Preliminary Exploring Of Optimized Primary Culture Method For Rabbit Limbal Stem Cells

Objective:Obtained the limbal epithelium from New-Zealand rabbits, digested them into signal cell suspension with DispaseⅡand trypsin-EDTA in 4℃ and 37℃ alternating hot and cold conditions respectively, then culured them in vitro.Compared with the conventional method that get the signal cell suspension only used DispaseⅡin 37℃.To investigate the optimized method of limbal stem cells(LSCs) in paimary culture by the study of the biological characteristics of them and then lay a better foundation for the clinical research of ocular tissue engineering.Methods:Obtained the limbal epithelium from rabbits under sterile conditions, divided into optimized group and conventional group. The optimized group digested it into signal cell suspension with DispaseⅡand trypsin-EDTA in 4℃ and 37℃ respectively and the conventional group digested it only with DispaseⅡin 37℃,then both of them were seeded in KSFM culture medium contain10% serum medium in it,then compared with the following three methods: 1.We analyzed the growth status of cultivated LSCs under the inverted microscope; 2. After the cells were passaged in 24-well plates, immunocytoch--emis-try was used to identify the LSCs on the next day.3. Crystal violet staining was used to detected their proliferative capacity on the sixth day after the cells were passaged in6-well plates.Results:The LSCs of the optimized group exhibited similar growth characteristics when compared with the conventional group.The optimized group had more ΔNp63 cells(P<0.05)the nuclei were stained green and less K3cells(P<0.05) the cytoplasm was stained red of the immunocytochemistry.The LSCs of the optimized group had a higher proliferative capacity than the conventional group(P<0.05).Conclusion:The LSCs isolated with Dispase Ⅱ and trypsin-EDTA(cold and hot alternating digestion) could be successfully cultured in vitro.It reduced the time required for conventional enzymatic digestion,thereby reduced the potential damage of LSCs which caused by the enzyme of a long time affection,thus provide a new approach for the culture of LSCs.