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B.V. Stanzel, S. Binder, M.S. Blumenkranz, M.F. Marmor; Culture of Human RPE From Aged Donors on a Potential Bruch's Membrane Prosthesis . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1407.
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Autologous RPE transplantation could be beneficial to patients with AMD, but may not be effective if Bruch's membrane has been damaged by age and/or surgery. We postulate that RPE autotransplantation might be facilitated by culturing, and thus rejuvenating, aged cells on a prosthetic Bruch's membrane. We have investigated growth of RPE from elderly donors on a candidate substrate.
Eight human eyes from donors ≥55 years old, preserved within 6 hours, were processed within 24 to 48 hours post mortem. RPE was isolated 1) as a full–thickness explant of RPE and choroid, 2) by mechanical scraping with a silicone spatula, or 3) by using enzymatic digestion (collagenase CLS I or Dispase II) prior to scraping. Cells collected from roughly 0.05cm2 were seeded onto 1.12 cm2. Primary cultures were initiated on semipermeable polyester filter inserts (Transwell®, 0.4 µm pore size, Corning) with or without laminin coating. Basal growth media were modified from the Hu medium (2001) containing 1g/L glucose concentration. With 6 eyes we compared the 3 harvesting methods above, the relevance of laminin coating, and low vs. normal calcium growth conditions. With 2 eyes, cells were grown in high (4.5 g/L) glucose medium on uncoated inserts. Culture media were refreshed 3 times weekly. Morphology and transepithelial resistance (TER) were monitored weekly. Immunostaining was performed for pancytokeratin (K8.13), vWF and ZO–1.
All explant cultures showed choroidal contamination as evidenced by positive vWF immunostaining. However, good epithelioid morphology was observed in cultures harvested mechanically or enzymatically, and this was maintained until about 1 week post–confluence. After that, fibroblast–like transdifferentiation led to cellular contracture and detachment from the substrate. There was no difference between laminin–coated and uncoated inserts, or between low and high calcium media. Data from one set of cultures grown in high glucose medium showed that epithelioid morphology was maintained until 2– 3 weeks post–confluence, with a concomitant increase in TER.
Aged human RPE grown on a thin permeable membrane, that may serve as a prosthetic Bruch's membrane, can achieve epithelioid morphology at confluence. However differentiation does not persist beyond 1– 3 weeks. Preliminary data from one subject (2 eyes) suggest that a high glucose medium may help to sustain the epithelial phenotype.
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