Objective To establish a method for culturing rabbit limbal stem cells (LSCs)

in vitro

and observe their biological characteristics. Methods The pieces of rabbit limbal tissue were cultured in the medium DMEM ∶ F12 (1 ∶ 1) containing 20% fetal bovine serum. The morphological feature of the cultured cells was observed by inverted phase contract microscopy and HE staining. LSCs were identified by immunocytochemical staining with AE5 and P63 antibodies. The ultrastructure of the cultured cells were studied by transmission

electron microscopy. Results The cells were emigrated from pieces of limbal tissue after cultured for 2 to 3 d and then formed a favorable monolayer in 6 to 8 d

with most of the cells were round or oval in shape. Under transmission electron microscope

the cultured cells were oval with few microvilli on the surface and few organelles in the cytoplasm; their nuclei were large and the proportion of nucleus to cytoplasm volume was larger

the nucleolus was obvious and 2 nucleolei were found in some cells. Immunocytochemical technique showed that a few of the cultured cells were positive to AE5 staining and most of them were positive to P63 in cultured for 7 d

and in 14 d

the number of AE5-positive cells were obviously increased and their average gray scale were significant decreased from 184.30 ± 9.85 in 7 d to 133.36 ± 25.00 in 14 d (

P

0.01). However

the P63 - positive cells were obviously diminished in 14 d and their average gray scale were significant increased from 108.13 ± 17.96 in 7 d to 177.59 ± 7.86 in 14 d (

P

0 . 01). Conclusion Our primary cultured rabbit LSCs have consistent biological characteristics with the normal LSCs

in vivo

. The peak growth period of LSCs is on the 7th day.