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Jarel Gandhi, Zahid Manzar, Hannah Schmidt, Benjamin Gilles, Travis Knudsen, Matthew Hill, Lori Bachman, Raymond Iezzi, Jose Pulido, Alan D Marmorstein; In vitro testing of fibrin as a temporary support for RPE transplantation. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2317. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) transplantation holds promise as a therapy for macular degeneration. While RPE transplanted without support appear safe, they form clumps in vivo. Using synthetic polymers to preserve the RPE monolayer phenotype necessitates an extended half-life for the support, which in turn can cause inflammation and fibrosis in vivo. Our goal in this study is to produce a natural support that is retained during RPE culture while being rapidly (hours to days) degraded following transplantation.
Hydrogels of various thickness and fibrinogen concentration were produced and mechanical rigidity tested qualitatively. The degradation kinetics of the hydrogel were studied by varying concentrations of fibrinogen, plasminogen and tissue plasminogen activator (tPA). The degraded fibrin was quantified by spectrophotometry over time, and a first order reaction was assumed to find the rate constant.Fibrin was polymerized and seeded with RPE to form a monolayer. RPE viability was studied before and after fibrin degradation using a live/dead assay. Immunofluorescence was used to confirm monolayer phenotype by staining for RPE markers.
Hydrogels, thinner than 500µm, required a high fibrinogen concentration to support their own weight. Qualitatively, these gels rebounded back to their original, flat shape after manipulation and bending. Implantation of the fibrin sheets into the subretinal space of an ex vivo eyecup was successful.Degradation of the hydrogel was only observed after the addition of plasminogen and tPA, with all tested conditions degrading within 48 hours. The degradation rate constant was independent of fibrinogen concentration (p=0.35) but dependent on both plasminogen (p=0.005) and tPA concentrations (p<0.001).On the hydrogel, the RPE reformed as a flat monolayer, with confirmation of ZO-1, a cell-cell junction marker, throughout the monolayer. RPE viability remained the same before and after complete degradation of the hydrogel (p=0.88). With the degradation of fibrin, the RPE monolayer folded onto itself. ZO-1 was similarly present after fibrin degradation.
Fibrin hydrogels were formed to mechanically support a flat RPE monolayer and were rapidly degraded, without harm to the RPE. Fibrin provides a great alternative for RPE transplantation, as this strategy maintains a flat, viable RPE monolayer without a persisting material between the choroid and RPE.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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