Fibrin hydrogels as a xenofree and rapidly degradable support for transplantation of retinal pigment epithelium monolayers.

Recent phase 1 trials of embryonic stem cell and induced pluripotent stem cell (iPSCs) derived RPE transplants for the treatment of macular degeneration have demonstrated the relative safety of this process. However, there is concern over clumping, thickening, folding, and wrinkling of the transplanted RPE. To deliver a flat RPE monolayer, current phase 1 trials are testing synthetic substrates for RPE transplantation. These substrates, however, cause localized inflammation and fibrosis in animal models due to long degradation times. Here we describe the use of thin fibrin hydrogels as a support material for the transplantation of RPE. Fibrin was formed into a mechanically rigid support that allow for easy manipulation with standard surgical instruments. Using fibrinolytic enzymes, fibrin hydrogels were degraded on the scale of hours. The rate of degradation could be controlled by varying the fibrinolytic enzyme concentration used. RPE cells degraded fibrin spontaneously. To preserve the fibrin support during differentiation of iPSCs to RPE, media was supplemented with the protease inhibitor aprotinin. iPSC-RPE on fibrin gels remained viable, generated monolayers with characteristic cobblestone appearance and dark pigmentation, and expressed mRNA and protein markers characteristic of RPE in the eye. Following differentiation of the cells, addition of fibrinolytic enzymes fully and rapidly degraded the fibrin support leaving behind an intact, viable iPSC-RPE monolayer. In conclusion, human fibrin hydrogels provide a xeno-free support on which iPSCs can be differentiated to RPE cells for transplant which can be rapidly degraded under controlled conditions using fibrinolytic enzymes without adverse effects to the cells. STATEMENT OF SIGNIFICANCE: Stem cell-derived retinal pigment epithelial (RPE) cell transplantation is currently in phase 1 clinical trials for macular degeneration (MD). A major obstacle in these studies is delivering the RPE as a living, flat sheets without leaving behind foreign materials in the retina. Here we investigate the suitability of using hydrogels made from human blood-derived proteins for RPE transplant. Our data shows that these fibrin hydrogels are rigid enough for use in surgery, support growth of stem cell-derived RPE, and are easily degraded within hours without damage to the RPE sheet. These fibrin hydrogels offer a promising solution to transplant RPE for patients with MD.

Differential Intraocular Pressure Measurements by Tonometry and Direct Cannulation After Treatment with Soluble Adenylyl Cyclase Inhibitors.

PURPOSE: To validate the increase in intraocular pressure (IOP) caused by soluble adenylyl cyclase (sAC) inhibitors and determine reasons behind variation in IOP measurements performed by tonometry. METHODS: C57BL/6J mice were administered DMSO solubilized sAC inhibitors (KH7 or LRE-1) by intraperitoneal injection. Two hours post-treatment, mice were anesthetized with avertin or ketamine/xylazine/acepromazine (KXA). IOP was measured by a rebound tonometer or direct cannulation of the anterior chamber. Spectral-domain optical coherence tomography was used to measure anterior chamber depth and corneal thickness in live mice. Outflow facility was measured in perfused, enucleated mouse eyes. RESULTS: Compared with DMSO controls, KH7 treatment caused an increased IOP in avertin- and KXA-anesthetized mice when measured by direct cannulation [avertin: 14.4 ± 2.1 mmHg vs. 11.1 ± 1.0 mmHg (P = 0.003); KXA: 14.4 ± 1.0 mmHg vs. 11.3 ± 0.8 mmHg (P < 0.001)] and tonometry [avertin: 10.8 ± 1.4 mmHg vs. 7.4 ± 0.6 mmHg (P < 0.001); KXA: 11.9 ± 0.9 mmHg vs. 10.3 ± 1.7 mmHg (P = 0.283)]. However, treatment with KH7 in nonanesthetized mice showed a significant decrease in IOP measured by tonometry and compared with DMSO-treated animals [13.1 ± 2.6 mmHg vs. 15.6 ± 0.5 mmHg (P = 0.003)]. Both KH7- and DMSO-treated groups anesthetized with avertin showed increased corneal thickness, whereas KH7-treated mice anesthetized with KXA exhibited a shallower anterior chamber compared with untreated mice. KH7 decreased outflow facility by 85.1% in nonanesthetized, enucleated eyes (P < 0.003). CONCLUSIONS: Systemically administered DMSO and anesthesia have significant effects on anterior chamber characteristics, resulting in altered IOP readings measured by tonometry. In the presence of DMSO and anesthesia, tonometry IOP readings should be confirmed with direct cannulation.