Isolation and Expansion of Primary Conjunctival Stem Cells (CjSCs) from Human and Rabbit Tissues

Conjunctival disorders are multivariate degenerative ocular surface diseases that can jeopardize ocular function and impair visual capacity in severe cases. The recent development of stem cell technologies has shed a new light on the treatment of conjunctival disorders as the regenerative medicine using endogenous stem cells becomes a potential therapeutic strategy. However, the efficient in vitro expansion of the endogenous stem cells dominating the conjunctival regeneration, the conjunctival stem cells (CjSCs), remains challenging. Existing protocols largely adopted primary culture using feeder layers, which has limited efficiency and risk of contamination. Here, we report a protocol for the isolation and expansion of primary CjSCs derived from human or animal tissues. This protocol adopts collagenase-based enzymatic digestion to release the primary cells from conjunctival tissues and utilizes a feeder-free culture strategy based on a small molecule inhibitor cocktail that stimulates the expansion of CjSCs. The CjSCs generated with this method were rapidly dividing and highly homogeneous. They also expressed characteristic stem cell markers and exhibited differentiation potency. These findings marked an important step forward in building stable CjSCs in vitro expansion, which will help researchers better understand the biology of ocular surface stem cells and develop innovative regenerative medicine approaches for ocular surface diseases. This protocol was validated in: Biomaterials (2021), DOI: 10.1016/j.biomaterials.2020.120462 Graphical abstract.