF powder materials, and a new type of polymer with carbon-carbon double bond(C=C) or three key (C=C) was obtained. Using 2-azobisisobutyronitrile (AIBN) as initiator, a series of PVDF-g-PNIP-AAm thermos-sensitive polymers with different grafting percentage were prepared with free polymerization method by changing the mass ratio of NIP A Am monnmer and alkali treatment PVDF. Fourier transform infrared spectroscopy (FTIR) and H-NMR spectrum were used as characterization to determine the PNIPAAm was grafted successfully. And the grafting percentage was calculated. Finally, under the better parameters for electrospinning PVDF (15 cm of receiving distance.15kv of spinning voltage,8:2 of solvent N,N-dimethyl formamide (DMF) and acetone volume ratio,12wt% of dope concentration) after exploring, a series of PVDF-g-PNIPAAm thermos-sensitive fiber membranes M21, M43, M11 and M45 were prepared by electrospinning with different PVDF-g-PNIPAAm polymer. The morphology of each fiber membrane was observed by scanning electron microscope, indicating that the grafting percentage of PNIPAAm has influenced on the spinnability of PVDF. the higher the grafting ratio, the worse the spinnability of PVDF-g-PNIPAAm polymer. Fourier transform infrared spectroscopy of PVDF and PVDF-g-PNIPAAm fiber membranes showed that electrostatic spinning would not change the structure of thermos-sensitive polymer. The static water contact angle of different thermos-sensitive fiber membranes tested in different temperature showed that, the PVDF-g-PNIPAAm fiber membranes with different grafting ratio all had thermosensitivity, and with the increase of grafting ratio of PNIPAAm, the hydrophilic shift of the surface caused by changing temperature is more obvious.PC 12 cells were seeded on the surfaces of MPVDF, M21, M43 and M11 for cell experiments. The cell adhesion, proliferation and growth on different fiber membranes were examined. Then harvest of cells on different fiber membranes with temperature reduction was compared, and the cells harvested from M11 was also compared with those harvested by trypsin digestion method. The cells were continuously cultured and observed for three days. And it was found that the cells on various fiber membranes surface all grew with good adhesion. The images of cell live/dead fluorescence staining showed that cells in different fiber membranes all had a high viability and the prepared thermos-sensitive fiber membranes had good biocompatibility. The results by MTT assay showed that the nanometer fiber structure was conducive to the proliferation and grown of cells. The cells growth on the surface of M21 were the best and relatively poor on the M11, which indicated that higher grafting ratio is not suitable for the adhesion and growth of cells, There should be a suitable range. Finally the cells cultivated on the three thermos-sensitive fiber membranes for three days were harvested with temperature reduction, and the stripping ratio from high to low is:M11, M43, M21, showing that high grafting ratio is advantageous to the detachment of cells. Compared with trypsin digestion method, the result also shows that the temperature reduction method has significant advantages.