Abstract
Purpose :
Vitreous macrophages called hyalocytes have been implied in the pathogenesis of vitreoretinal interface disease. Due to their scarcity, hyalocyte investigation was hampered in the past, partly owing to the lack of an appropriate in vitro model. In this study, we aimed to establish conditions for successful cultivation of porcine hyalocytes, which would enable a thorough elucidation of their properties in health and disease.
Methods :
Since hyalocytes belong to the myeloid cell lineage, we first adapted a protocol described for cultivation of brain microglia by Bohlen et al (Curr Protoc Immunol 2019; referred to as BM here) and compared it to a novel protocol developed specifically for this project and comprising DMEM supplemented with 10% FCS, with or without m-CSF. For preparation of porcine eyes, an initial scleral incision along the equator was created, followed by transfer of the vitreous while maintaining correct spatial orientation with the posterior vitreous cortex abutting the surface of a culture dish. Freshly extracted vitreous samples were placed in BM or DMEM (±m-CSF) and observed by phase-contrast microscopy over the span of 14 days (d). Factors, such as cell number, expansion of cell protrusions, adherence to the dish bottom and overall cell survival were evaluated. Protein expression of cultured hyalocytes was examined on d1, 2, 4 and 8 by immunohistochemistry for IBA1, an immune cell marker, and α-SMA, a myofibroblast marker, of both cells adherent to the surface and jelly-like vitreous above.
Results :
Hyalocytes in DMEM showed an overall better survival than hyalocytes in BM over the observation span. Viable cells were observed consistently until at least d14 in DMEM, as opposed to BM (165±20 at d2 vs. 108±27 at d14 in DMEM vs. 170±26 at d2 vs. 64±20 at d14 in BM). Spatial orientation of the vitreous cortex proved crucial for cell adherence and future survival rates. The addition of m-CSF did not influence cell vitality. For the duration of the experiment, hyalocytes in both conditions stained positive for IBA1. Early fibroblast contamination was excluded by staining for α-SMA.
Conclusions :
We report here the design of an in vitro protocol for cultivation of porcine hyalocytes. This knowledge can be transferred to in vitro studies of human hyalocytes extracted from vitrectomized or enucleated eyes. Our model further enables the profound examination of questions arising from preclinical studies of hyalocytes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.