A promising strategy to rapidly expand high-quality human limbal stem cells for tissue engineering and cornea reconstruction.

The present study investigated a promising strategy for scaling up the expansion of high-quality human limbal stem cells (LSCs) in vitro. The LSCs isolated from human cornea biopsies were cultured with Mitomycin-C (MMC)-treated Swiss-3T3 feeder layers using three different methods: LSCs with MMC-treated Swiss-3T3 (L with MS), LSCs on MMC-treated Swiss-3T3 (L on MS), and LSCs on reverse MMC-treated Swiss-3T3 (L on RMS). Cell morphology, purity, and differentiation during culture were examined through light microscopy, flow cytometry, immunofluorescence staining, and reverse transcription-polymerase chain reaction (RT-PCR). Results showed that the LSCs continued to grow and exhibited the highest expression of p63 + cells (99.5 ± 0.5 %) in the L on RMS method. After 7 days of culture, the L on RMS method exhibited a significantly higher cell expansion rate than the L with MS method (**p < 0.01). Moreover, RT-PCR demonstrated that the cultured LSCs could naturally differentiate into epithelial cells, indicated by K3 expression. The cells could also efficiently form colonies while maintaining their stem cell markers. Therefore, the L on RMS method is promising for rapidly expanding high-quality LSCs and preserving their growth and stemness for corneal tissue engineering and reconstructive surgery.