·AIM:To investigate the effect of serum free organ culture on corneal endothelial cells viability by comparing two specific endothelial cell media—endothelial cell medium(ECM)and animal compound free medium(ACF)with the conventionally used basic minimum essential mediun(MEM).·METHODS:Rat corneas were cultured in ECM and ACF test media with or without 20mL/L fetal bovine serum(FBS)for 4 weeks and then dehydrated in dextran T500 for 48 hours.The viability evaluation parameters of corneal endothelial cells included:endothelial cell density before and after preservation;The immunohistochemistry and semi-quantitative expression of zonula occludens-1(ZO-1)using RT-PCR in rat corneas whose epithelium had been predissected.·RESULTS:After cultivation,the grafts cultured in MEM+20mL/L FBS control group exhibited the lowest endothelial cells density(1875±162 cells/mm2).The highest mean corneal endothelial cell density was found in ECM+20mL/L FBS medium group(2250±202 cells/mm2)and the corneas cultured in ECM and ACF media achieved similar endothelial cells loss(F=28.965,P=0.000).Vital staining revealed the lower numbers of damaged corneal endothelial cells in ECM(with or without FBS)and ACF groups.ZO-1 could be seen in corneal endothelia of frozen sections.ZO-1mRNA expression in rat corneas without epitheliums exhibited the lowest level in MEM medium and the highest level in ECM(with or without FBS)and ACF media(F=592.751,P=0.000).·CONCLUSION:Serum-free ECM and ACF media can preserve rat corneal endothelial cells viability and tight junction integrity better than conventional MEM medium with low FBS.They show great superiority and may play an important part in serum free organ culture corneas in the future.·