Abstract: Space station is important to space biology and it is difficult to provide CO<sub<2</sub< environment,a crucial element to cell culture.Meanwhile,a controllable and automatic method of cell culture is needed to perform the research in space station.Consequently,the establishment of cell culture based on microfluidic chip in a CO<sub<2</sub<-free environment is extremely important.Firstly,changed the component of medium (Initial pH of the medium was changed,then adding up exogenous CO<sub<2</sub< into the medium,finally adding up buffer in culture medium),and performed the cell culture in CO<sub<2</sub<-free environment (the co-culture of U87 MG and SHSY-5Y and THP-1),then used MTS method to determine the cell viability.Secondly,after adding different proportions of HEPES, performed the cell culture in CO<sub<2</sub<-free environment,the cell viability was determinated by MTS method.Finally,according to the optimal proportion of HEPES,the cells were cultured in microfluidic chip.It was concluded that in CO<sub<2</sub<-free environment,cells in microfluidic chip would survive in the medium with 3% HEPES for 5 days.