Fabricationof High-sugar-induced 3DINS-1 Cell Damage Model Based On Microfluidic Chip

Objectives:TypeⅡdiabetes is a metabolic disease characterized by hyperglycemia,in which islet cell damage leads to inadequate insulin secretion is an important pathogenesis and significant features of type 2 diabetes mellitus.At present,the animal model and the orifice plate model are used as the research object in the research and development of the hypoglycemic drugs at home and abroad.The animal model has the problems of long experimental period and complicated experiment process.The cells in the plate model are in a two-dimensional state and are static culture,which is different from the internal environment of the human body and can not achieve the screening of high-throughput drugs.It is easy to produce the local concentration of drugs when the drug is too high.Therefore,in this study,a microfluidic chip with a concentration gradient generating device was used as a technical platform to construct a high glucose-induced three-dimensional rat pancreatic islet tumor cell(INS-1)injury model and to observe the effect of glipizide on the model for screening.Materials and Methods:The microfluidic chip model consists of two layers of PDMS substrate and one layer of polycarbonate film.The upper PDMS chip consists of a concentration gradient generating device,the lower PDMS chip consists of INS-1 cell culture chambers,each liquid outflow channel and INS-1 cell culture chamber in the concentration gradient generating apparatus were separated by the 10μm polycarbonate film.In this study,basement membrane-like substance(BME)was used as an alternative to extracellular matrix(ECM).INS-1 cell were coated in BMEand seeded in the culture cells of the lower INS-1cell for three-dimensional culture to simulate islet cells in vivo growth pattern.INS-1 cell culture medium containing high concentration of glucose was injected from the chip inlet at a flow rate of 1.0 μL / min under the driving of a syringe pump to construct high glucose injury to three-dimensional INS-1 cell on the chip model.After the model was successfully constructed,INS-1 cellculture medium containing glipizide from the chip inlet at a flow rate of 1.0 μL / min under the driving of the syringe for screening.Results:The microfluidic chip consists of two functional units,including a concentration gradient generating device and a three-dimensional growth space of INS-1 cell.Under the driving of the syringe pump,the concentration gradient generating device designed in this experiment can generate five different concentrations,each of which has four different channel to characterize.It is necessary to set three sets of parallel experimental requirements for one experiment,so this model shorten the experimental cycle.INS-1 cell were encapsulated in a cell culture chamber on a microfluidic chip with BME to meet the requirements of the flow and three-dimensional growth space,making the whole model a multidimensional biomimetic model and can be used to investigate the effect of the glee series of drugs on 3D cultured INS-1 cell.Microfluidic chip model studies have shown that INS-1 cell in the 3D matrix can form a multi-cell sphere structure.The effect of glibenclamide on the isolation and promotion of insulin secretion in INS-1 cells induced by microfluidic hyperglycemia in the three-dimensional INS-1cell concentration within the test concentration increases with the drug concentration,the better the protection and promotion of insulin secretion.The effect of glipizide on INS-1cell under 2D and 3D culture conditions showed that high glucose-induced three-dimensionalINS-1cellwere constructed on microfluidic chip platforminjury model is more effective than the conventional 2D model and is more prominent at low concentration.Conclusions:In this study,we constructed a multi-dimensional and multi-concentration microfluidated high glucose-induced three-dimensional INS-1cell injury model,which modeled the model of islet cell injury and examined the efficacy of insulin secretagogue.This model provides a basis for studying the pathogenesis of diabetes,drug development and drug screening of diabetes.