Abstract
Purpose :
Retinal endothelial cells (ECs) and pericytes are involved in the pathogenesis of retinal vasculopathies, including diabetic retinopathy. Previously, a protocol has been presented that allows the isolation of retinal pericytes from the bovine retina. However, protocols are needed to isolate retinal vascular cells from species with a physiological response potential of retinal vessels closer to that of humans, such as the porcine retina. Additionally, it is important to generate monocultures of ECs and pericytes simultaneously. Therefore, we have developed a simple and adaptable method for isolation of vascular cells from porcine eyes that can selectively isolate ECs and pericytes from the same retinal solution.
Methods :
Porcine eyes obtained from a local slaughterhouse were disinfected and dissected, and the retinas were harvested for the primary cell isolation. The tissue was digested enzymatically with collagenase type II, followed by steps of filtration and centrifugation. Subsequently, cultures of ECs or pericytes were obtained using selective culture conditions adapted to the cell type of interest. For ECs, this included ECGM2-medium supplemented with puromycin, while for pericytes, DMEM with 5-20% FCS was used. To minimize the risk of contaminating cells, medium was changed 2 h after seeding. Purity of the monocultures were validated with immunofluorescent staining using antibodies targeting cell-specific markers.
Results :
Pericytes and ECs from porcine eyes could be isolated by modifying an already described protocol for isolation of bovine retinal pericytes. The modifications included optimization of the digestion solution, lysis of red blood cells, and enhancing the culture conditions. We observed an estimated doubling in the number of cells when the tissue was digested in medium compared to PBS, and furthermore cell viability was increased. When selecting for ECs, we found that ECGM2 increased the number of EC-colonies, and that addition of puromycin to the medium was favourable to eliminate pericyte-contamination. In summary, we have seen that the adjustments not only increase the viability of the cells, but also purity and number of cells.
Conclusions :
We have managed to isolate ECs and pericytes separately from the same retinal digestion. This may provide an important tool for studying the interaction between these cell types in the vascular wall that occurs in vivo.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.