Study Of Establishing Three-Dimensional Cell Culture Models For MRC-5 Cells

Human embryonic lung fibroblasts(MRC-5)are derived from lung tissue of 14-week-old human embryos.MRC-5 cells are cultured in vitro for more than 40 generations,and the characteristics of diploid fibroblasts are maintained.MRC-5 cells are susceptible to many viruses,such as human cytomegalovirus,herpes simplex virus,enterovirus 71 and so on.Generally,MRC-5 cells are cultured using traditional two-dimensional techniques,in which cells are grown in flat layers on plastic culture bottles or petri-dishes.However,biological cells of the in vivo state grow in a three-dimensional environment,which show complex and dynamic features.Due to limitations on two-dimensional cell cultures,cellular physiological activities couldn't fully be showed.Some deficiencies of two-dimensional cell cultures are supplied by three-dimensional cell culture models.The three-dimensional models could provide a more relevant physiological environment for researching cell biology and physiology,cytopathology and so on.In this study,rat tail tendon collagen type I and polystyrene scaffold(PS)are selected respectively to establish two different three-dimensional cell culture models for MRC-5 cells.The effects of conventional culture medium,PDGF(platelet-derived growth factor)medium and BEGM(bronchial epithelial cell growth medium)medium for the MRC-5 cell growth were investigated in two three-dimensional cell culture models.The growth statuses of MRC-5 cells were analyzed in the two models.The cytopathic effects of MRC-5 cells infected with HCMV(human cytomegalovirus)were observed in the two three-dimensional culture models.Our results showed that conventional culture medium,PDGF medium and BEGM medium promoted MRC-5 cells to overlap each other and to form a multilayer three-dimensional network structure,respectively.In the 3D collagen-MRC-5 cell culture model,PDGF medium promoted cells to produce extensions of dendrites at early stage.Conventional culture medium and BEGM medium helped cells to keep multilayer network structure for longer time.Furthermore,MRC-5 cells in conventional culture medium were contracted into clusters,whereas cell migration in PDGF medium and BEGM medium were observed as individual cells.In the 3D PS-MRC-5 cell culture model,MRC-5 cells cultured in BEGM medium could form more obvious 3D reticular or "nest-like" structure compared with cells cultured in conventional culture medium and PDGF medium,whereas cell migrations in PDGF medium and BEGM medium were observed as individual cells.In the 3D PS-MRC-5 cell culture model,MRC-5 cells cultured in BEGM medium could form more obvious 3D reticular or "nest-like" structure compared with cells cultured in conventional culture medium and PDGF medium.The multilayer network structure was maintained for longer time.Compared with MRC-5 cells grown in 3D collagen scaffold,the rate of cell expansion and extension in the 3D PS scaffold is relatively fast,but the time for maintaining 3D structure of MRC-5 cells is shorter.HCMV could infect MRC-5 cells cultured in two 3D culture models,which showed that there was no change in susceptibility to HCMV for MRC-5 cells in 3D culture models.Compared with MRC-5 cells in 3D collagen matrix,the cytopathic effect of MRC-5 cells induced in the PS scaffold was earlier,longer and stronger.According to our study,two different types of 3D MRC-5 cell culture models were successfully established by using rat tail tendon collagen type I and polystyrene scaffold.There was no change in the susceptibility to HCMV for MRC-5 cells in the two different types of 3D culture models.It is significant to optimize the 3D MRC-5 cell culture models in the future.It also provided experimental data using the 3D MRC-5 cell culture models for the further study of viral infection,replication,proliferation and antiviral drugs.