Abstract：Objective To investigate whether murine corneal stroma cells (CSCs) expand in a KSFM medium and still hold their original biological characteristics. Methods Experimental study After central cornea were incubated in an EDTA solution (20 mmol/L) for 45 minutes， the corneal epithelium and endothelium were carefully peeled away from the corneal stroma with fine forceps. Central corneal stromas were then digested with collagenase I (300 U/ml) for 4 hours. Following centrifugation， isolated single cells were harvested， and seeded on plastic in a DMEM basic medium (serum-free)， a DMEM complete medium (containing 10% FBS)， or in a KSFM medium. The cells were cultured at 37℃ in a 5% CO2 atmosphere， and subcultured with EDTA solution (containing 1 U/ml dispase). Meanwhile， the cells were observed and a cell growth curve was drawn； the gene expression of keratocan and aldehyde dehydrogenase (ALDH) was examined by reverse transcription polymerase chain reaction (RT-PCR)； the protein expression of keratocan was analyzed by immunofluorescence and Western Blot. An independent samples t test was performed to evaluate the significance of the results. Results After collagenase digestion， cell suspension obtained from two murine corneal stromas yielded about 1×104 single cells. The data of RT-PCR indicated that the primary cells exhibited positive expression of keratocan and ALDH， which were considered hallmarks for keratocytes； the data of immunofluorescence and Western Blot further showed that these cells expressed keratocan protein. Thus， the primary cells in this study were of stromal origin. In the DMEM basic medium， primary CSCs could not proliferate； in the DMEM complete medium， CSCs proliferated， but passage 3 cells lost the gene expressions of ALDH and keratocan and the protein expression of keratocan； in the KSFM medium， CSCs also proliferated， and passage 3 cells still maintained the gene expressions of ALDH and keratocan and the protein expression of keratocan， with no significant difference when compared to primary CSCs. Conclusion KSFM medium can maintain the biological characteristics of murine CSCs while promoting cell proliferation.
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