Development Of Thermoresponsive Microspheres For Cell Attachment And Detachment

Traditional cell harvesting method uses trypsin for digestion.However,trypsin digestion inevitably degrades cell membrane proteins and deposited extracellular matrix(ECM),resulting in reduced cell viability and even loss of function,which affects the cell quality.Poly(N-isopropylacrylamide)(PNIPAM)is a temperature-sensitive intelligent material that can be used for cell culture and temperature control attachment.However,PNIPAM temperature-sensitive materials have the problems of slow cell detachment rate and cell shedding.To this end,in this paper,temperature-sensitive microspheres were prepared by grafting PNIPAM with microspheres of different substrates,and the attachment,expansion and detachment of cells on the microspheres were achieved by controlling the temperature.In this paper,Cytodex 3 and SiO2 microspheres were first used as substrates,and the PNIPAM-Cytodex 3 microspheres and PNIPAM-SiO2 microspheres were successfully prepared by the "Grafting to "method and the "Grafting from" method,respectively.The effect of PNIPAM grafting on Hela cells attachment,growth,and detachment was evaluated.It was found that these two microspheres can support Hela cells attachment and growth to a certain extent,but failed to achieve temperature-controlled detachment of cells.For this purpose,using polystyrene microspheres as the substrate,polystyrene microspheres with different PNIPAM grafting amounts were prepared by the "Grafting from"method.Using Hela cells to evaluate the effect of polystyrene microspheres with different PNIPAM grafting amounts on cell attachment,growth and detachment,it was found that grafting PNIPAM on polystyrene microspheres can promote the attachment and expansion of Hela cells;Polystyrene microspheres with PNIPAM grafting amount of 67.45 ?g/cm2 and 154.16 ?g/cm2 can achieve temperature-controlled desorption of cells,and the temperature-sensitive detachment rate of cells will vary with the amount of PNIPAM grafting on polystyrene microspheres increase and increase.The CCK8 method was used to examine the effect of the temperature-sensitive desorption process on the cell reproliferation ability.The results showed that the cells detachment from the temperature-sensitive microspheres had good reproliferation ability.Further,the selected temperature-sensitive polystyrene microspheres were used for the in vitro culture of MSCs cells.According to the MSCs cell attachment,expansion,detachment,and harvested MSCs cell reproliferation and differentiation capabilities,the application of prepared microspheres in MSCs culture found that temperature-sensitive polystyrene microspheres can support cell attachment,promote cell expansion,and achieve temperature-controlled detachment of cells.MSCs cells have a good ability to reproliferate and differentiate.