Serum-free Culture And Biological Characteristics Preliminary Research Of Rabbit Limbal Stem Cells In Vitro

Aim:To culture New-Zealand Rabbit limbal stem cells without fetal bovine serum (replaced by2%B27),observe the biological characteristics and compared with the traditional culture method with10%fetal bovine serum(FBS),and then discusses the culture method without FBS,accumulating information for reconstruction of tissue cornea and the basic research study of cornea.Method:(1)Obtain rabbit limbal tissue under sterile condition and use enzymatic disgestion method(Dispase Ⅱ) for single cell suspension.And then,cell suspensions were diluted into the same density respectively by DMEM/F12(1:1) culture medium containing2%B27and10%FBS.(2)Cells were directly inoculated in culture bottles for cell growth and96-well plates for clonal proliferation.(3)Do immunofluorescence staining of⊿Np63and K3(AE5) in primary culture to the three and five day,then compare the two sets of results.Results:(1)Observing under the inverted microscope,cells cultured in medium containing2%B27exhibit the same growth characteristics with that in medium containing10%FBS.(2)Cell proliferation ability was detected by CCK-8,and then we draw cell growth curve.The cell growth curve showed that the two groups of cells have a similar proliferative capacity:cells entered their logarithm proliferative phase at the fourth to sixth day,then they showed down forming the platform phase.(3) Immunofluorescence staining of⊿Np63and K3(AE5) showed that:majority cells cultured in medium containing2%B27express⊿Np63and little express K3(AE5).At the third day,⊿Np63expression rate was similar,and then slightly higher in group containing2%B27at the fifth day.Conclusion:(1)New-Zealand Rabbit limbal stem cells can be separated successfully in vitro using enzymatic disgestion method;(2)we can culture limbal stem cells successfully by using2%B27replace FBS. it can reduce the influence of unknown components in FBS on limbal stem cells, and provides new options for limbal stem cells in vitro culture, amplification and purification.