Preparation Of Thermoresponsive Surface And Its Application In The Cultivation And Harvest Of Anchorage-dependent Cells

Anchorage-dependent cells which to be cultured and expanded in vitro have to undergo repeated trypsin enzyme-digesting processes for the need of subculture. But enzymatic treatment in these processes can induce the fractures of film proteins and often lead to cell dysfunction and disease, which will weaken the potential of cells in therapeutics application finally. So it is necessary to select a more moderate way for cell recovery. Recent studies have shown that temperature-responsive Poly (N-isopropylacrylamide)(PNIPAAm) can be potentially applied to culture and recover anchorage-dependent cells. Compared to enzymatic treatment, the extracellular matrix (ECM) proteins of cells cultured on PNIPAAm-containing materials can be more effectively protected after cooling treatment, thereby cell physiological functions will be maintained. If PNIPAAm based materials are used for the harvest of in-vitro cultured stem cells, it is possible to provide ideal seed cells with high quality for tissue engineering.In this study, thermoresponsive microcarriers, chitosan (CS)-g-polymer double layer film and P(CS-g-TMSPM-g-NIPAAm) copolymer film were prepared by grafting thermoresponsive monomer N-isopropyl acrylamide (NIPAAm) to microcarriers, CS film and CS molecular chain respectively.First, microcarriers were modificated by3-trimethoxysilylpropyl methacrylate (TMSPM), a silane coupling agent which contained carbon carbon double bond. Then thermoresponsive microcarriers were prepared by grafting NIPAAm and hydroxypropyl methacrylate (HPM) with the carbon carbon double bond on the surface of microcarriers by free radical polymerization initiated by2-azobisisobutyronitrile (AIBN). The images of scanning electron microscopy (SEM) showed the change of surface morphology and the results of energy dispersive spectrometer (EDS) showed the change of the elementary composition. To study if the prepared thermoresponsive microcarriers are suitable for the adhesion, growth and harvest of bone marrow mesenchymal stem cells (BMMSCs). the purified fourth passage of BMMSCs was seeded on the microcarriers. The adhesion rate and growth curve of BMMSCs adhered on microcarriers were determined. The live/dead situation of BMMSCs were stained by fluorescent dyes before and after being harvested by lowing temperature, and the harvested cells were cultured again. The results showed that the prepared thermoresponsive microcarriers had good cell compatibility. The adhesion rate of BMMSCs could achieve 87.73%after seeded for24h. BMMSCs reached plateau phase on the day5and day6, and can be harvested from grafted microcarriers by lowing temperature. The harvested cells retained biological activity well and could adhere on culture plate after seeded for12h.To prepare thermoresponsive CS film, polymer P (NIPAAm-co-HPM-co-TMSPM) was grafted on CS film surface. Fourier transform attenuated total reflection infrared spectrometry (ATR-FTIR) confirmed the existence of polymer on CS film surface. To examine the thermosensitivity of P(NIPAAm-co-HPM-co-TMSPM) grafted CS film and select optimal films for different types of cells, the attachment at37℃and detachment at20℃of anchorage-dependent cells cultured on grafted CS film were observed.Finally, CS molecular chains were modified with TMSPM and then P(CS-g-TMSPM-g-NIPAAm) copolymer was synthesized by free radical polymerization initiated by Potassium persulfate (PPS)/N, N, N’, N’-tetramethyl ethylenediamine (TEMED). ATR-FTIR confirmed the existence of NIPAAm and TMSPM in copolymer film composition. The Lower critical solution temperature (LCST) of copolymer in aqueous solution was34℃, which was determined by observing the variation of films’characteristics in aqueous solution at different temperature. The cellular compatibility of polymer films was investigated by the attachment of osteoblasts (OB) and ADSCs transfected by bone morphogenetic protein-2(BMP-2) on the film surface.