Zhu M, Provis J M, Penfold P L
Department of Clinical Ophthalmology, University of Sydney, New South Wales, Australia.
Aust N Z J Ophthalmol. 1998 May;26 Suppl 1:S50-2. doi: 10.1111/j.1442-9071.1998.tb01371.x.
Current methods for the isolation and culture of adult retinal pigment epithelium (RPE) provide successful primary culture. However, most methods result in contamination with other cell types and low cell yields. The isolation and culture of human foetal RPE presents further problems associated with the limited size of the eye cup and adherence among cells. Reliable methods are necessary for the culture of human RPE and subsequent functional studies.
The present procedure is based on mechanical peeling of the whole RPE layer under the dissecting microscope. Dissected pieces are subsequently explanted to a 35 mm culture dish and are cultured with Dulbecco's modified Eagle's medium containing 10% foetal bovine serum. Peroxidase immunohistochemical methods were used to investigate cell phenotypes.
Primary cultures were obtained within 10-14 days with high yields, good viability and purity in subsequent culture. Cultured cells were vimentin and cytokeratin positive and CD31 negative.
This mechanical dissection technique is recommended for the isolation of foetal and young adult RPE cells, while the enzyme digestion method is preferred for aged adult tissue.
目前用于分离和培养成人视网膜色素上皮（RPE）的方法能够成功进行原代培养。然而，大多数方法会导致其他细胞类型的污染以及细胞产量较低。人胎儿RPE的分离和培养存在与眼杯尺寸有限以及细胞间黏附相关的更多问题。可靠的方法对于人RPE的培养及后续功能研究是必要的。
本操作基于在解剖显微镜下机械剥离整个RPE层。随后将解剖后的组织块接种到35毫米培养皿中，并用含有10%胎牛血清的杜氏改良 Eagle 培养基进行培养。采用过氧化物酶免疫组织化学方法研究细胞表型。
在10 - 14天内获得了原代培养物，后续培养中产量高、活力好且纯度高。培养的细胞波形蛋白和细胞角蛋白呈阳性，CD31呈阴性。
推荐使用这种机械解剖技术分离胎儿和年轻成人的RPE细胞，而酶消化法更适用于老年成人组织。
