University of Münster Medical School, Department of Ophthalmology, Münster, Germany.
University of Münster Medical School, Department of Ophthalmology, Münster, Germany.
Exp Eye Res. 2021 Jun;207:108584. doi: 10.1016/j.exer.2021.108584. Epub 2021 Apr 25.
Microglia are the resident immune cells in the retina. To investigate their properties and behaviour, a reliable and yielding procedure to culture them is necessary. We here describe a way of isolation of microglial cells from the porcine retina, as pig eyes are similar to human eyes in size, structure and vasculature, including similarities in proteins and pathways. Retina was isolated from fresh pig eyes, dissociated by a mixture of collagenase, hyaluronidase and DNAse, and passed through a cell strainer. After triple centrifugation with decreasing velocity and re-suspension, cells were seeded into poly-d-lysine coated culture flasks and cultured using DMEM and macrophage-colony stimulating factor (M-CSF). Number of cells increased gradually during the first 10-14 days, till they could be split and used for experiments. Identity of isolated cells as microglia was assessed by immunostaining against the microglia/macrophage markers Iba1, CD11b, CD68, CD45 and TMEM119. Phagocytic function of microglia could be demonstrated by phagocytosis of fluorescence beads and their response to lipopolysaccharide (LPS). As a conclusion, we developed a protocol for isolation and cultivation of pig retinal microglial cells that are suitable for research in the laboratory.
小胶质细胞是视网膜中的固有免疫细胞。为了研究它们的特性和行为，有必要建立一种可靠且高效的培养方法。我们在此描述了一种从猪眼视网膜中分离小胶质细胞的方法，因为猪眼在大小、结构和脉管系统方面与人眼相似，包括蛋白质和途径的相似性。将视网膜从新鲜的猪眼分离出来，用胶原酶、透明质酸酶和 DNA 酶的混合物进行解离，并通过细胞筛过滤。经过三次离心（速度逐渐降低）并重新悬浮后，将细胞接种到多聚-d-赖氨酸包被的培养瓶中，并使用 DMEM 和巨噬细胞集落刺激因子（M-CSF）进行培养。在最初的 10-14 天内，细胞数量逐渐增加，直到可以进行分裂并用于实验。通过对小胶质细胞/巨噬细胞标志物 Iba1、CD11b、CD68、CD45 和 TMEM119 的免疫染色，评估分离细胞的身份。可以通过吞噬荧光珠和对脂多糖（LPS）的反应来证明小胶质细胞的吞噬功能。总之，我们开发了一种适合实验室研究的猪视网膜小胶质细胞分离和培养的方案。
