Two Methods for the Isolation and Cultivation of Porcine Primary Corneal Cells.

In ophthalmic research, there is a strong need for in vitro corneal cell models. Here, we describe different protocols for the cultivation of primary corneal cells that were isolated from porcine eyes. This primary cell culture can be used to test new therapeutic options for corneal diseases, such as dry eye disease, traumatic injuries, or corneal infections, and to study limbal epithelial stem cell (LESC) expansion. Two different isolation methods were performed: the outgrowth and the collagenase method. To perform the outgrowth protocol, small explants of the corneal limbus were generated and incubated in culture flasks in an incubator for 4-5 weeks. Regarding the collagenase method, to extract corneal cells, porcine corneas were removed, cut into small pieces, and incubated with collagenase. After incubation and centrifugation, the cells were seeded in 6- or 12-well plates and incubated in an incubator for 2-3 weeks. The differences between corneal cell cultivation with fetal bovine serum (FBS) and without it are also discussed. Therefore, the main advantages of the outgrowth method are that it requires fewer porcine eyes, and it takes less time to be performed compared to the collagenase method. On the other hand, with the collagenase method, mature cells are obtained earlier, at about 2 to 3 weeks.

Porcine Corneas Incubated at Low Humidity Present Characteristic Features Found in Dry Eye Disease.

Dry eye is a multifactorial disease that affects the ocular surface and tear fluid. Current treatment options include lubricant eye drop application several times a day. However, these eye drops often cause local side effects like ocular allergies or blurred vision after the application. To test new treatment options, a robust dry eye model is needed. Here, a porcine ex vivo model was established by means of incubation of porcine corneas in low humidity (LH) and characterized by histological damage evaluation, epithelial thickness and by relevant dry eye markers, such as interleukin 1 beta (IL-1ß), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), occludin and galectin-3. In the dry eye model proposed, an increased secretion of IL-1ß was observed, as well as an upregulation of NF-κB, occludin and galectin-3 mRNA expression. Moreover, the model presented a higher rate of cell death in comparison to the controls. These effects could be reversed with successful treatment of dexamethasone (dexa) and partially reversed with hyaluronic acid (HA) containing eye drops. Furthermore, medium-molecular-weight HA stimulated an increase in IL-1ß in the model proposed. In conclusion, this dry eye model mimics the in vivo condition and hence allows for animal-free testing of novel dry eye treatments.