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Nikolaos Kopsachilis, Theofilos Tourtas, Konstantinos Tsaousis, Friedrich E. Kruse, Ulrich Welge-Luessen; A Novel Bruch’s Membrane Substrate from Human Donor Lens Anterior Capsule. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4020.
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© ARVO (1962-2015); The Authors (2016-present)
To study the potential use of human donor anterior lens capsule (AC) as a Bruch’s membrane substrate for human retinal pigment epithelium cell transplantation.
AC’s were recovered from the lenses of 30 cornea donors 24 to 72 hours post mortem. Human retinal pigment epithelium cells (RPE) were recovered from the remaining retinal fragments of 15 eye donors. The capsules were treated with Trypsin/EDTA solution and stored in distilled water for 12 hours to eliminate the crystalline epithelium. After initial processing, primary cultured RPE’s were added on the AC’s and allowed to attach in culture at 37°C. Samples were sorted into 3 groups. Group 1 consisted of 10 samples in which the RPE cells were allowed to grow on corresponding capsules. Group 2 consisted of RPE cells that were allowed to grow on a collagen membrane. Group 3 consisted of 10 samples in which human RPE cells were allowed to grow on polystyrene culture plates (Control). All specimens were incubated for 2 weeks at 37°C and 5% CO2. Cell density, morphology, and adherence of the cell-capsule complex were evaluated at 1, 4, 7 and 14 days with a phase-contrast microscope, a scanning electron microscope and by histology. Cell viability was quantified by a microscopic live-dead assay. Expression of zonula occludens-1 (ZO-1), Na+/K+-adenosine triphosphatase (Na+/K+-ATPase), tissue transglutaminase (tTgase) and vimentin were investigated by immunohistochemistry.
A mean diameter of 10,05 ± 0,13 mm of anterior capsule was obtained as a substrate for cell culture. The rate of cell growth in all three groups was comparable. Cell viability was 96% or superior in all groups and multiple cellular interconnections developed between growing cells. Immunohistochemical analysis demonstrated strongly positive staining for ZO-1, tTgase, vimentin and Na+/K+-ATPase. Electron microscopy confirmed the adherence and monolayer growth of the RPE cells on the non epithelial side of the lens capsule.
Phenotypical properties of the cell-capsule complex imply that the RPE-capsule complex is capable of maintaining intact barrier and ionic pump functions in vitro. Human donor anterior lens capsule might therefore be a potential scaffold for the ex vivo expansion of human RPE cells and could be used as a potential Bruch’s membrane substrate in retinal surgery.
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