Gandhi, Jarel K.

Death or dysfunction of retinal pigment epithelium (RPE) cells occurs in age-related macular degeneration (AMD) and certain inherited retinal dystrophies (IRDs). Induced-pluripotent stem cell (iPSC) derived-RPE have been used in early-stage clinical trials to treat AMD and IRDs by injecting them as a cell suspension or monolayers. While RPE transplant shows therapeutic potential, issues ranging from failure to repopulate the entire treatment area, clumping and monolayer folding, and a foreign body response to the support have been reported. We’ve shown that RPE can be grown on high concentration (>30 mg/mL) degradable fibrin hydrogels, and that cell free fibrin hydrogels implanted in the subretinal space degrade without causing inflammation. Here we describe manufacture and surgical implantation of degradable fibrin hydrogels carrying iPSC-RPE into a porcine model of geographic atrophy (GA). Large (15.25 × 58.42 × 0.2 mm) fibrin gel blanks were produced by injection molding, and iPSC-RPE were grown on their surface. Using a mechanical punch, the blank was subdivided into 1.5 × 5.0 × 0.2 mm doses, which fit a custom tool used for storage and surgical placement. Following aseptic packaging, RPE and gels were stable at 37 °C for at least 7 weeks. When transplanted into a pig model of GA, the fibrin scaffold degraded in <1 month and the iPSC-RPE provided partial rescue from GA as assessed by preservation of photoreceptors and blood flow in the choriocapillaris. We conclude that iPSC-RPE delivered on degradable fibrin hydrogels represent a potentially safe and effective approach to RPE transplantation.

AbstractFibrin is a degradable biopolymer with an excellent clinical safety profile. Use of higher mechanical strength fibrin hydrogels is limited by the rapid rate of fibrin polymerization. We recently demonstrated the use of higher mechanical strength (fibrinogen concentrations >30 mg/ml) fibrin scaffolds for surgical implantation of cells. The rapid polymerization of fibrin at fibrinogen concentrations impaired our ability to scale production of these fibrin scaffolds. We serendipitously discovered that the azo dye Trypan blue (TB) slowed fibrin gelation kinetics allowing for more uniform mixing of fibrinogen and thrombin at high concentrations. A screen of closely related compounds identified similar activity for Evans blue (EB), an isomer of TB. Both TB and EB exhibited a concentration dependent increase in clot time, though EB had a larger effect. While gelation time was increased by TB or EB, overall polymerization time was unaffected. Scanning electron microscopy showed similar surface topography, but transmission electron microscopy showed a higher cross‐linking density for gels formed with TB or EB versus controls. Based on these data we conclude that addition of TB or EB during thrombin mediated fibrin polymerization slows the initial gelation time permitting generation of larger more uniform fibrin hydrogels with high‐mechanical strength.

Abstract Human fibrin hydrogels are a popular choice for use as a biomaterial within tissue engineered constructs because they are biocompatible, nonxenogenic, autologous use compatible, and biodegradable. We have recently demonstrated the ability to culture induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium on fibrin hydrogels. However, iPSCs themselves have relatively few substrate options (e.g., laminin) for expansion in adherent cell culture for use in cell therapy. To address this, we investigated the potential of culturing iPSCs on fibrin hydrogels for three-dimensional applications and further examined the use of fibrinogen, the soluble precursor protein, as a coating substrate for traditional adherent cell culture. iPSCs successfully adhered to and proliferated on fibrin hydrogels. The two-dimensional culture with fibrinogen allows for immediate adaption of culture models to a nonxenogeneic model. Similarly, multiple commercially available iPSC lines adhered to and proliferated on fibrinogen coated surfaces. iPSCs cultured on fibrinogen expressed similar levels of the pluripotent stem cell markers SSea4 (98.7% ± 1.8%), Oct3/4 (97.3% ± 3.8%), TRA1-60 (92.2% ± 5.3%), and NANOG (96.0% ± 3.9%) compared with iPSCs on Geltrex. Using a trilineage differentiation assay, we found no difference in the ability of iPSCs grown on fibrinogen or Geltrex to differentiate to endoderm, mesoderm, or ectoderm. Finally, we demonstrated the ability to differentiate iPSCs to endothelial cells using only fibrinogen coated plates. On the basis of these data, we conclude that human fibrinogen provides a readily available and inexpensive alternative to laminin-based products for the growth, expansion, and differentiation of iPSCs for use in research and clinical cell therapy applications. Stem Cells Translational Medicine 2019;8:512–521