Ishida M, Lui G M, Yamani A, Sugino I K, Zarbin M A
Department of Ophthalmology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark 07103-2499, USA.
Curr Eye Res. 1998 Apr;17(4):392-402. doi: 10.1080/02713689808951220.
We studied various methods for harvesting retinal pigment epithelium (RPE) biopsies from cadaver human eyes of donors over age 60 years. Our goal was to harvest cells for possible autologous RPE cell transplantation in patients with age-related macular degeneration and to test the viability of the RPE after isolation by evaluating explant growth in culture.
Choroid-RPE biopsies were excised from enucleated human eyes. The RPE was separated from the choroid by treatment with type IV collagenase. RPE patches were cultured. After 100-500 cells had grown out from the explant, the primary cultures were passaged.
There was no clear effect of donor age on the ability to establish primary RPE cultures with good morphology from biopsies 2 x 2-10 x 10 mm2 in size. Biopsies 6 x 6 mm2 or larger produced satisfactory primary cultures more than 70% of the time. The number of viable RPE cells (defined as the number of cells adherent to the culture dish 24 h after plating) obtained after enzymatic separation of the RPE and choroid was an important determinant of our ability to establish primary cultures and passage the cells. Primary cultures with good cellular morphology were obtained 100% of the time when RPE explants > 4 mm2 in size were obtained from the biopsy specimen. Seventy-three percent of the biopsies yielding explants > 4 mm2 in size were successfully passaged.
These results suggest that peripheral scleral flap biopsies in aging donors can be used to establish RPE explant primary cultures. These cultures may be suitable as a source for autologous RPE transplantation in patients.
我们研究了从60岁以上供体的尸体人眼中获取视网膜色素上皮（RPE）活检组织的各种方法。我们的目标是获取细胞，用于年龄相关性黄斑变性患者可能的自体RPE细胞移植，并通过评估培养物中外植体的生长情况来测试分离后RPE的活力。
从摘除的人眼中切除脉络膜-RPE活检组织。通过用IV型胶原酶处理将RPE与脉络膜分离。培养RPE贴片。当100-500个细胞从外植体生长出来后，对原代培养物进行传代。
供体年龄对从2×2-10×10mm²大小的活检组织建立形态良好的原代RPE培养物的能力没有明显影响。6×6mm²或更大的活检组织在超过70%的时间内产生了令人满意的原代培养物。RPE和脉络膜酶分离后获得的存活RPE细胞数量（定义为接种后24小时附着在培养皿上的细胞数量）是我们建立原代培养物并传代细胞能力的重要决定因素。当从活检标本中获得大小>4mm²的RPE外植体时，100%的时间都能获得细胞形态良好的原代培养物。产生大小>4mm²外植体的活检组织中有73%成功传代。
这些结果表明，老年供体的周边巩膜瓣活检可用于建立RPE外植体原代培养物。这些培养物可能适合作为患者自体RPE移植的来源。
