Age-related macular degeneration (AMD) is a leading cause of blindness. It begins in the outer blood-retinal barrier (oBRB) formed by the retinal pigment epithelium (RPE), Bruch’s membrane, and choriocapillaris. AMD initiation and progression mechanisms still need to be better understood due to the lack of physiologically relevant human oBRB models.

The research team at the National Eye Institute (NEI), part of the National Institutes of Health, used patient stem cells and 3D bioprinting to produce eye tissue that will advance understanding of the mechanisms of blinding disease. Scientists have printed a combination of cells that form the outer blood-retinal barrier.

The retinal pigment epithelium (RPE), separated from the blood vessel-rich choriocapillaris by Bruch’s membrane, forms the outer blood-retinal barrier. The choriocapillaris and RPE exchange nutrients and waste under the control of Bruch’s membrane. Drusen, which are lipoprotein aggregations, develop outside Bruch’s membrane in AMD and impede its function. RPE degradation over time causes photoreceptor deterioration and vision loss.

Scientists combined three immature choroidal cell types in a hydrogel: pericytes, endothelial cells and fibroblasts. They then pressed the gel onto a biodegradable scaffold. Within days, the cells began to mature into a dense capillary network.

On day nine, the scientists seeded retinal pigment epithelial cells on the other side of the scaffold. The printed tissue reached full maturity by day 42. Tissue analyses, genetic and functional testing showed that the printed tissue looked and behaved similarly to the natural outer blood-retinal barrier.

outer blood-retina barrier
a. The outer blood-retinal barrier is the interface of the retina and choroid, including Bruch’s membrane and choriocapillaris. Image credit: National Eye Institute. b. The outer blood-retinal barrier of the eye consists of retinal pigment epithelium, Bruch’s membrane, and the choriocapillaris. Image credit: National Eye Institute. c. Growth of blood vessels over printed rows of an endothelial-pericyte-fibroblast cell mixture. On day 7, blood vessels fill the space between the rows and form a network of capillaries. Image credit: Kapil Bharti.

When imprinted tissue was exposed to stress, it showed early-stage AMD features such as drusen deposits below the RPE and progressed to late-stage dry-stage AMD, where tissue degradation was observed. Low oxygen levels caused a wet AMD-like look with choroidal vascular hyperproliferation moving into the sub-RPE zone. When used to treat AMD, anti-VEGF drugs slowed blood vessel formation and migration while also improving tissue shape.

Kapil Bharti, Ph.D., head of the NEI Section on Ocular and Stem Cell Translation Research said: “By printing cells, we facilitate the exchange of cellular signals necessary for normal anatomy of the outer blood-retinal barrier. For example, the presence of RPE cells induces gene expression changes in fibroblasts that contribute to the formation of Bruch’s membrane – something that was suggested many years ago but only proven in our model.”

Scientists have tackled two technological problems: creating a suitable biodegradable scaffold and achieving a consistent printing pattern. They developed a temperature-sensitive hydrogel that produced clear rows while the gel was cold, but dissolved when the gel warmed up. A more accurate system for assessing tissue architecture was enabled by good row consistency. In addition, they optimized the proportion of fibroblasts, endothelial cells and pericytes in the cell combination.

Co-author Marc Ferrer, Ph.D., director of the 3D Tissue Bioprinting Laboratory at NIH’s National Center for Advancing Translational Sciences, and his team provided expertise for the biofabrication of the outer blood-retina barrier tissues “in-a-well, along with analytical measurements to enable drug screening.

“Our collaborative efforts have resulted in highly relevant retinal tissue models of degenerative eye disease,” Ferrer said. “Such tissue models have many potential applications in translational applications, including therapeutic development.”

Magazine reference:

  1. Min Jae Song, Russ Quinn et al. Bioprinted 3D outer retinal barrier reveals RPE-dependent choroidal phenotype in advanced macular degeneration. Nature Methods, 2022; DOI: 10.1038/s41592-022-01701-1