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L. A. Bachman, M. P. Fautsch, S. V. Patel; Viability and Magnetic Attraction of Cultured Human Corneal Endothelial Cells Labeled With Superparamagnetic Microspheres. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3951.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the viability of cultured human corneal endothelial cells (HCECs) in vitro after uptake of superparamagnetic microspheres (SPMs), and to demonstrate the attraction of HCECs labeled with SPMs to a magnetic source.
Human corneas unsuitable for transplantation were received in Optisol-GS preservation medium. Donors were selected for age (<50 years), endothelial cell density (>2500 cells/mm2), death to preservation time (<18 hours), acute cause of death, and absence of chronic systemic disease. Descemet’s membrane with intact endothelium was stripped from the corneas and incubated overnight in 8% fetal bovine serum (FBS) at 37°C. Endothelial cells were released from Descemet’s membrane by incubating in 0.02% EDTA for 1 hour with gentle agitation and plated into a single well of a 6-well plate coated with collagen type IV. Cells were cultured in medium containing 8% FBS supplemented with EGF, FGF and NGF. Primary cultures and subsequent passages were grown to confluence to confirm the typical morphology of endothelial cells. Confluent passage 2 cells of 6 eyes (5 donors) were incubated overnight with 0.9 µm-diameter SPMs at concentrations of 0 (control), 250, 500, 1000, 2000 and 4000 SPMs per cell plated. After incubation with SPMs, cells were rinsed and left in culture for 2-6 days. Cellular uptake of SPMs was determined by electron microscopy. The percentage of non-viable cells was measured by trypan blue staining. From the total number of cells retrieved after incubation with SPMs, the percentage of cells that were attracted to a 5000-Gauss magnet in a separation column was determined.
With careful donor selection criteria, HCECs were consistently cultured in vitro and primary cultures reached confluence in 10-22 days. Confluent HCECs formed a monolayer with a near-hexagonal morphology similar to that of corneal endothelium in vivo. SPMs were detected within the cytoplasm of cells by electron microscopy after overnight incubation. The percentage of non-viable cells (maximum, 6 ± 4 %; n=6) was not affected by the concentration of SPMs and did not differ from control (5 ± 2 %, n=6, P>0.71). Cell migration to a magnetic source was higher for HCECs containing SPMs (minimum, 50 ± 10 %; n=6) compared to control HCECs (4 ± 4 %, n=6, P<0.004). Peak magnetic attraction was for cells labeled at 1000 SPMs per cell plated (64 ± 30 %).
Cultured HCECs labeled with SPMs remain viable and can be localized to a magnetic source. This technique might be useful for localizing HCECs to the posterior cornea in vivo for endothelial cell transplantation.
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