Abstract
Purpose :
The retinal pigment epithelium (RPE) is a cell monolayer present in the human retina. Its most relevant role is providing support for the neural retina. Impairment of the RPE, and thus of the photoreceptors, is often seen in vision loss-related diseases such as age-related macular degeneration, retinitis pigmentosa and Stargardt disease. Current treatments are focused on stopping disease progression instead of repairing the damaged tissues. Recent studies have proposed the use of biofabrication technologies for creating transplantable models of human RPE. Here we propose the use of porcine amniotic membrane-derived extracellular matrix (ECM)-based hydrogels for fabricating in vitro RPE models.
Methods :
Since hydrogels made of solely tissue-derived ECMs have failed to provide appropriate mechanical properties for tissue engineering, a methacryloyl modification was performed and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was used as photoinitiator to yield photocrosslinkable hydrogels. Successful methacryloyl modification was confirmed by nuclear magnetic resonance (NMR) spectroscopy. The biochemical composition of the modified hydrogels was characterized by means of total protein content, sulfated glycosaminoglycans content and growth factor composition. Printability of the hydrogel was assessed by manual extrusion experiments and via rheology with flow sweep and post-printing recovery tests. For biofabrication experiments, a novel concave and porous microplate insert was 3D printed with PLA and used as a support for the model, which consisted of a thin ECM hydrogel layer with RPE primary cells laying on top. After 7 and 14 days in culture, the integrity and differentiation level of the RPE models was assessed via immunofluorescence, histology and transepithelial electric resistance (TEER).
Results :
Results showed a higher expression of the junction-specific protein ZO-1 and a higher TEER after 14 days of culture, when compared to models cultured for 7 days. Moreover, hematoxylin and eosin staining showed a highly organized epithelial layer with a clear separation from the ECM region.
Conclusions :
The preliminary results herein shown demonstrate the potential of these 3D constructs in replicating native tissue structures which could be employed for treating disease in a personalized manner.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.