A Novel Selective Strategy for Bioactive Limbal Stem Cells Primary Culture Using Deep Cryopreservation and IL-1β Precondition.

Limbal stem cell (LSC) transplantation is an important treatment for limbal stem cell deficiency (LSCD), but low efficacy in maintaining LSC stemness during in vitro expansion greatly affects its wider application. The primary contributing factors include a low proportion of stem cells and the lack of a stable, supportive microenvironment over prolonged culture. Rabbit corneal tissues preserved under deep cryogenic conditions for more than six months retain viable limbal stem cells (LSCs), and primary LSCs isolated from these tissues exhibit robust stem cell characteristics. It is noteworthy that the NLRP3/Caspase-1/IL-1β signaling axis was activated in corneal epithelial cells, and outer limbal layers preserved for one or two years. Based on these findings, a combined strategy integrating deep cryopreservation with IL-1β induction was established for the processing of limbal tissues. The combined cryogenic and IL-1β preconditioning yielded primary LSCs with maintained p63+ cell proportions, a reduction in K3+ differentiated cells from approximately 80% to 60%, and a 6.25-fold increase in colony-forming efficiency. In addition, an increased proportion of cells in the G2/M phase and enhanced proliferative capacity were observed. The enriched LSC population also exhibited improved stratified epithelial reconstruction potential. These findings identify an effective strategy for preserving and enriching LSCs from limbal tissue, providing a practical and efficient approach for LSC preparation prior to transplantation. Further in vivo studies will be important to validate the functional performance of these cells in ocular surface reconstruction.