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

Abstract

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.

Keywords: cornea; corneal cells; epithelial cells; porcine corneas; primary culture.

Figure 1

Step-by-step corneal cell outgrowth method. (A) Muscles and tissues around the eye were removed. (B,C) To extract the cornea, an incision was made in the sclera with a scalpel, and the cornea was finally cut out with scissors. (D) The cornea was cut in half. (E,F) Each cornea half was cut in pieces, and the center of the cornea was discarded, so that a strip of approximately 2 mm of corneal tissue and 2 mm of sclera was produced. (G) The small cornea explants were placed in a culture flask with a ventilator screw cap. After waiting for 5 min until the cornea explants adhered to the surface of the flask, 1 mL of culture medium was added (CnT-Prime + 2% PS), one drop per explant. Then, 10% FBS was added to the medium, depending on the purpose of the culture.

Figure 2

Step-by-step corneal collagenase method. Porcine eyes were received from a local abattoir, muscles and tissues around the eye were removed, and the corneas were extracted. The corneas were cut into small pieces and incubated with medium and collagenase in Eppendorf tubes on a shaker for 4 h. After centrifuging the tubes, the supernatant was transferred to another Eppendorf, and the corneal pieces were discarded. The tube was centrifuged again, and the supernatant was disposed. Then, 1 mL of culture medium was added to the cells remaining at the bottom of the tube, the cells were resuspended, seeded in well plates, and incubated at 37 °C and 5% CO₂. WP: well plate. Created in BioRender.com.

Figure 3

Corneal cell cultivation with the outgrowth and the collagenase methods. (A) Cell outgrowth was visualized from corneal explants two days after corneal isolation. After 3–4 weeks of cultivation, most cells were morphologically like epithelial cells and some like keratocytes. After 4 to 5 weeks, cultures achieved 70–80% confluence and could be split. (B) Collagenase method: After two days of isolation, the cells attached to the surface of the well plate. After two weeks, homogenous cells, which resemble corneal epithelial cells and some stellate cells, similar to keratocytes, were present. Cell cultures were 70–80% confluent after 2–3 weeks of cultivation. Scale bar = 200 μm.

Figure 4

Average number of cells at each week of cultivation with the outgrowth method. The graph confirmed that the number of cells in the cultures incubated with FBS was higher than in the ones without serum. The increase in the number of cells was substantially greater in FBS-containing cultures. The mean values and the SEM are shown.

Figure 5

Corneal cell isolation with the outgrowth and the collagenase methods and the use of 10% FBS. (A) Cells were observed to grow from corneal explants after two days of isolation. More cells were growing in the serum culture from day 4 until 2 weeks, and some cells resembled fibroblasts morphologically. After 4 to 5 weeks, the culture achieved 70–80% confluence and could be split. (B) After two days of isolation, the cells attached to the surface of the well plate. A higher number of cells in the culture cultivated with serum was noticed, and some cells resembled fibroblasts. The cell culture was 70–80% confluent after 2–3 weeks of cultivation. Scale bar = 100 μm and 200 μm.