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Q. Sun, A. Rapista, J. Wang, C. Nunes, N. Cheewatrakoolpong, I. K. Sugino, M. A. Zarbin; A Method to Improve Cell Survival on Aged and Amd Bruch's Membrane. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5249.
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© ARVO (1962-2015); The Authors (2016-present)
To determine if conditioned media harvested from cultures of bovine corneal endothelial cells (BCE-CM) improves cell survival on aged and AMD submacular Bruch's membrane. In previous studies, we showed that resurfacing the inner collagenous layer (ICL) of aged Bruch's membrane with extracellular matrix deposited by bovine corneal endothelial cells (BCE) improves long-term cell survival. Since BCE have been shown to secrete extracellular matrix proteins (e.g., collagens and glycosaminoglycans) into the media, we determined if this conditioned media (BCE-CM) could support cell survival on aged and AMD Bruch's membrane.
BCE-CM was collected from confluent cultures of BCE after 3 days exposure to serum-free media (MDBK-MM), centrifuged to remove cells and cellular debris, and stored frozen; 6mm trephine punches consisting of submacular choroid/sclera from aged human eyes (mean donor age 79.7 ± 6.8 yrs.) were debrided to expose the ICL. ~90,000 fetal RPE (fRPE, N=13) or RPE derived from human embryonic stem cells (hES-RPE, N=8; provided by Advanced Cell Technology) were seeded on paired explants from fellow eyes and cultured in BCE-CM or MDBK-MM without prior exposure to BCE. At day-21, explants were fixed and processed for light and scanning electron microscopy. Nuclear density counts were performed on tissue sections and compared by paired t-tests.
Explants cultured in BCE-CM showed significantly higher nuclear density counts than explants cultured in MDBK-MM alone for both fetal RPE and hES-RPE (P=<0.001 for both). Fetal RPE showed a ~11 fold increase in nuclear density, hES-RPE a ~3 fold increase in nuclear density. Improvement in cell survival was not related to submacular pathology; included in both studies were donor eyes with drusen, confluent soft drusen, geographic atrophy, and neovascular membranes. In explants with the highest degree of resurfacing and nuclear densities, cells exhibited or began to exhibit features of mature RPE (apical processes on the entire cell surface, uniformly small, plump (not flat) cells). With few exceptions, cells cultured in MDBK-MM only, when present, were large and flat with smooth surfaces.
BCE-CM, when used as media for cells transplanted onto aged and AMD Bruch's membrane, enhances cell survival and enables some degree of differentiation in both fRPE and hES-RPE.
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