Using ovine eyes as a readily available source for the isolation and 2D or 3D cultivation of uveal melanocytes

Background: Uveal melanocytes can be difficult to isolate and culture in vitro due to the number of cell types present in the uvea. Methodologies have been described to isolate these cells from humans, macaques and canines. This process has, however, not been described from other more readily available sources of uveal melanocytes, including the modification and establishment of protocols for 2D and 3D growth of isolated uveal melanocytes. Objectives: To establish a method for isolation and culture of ovine uveal melanocytes. Methods: Donor globes were dissected, and the uveal tract isolated. Successive enzymatic disaggregation of cells using were used to extract cells. These isolates were confirmed as being uveal melanocytes by the active production of melanin and co-expression of genes specific to melanocytes, in the pigmentation pathway. Uveal melanocytes were cultured in modified F12 media or smooth muscle growth media (SMGM) and assessed for growth rates and methods for establishment of spheroid cultures were examined. Results: Melanocytes and retinal pigment epithelial (RPE) cells were successfully isolated from uveal explants. Contaminating cell types were not observed and the uveal melanocytes could be cryopreserved, thawed, and cultures successfully re-established. The cells were confirmed as melanocytes. It was found that ovine uveal melanocytes grew better in SMGM than the modified F12 media used for human uveal melanocytes. Methods were established for successfully culturing cells in 3D spheroids. Conclusions: This extraction technique allows for generation of large populations of ovine uveal melanocytes in a relatively short period of time. In many regions, ovine eyes are a highly available source of primary cells from the eye, allowing for better research into the cellular characteristics of uveal melanocytes. This technique is therefore a useful tool for future studies into uveal melanocyte biology as a model for other less available species.