A cocktail of small molecules maintains the stemness and differentiation potential of conjunctival epithelial cells.

Purpose: The conjunctival epithelial cells cultured with bovine serum or feeder cells were not suitable for clinical application. Therefore, we developed a novel serum-free and feeder cell-free culture system containing only a cocktail of three chemicals (3C) to expand the conjunctival epithelial cells.

Methods: The cell proliferative ability was evaluated by counting, crystal violet staining and Ki67 immunostaining. Co-staining of K7 and MUC5AC was performed to identify goblet cells. PAS staining was used to assess the ability of cells to synthesis and secrete glycoproteins. In vivo, eye drops containing 3C was administered to verify the role of 3C in the mouse conjunctival injury model. PAS, HE and immunofluorescence staining were performed to show conjunctival epithelial repair.

Results: Compared with other small molecule groups and the serum group, the cells in 3C group showed superior morphology and proliferative ability. Meanwhile, 3C maintained the well-proliferative capacity of cells even after fifth passage. The 3C group also exhibited more K7 and MUC5AC double positive cells, and the PAS staining positive areas were present in both the cytoplasm and extracellular matrix. The cell sheets treated with 3C in air-lifted culture were obviously stratified. In vivo, more goblet cells in the conjunctival epithelium were observed in the 3C group.

Conclusion: Overall, our culture system can expand the conjunctival epithelial cells and retain their potential to differentiate into mature goblet cells, which provided a promising source of seed cells for conjunctival reconstruction. Furthermore, this system provides new insights for the clinical treatment of ocular surface diseases.