Heller Janosch P, Kwok Jessica C F, Vecino Elena, Martin Keith R, Fawcett James W
John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge Cambridge, UK ; Department of Clinical and Experimental Epilepsy, Institute of Neurology, University College London London, UK.
John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge Cambridge, UK.
Front Cell Neurosci. 2015 Nov 20;9:449. doi: 10.3389/fncel.2015.00449. eCollection 2015.
Diseases such as age-related macular degeneration (AMD) affect the retinal pigment epithelium (RPE) and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 min yielded 4 × 10(4) viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch's membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch's membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases.
年龄相关性黄斑变性（AMD）等疾病会影响视网膜色素上皮（RPE），导致上皮细胞死亡并最终失明。RPE移植目前是眼部研究的主要焦点，使用人类干细胞衍生的RPE细胞的临床试验正在进行。然而，移植用RPE细胞的来源在多大程度上影响其治疗效果仍有待确定，这需要在动物模型中进行探索。RPE细胞自体移植作为一种治疗方法具有吸引力，但现有的从啮齿动物中分离成年RPE细胞的方案技术难度大、耗时、产量低且未针对长期细胞培养进行优化。在此，我们报告了一种新设计的方案，该方案有助于从成年大鼠中可靠且简单地分离和培养RPE细胞。将整个大鼠眼球在20 U/ml木瓜蛋白酶溶液中孵育50分钟可产生4×10⁴个存活的RPE细胞。这些细胞在培养时呈六边形且有色素沉着。通过免疫染色，我们证明这些细胞表达RPE细胞特异性标记蛋白，包括细胞角蛋白18和RPE65，类似于体内的RPE细胞。此外，这些细胞能够产生和分泌类似于体内情况的布鲁赫膜基质成分。同样，培养的RPE细胞如先前报道的那样粘附于分离的布鲁赫膜。因此，本文所述的方案提供了一种高效方法，可快速简便地分离大量成年大鼠RPE细胞。这为研究治疗靶点、在临床前设置中测试药物效果以及进行体外和体内移植实验以研究视网膜疾病提供了一个可靠的平台。
