Corneal lenticules as an ex‐vivo model to study keratocyte biology

PurposeKeratocytes show differential gene expression in culture media; and are extensively used to study wound healing, corneal disease biology and response to topical drugs. However, mono‐layer culture cannot replicate the 3‐dimensional biological environment of corneal stroma. Hence, we propose to establish corneal lenticule as an ex‐vivo model to study keratocyte biology for corneal diseases, drug response studies and in wound healing experiments.MethodsSMILE surgery was performed using the VisuMax femtosecond laser system (Carl Zeiss Meditec AG, Jena, Germany). After the refractive lenticule of intra‐stromal corneal tissue was created using the femtosecond cutting procedure, it was dissected and separated through the side‐cut opening and removed manually. SMILE lenticules from patients were obtained in MK media and were transferred to DMEM F12 with 10% FBS and 1% PSA. The media was replenished every 24 h and lenticules were harvested at 0 h, 24 h, 48 h, 78 h and 96 h. Gene Expression analysis was performed for pro‐fibrotic genes (fibronectin, ɑ‐sma, vimentin, TGF‐β and TGF‐βR2), pro‐inflammatory (IL‐6 and TNF‐ɑ) and structural genes (Col1‐A1, Col4‐A1and Col5‐A1).ResultsOur results demonstrate that lenticules remain metabolically active in culture media for long periods of time as evident from the varying expression of different pro‐fibrotic, pro‐inflammatory and structural genes after 0 h, 24 h, 48 h, 78 h and 96 h of culture. Furthermore, linear regression analyses show that clinical parameters like lenticule thickness do not affect the expression profile of the various genes by the keratocytes contained in the lenticule.ConclusionsIn conclusion, lenticule can be used as an ex‐vivo model to study keratocyte biology in various corneal diseases and for drug testing.