Thermo-responsive Surfaces Based On Poly(2-oxazoline)s For Non-enzymatic Cell Harvesting

At present,the main harvesting method of anchorage-dependent animal cells in vitro is trypsin digestion.This method is simple and efficient,but it will damage the cell membrane and extracellular matrix during the digestion process,resulting in the decrease of cell viability and adhesion efficiency.Therefore,various non-enzymatic cell harvesting techniques have been developed to minimize the cell damage caused by harvesting.According to different design principles,the non-enzymatic cell harvesting technology is mainly divided into four types,including photo-responsive system,magnetism-responsive system,electro-responsive system and thermo-responsive system.Among those,thermo-responsive system based on LCST(Lower Critical Solution Temperature)polymers exhibits the merits of good practicality and convenience,and it may be the promising candidate in the field of tissue engineering,cell sheet engineering and non-enzymatic cell harvesting.There are four main types of LCST polymers that have been applied to construct the thermo-responsive system,including poly(Nisopropylacrylamide),poly(oligo(ethylene glycol)methacrylate)s,poly(glycidyl ether),and poly(2-oxazoline)s.Poly(2-oxazoline)s is also known as pseudopeptide.Compared with other LCST polymers,it has the advantages of excellent biocompatibility,stable physicochemical properties,adjustable structure and functions,and it's an ideal material for biomedical use.The current research on the application of poly(2-oxazoline)s is mainly focused on the field of antifouling,drug and gene delivery,etc.Only a few reports of poly(2-oxazoline)s on non-enzymatic cell harvesting has been published.However,in these related reports,due to the complexity and instability of the grafting method,poly(2-oxazoline)s barely meets the requirements of practical use.Therefore,in order to solve the above-mentioned various problems,in this work,poly(2-propyl-2-oxazoline)s and poly(glycidyl methacrylate)were chosen as the molecular bases,and poly(2-propyl-2-oxazoline)s were immobilized onto the glass wafers with a facile,reliable dipcoating and annealing procedure to construct the thermo-responsive system for non-enzymatic cell harvesting.The main research contents are as follows:(1)Preparation of polymers involved in the construction of thermo-responsive surfaces,including carboxyl functionalized poly(2-propyl-2-oxazoline)s and poly(glycidyl methacrylate).The monomers of 2-propyl-2-oxazoline and 2-(2-methoxycarbonylethyl)-2-oxazoline were prepared from nitrile and acid chloride,respectively,and carboxyl functionalized poly(2-propyl-2-oxazoline)s were obtained by cationic ring-opening polymerization.Using glycidyl methacrylate as the monomer,poly(glycidyl methacrylate)was prepared by free radical polymerization.(2)Construction of the thermo-responsive surfaces.Prior to coating,glass wafers were immersed in piranha solution to remove organics and activate the surface.First,the activated glass wafers were immersed into the chloroform solution of 0.2 wt.% poly(glycidyl methacrylate)for three times,taken out and annealed for 20 minutes.Poly(glycidyl methacrylate)was covalently immobilized onto glass wafers as thin film.Then,the preliminary modified glass wafers were immersed into the chloroform solution of 1 wt.% carboxyl functionalized poly(2-propyl-2-oxazoline)s for three times,taken out and annealed for 16 hours.Poly(2-propyl-2-oxazoline)s were covalently immobilized onto the layer of poly(glycidyl methacrylate).(3)Evaluation of the biocompatibility of thermo-responsive surfaces.L929 cells(Mouse Fibroblast)were seeded on the thermo-responsive surfaces of poly(2-propyl-2-oxazoline)s,and the cell viability was determined at 24,48,and 72 hours by CCK-8(Cell Counting Kit-8).The survival status of cells was evaluated by FDA / PI(Fluorescein Diacetate / Propidium Iodide)staining.(4)Thermal-induced cell detachment and the subculture of harvested cells.L929 and Vero cells(African Green Monkey Kidney Cells)were cultivated on the thermo-responsive surfaces of poly(2-propyl-2-oxazoline)s,and when the 80% confluence of cells was reached,the coated glass wafers were transferred to the environment of 8 ? to complete the detachment of cells in2 hours.The harvested cells were seeded into 96-well plates,and the morphology and survival status of harvested cells were observed after 24 hours of culture.