Preparation Of Novel Temperature-responsive Film And Stem Cell Culture And Recovery On It

Stem cells possess self-renewal ability and multilineage differentiation potential so that they are ideal target cells for tissue engineering, gene engineering and other biological medicine domains. But the number of primary stem cells derived from tissue or body fluids is insufficient, thus they need to be cultured and expanded in vitro to satisfy the clinical requirements where the cells have to undergo repeated plating-harvesting processes. The enzymatic treatment is still widely used in cell recovery, but the involvement of enzymes in these processes can induce the fractures of membrane proteins and any alterations in these proteins often lead to cell dysfunction and disease. Therefore, enzymes can impair cell characteristics and accordingly weaken the potential of stem cells in therapeutics application. So it is necessary to select a more reasonable system for stem cell culture and recovery. Poly (N-isopropylacrylamide)(PNIPAAm), a temperature-responsive polymer, can be potentially applied to replace enzymes to recover attached cells. The membrane proteins or related receptors 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 in vitro culture of stem cells, it is possible to provide ideal seed cells with high quality for tissue engineering.A novel PNIPAAm-containing copolymer was synthesized, and its temperature-sensitive properties, chemical structure, molecular composition and molecular weight were investigated. Poly (N-isopropylacrylamide-co-hydroxypropyl methacrylate-co-3-trimethoxysilylpropyl methacrylate)(abbr. P(NIPAAm-co-HPM-co-TMSPM) copolymer), was synthesized in absolute alcohol by free radical polymerization initiated by2-azobisisobutyronitrile (AIBN). The thermal responses of the aqueous copolymer solutions were demonstrated by dynamic light scattering (DLS) through detecting the sensitive changes of copolymer aggregation against temperature, with a lower critical solution temperature (LCST) of approximately29℃observed. The Fourier transform infrared spectroscopy showed chemical structure of copolymers and the1H nuclear magnetic resonance results confirmed the monomer molar ratio of final product with NIPAAm:HPM:TMSPM of18:1:1. The molecular weight was determined by gel permeation chromatography, with Mw of192kD, Mn of119kD, and polydispersity index Mw/Mn of1.6, and furthermore degree of polymerization of45. The experimental results showed that P(NIPAAm-co-HPM-co-TMSPM) copolymer was successfully synthesized with remained temperature sensitivity, but the addition of HPM and TMSPM decreased2-3℃in LCST.On the basis of P(NIPAAm-co-HPM-co-TMSPM) copolymer study, the fresh copolymer films were prepared on the substrates containing hydroxyl groups and their performances were also tested. The copolymer solutions with various concentrations in absolute alcohol were spun coated on silicon wafers and glass coverslips under the same rotation speed and coating time, and annealed under vacuum, followed by washing and drying. The stability, reversibility and controllability with respect to temperature switches and copolymer concentrations were examined by contact angle measurements, atomic force microscopy and spectroscopic ellipsometry. The experimental results indicated that the water contact angles of the copolymer films initially increased, reached a maximum at2mg·mL-1, and then decreased with the increasing copolymer concentration at the given temperature and at the same state, and the contact angles of the copolymer films at37℃were greater than those at20℃, and dry films had greater contact angles than the wet ones at each temperature; the dry film thicknesses and surface roughness in air under room temperature increased with the increasing copolymer concentration; the copolymer films in ultrapure water would occur in swelling at20℃, when the temperature rised to37℃, they presented shrinkage state, if the temperature were repeatedly changed between20℃and37℃, the copolymer films exhibited reversible changes. The variations indicated that P(NIPAAm-co-HPM-co-TMSPM) copolymer particles composed of3-trimethoxysily and hydroxyl groups for anchoring onto substrate and forming macromolecular network. The surface wettability, surface structure and thickness of copolymer films could be controllable by adjusting copolymer concentration, and the fresh copolymer films present the fascinating performances of temperature sensibility, controllability, reversibility, stability and so on.Different types of cells including HeLa cells, HEK293cells and mesenchymal stem cells were cultured on the temperature-responsive films with different film properties to select respective optimal films. The attachment at37℃and detachment at20℃were observed for each kind of cells. The experimental results demonstrated that the suface wettability and microstructure of P(NIPAAm-co-HPM-co-TMSPM) copolymer films could affect cell adhesion and subsequent detachment. Therefore, screening suitable culture substrate is of necessity prior to formal use.The purified bone marrow mesenchymal stem cells (BMMSCs) or adipose-derived stem cells (ADSCs) were cultured on the suitable P(NIPAAm-co-HPM-co-TMSPM) copolymer films or glass coverslips in24-well culture plates at the same density. When reaching confluence, the cells were recovered by temperature reduction and trypsinization worked as control groups correspondingly and then replanted on the coverslips with or without copolymer films in other culture plates. After BMMSCs and ADSCs were passaged using the two recovery methods for three times, the cell morphology observation, cell viability profile, immunophenotype analysis, colony-forming assay, differentiation inspection and protein examination were performed. During the short-term culture and passage, the MSCs harvested by temperature drop showed no significantly different from the control groups in cell morphology, immunophenotype and differentiation of osteogenesis and chondrogenesis (for BMMSCs) or adipogenesis (for ADSCs), but had higher viability, stronger proliferation ability and higher adipogenic differentiation (for BMMSCs) or higher osteogenic and chondrogenic differentiation (for ADSCs), and further more proteins were remained around or within the cell membranes. This shows that the temperature-responsive copolymer films and their matched cooling treatment more preferably maintain proliferation ability and cell characteristics than trypsinization.The P(NIPAAm-co-HPM-co-TMSPM) copolymer and copolymer films were prepared, and successfully applied in culture and harvesting of stem cells in this study. It opens up an extremely convenient interfacial mediation for temperature-sensitive film production and stem cell cultivation in vitro. Therefore, if this novel copolymer is used by grafting onto microcarriers and hollow-fiber membranes, large scale in vitro expansion of stem cells may be achieved in combination with bioreactors.