3D Bioprinting Eye Tissue to Advance Degenerative Eye Disease Research

January 31, 2023 - Data from the CDC notes that age-related macular degeneration (AMD) affected approximately 19.8 million people over 40 in the United States in 2019, with nearly 1% of patients having AMD that threatens their vision. Considering the prevalence of AMD, researchers are constantly looking to learn more about the disease, how to prevent it, and new treatments. In a recent study published in Nature, Kapil Bharti, PhD, and his colleagues, 3D bioprinted eye tissue to advance research on degenerative eye disease.

Bharti is a stem cell development biologist at the National Eye Institute (NEI), a subset of the NIH. “Stem cell developmental biologists use induced pluripotent stem cells (IPS) to study human diseases in a dish,” explained Bharti. “The advantage of these IPS cells is that they can be made from any individual.”

“The other advantage of IPS cells is that they can be differentiated into any cell type, for example,  eye tissue,” he added. “We can recreate a patient's eye tissue, which allows us to recreate their disease in a dish.”

AMD

Bharti told LifeSciencesIntelligence that his lab is particularly interested in exploring diseases that affect the back of the eye, the retina, and the choroid. His lab is focused on AMD, which impacts the retinal pigment epithelium (RPE) and the blood vessels in the choroid.

The NIH states that AMD is associated with disruptions to a patient’s central vision due to damage in the macula. Despite being one of the leading causes of blindness, disease progression is poorly understood, with varying degeneration rates.

“The most recent data suggests that the disease starts with degenerative changes in the RPE tissue. As RPE cells start to die, the choriocapillaris underneath that brings all the nutrients and blood supply to the retina, which is above the RPE, start to die off because the RPE, which is in between the retina and the choriocapillaris, is maintaining both tissues,” explained Bharti.

Bharti described that RPE cells control the nutrients going to the retina and the metabolites returning to the choriocapillaris and the bloodstream. He described the two versions of AMD, noting that dry AMD happens when RPE cells die off, causing the blood vessels to die off. Data from Genentech suggests that dry AMD accounts for 85–90% of all AMD cases.

The other form of AMD, wet AMD, occurs when the blood vessels hyper-proliferate, go through the RPE, and penetrate the retina. At this point, they can leak blood and fluid, causing blindness. While this form is not as prevalent, it is more likely to lead to blindness, accounting for 90% of all AMD-related blindness cases.

“In this case, the thinking is that there's hypoxia in the RPE, which causes the blood vessels to proliferate in the wrong direction,” expanded Bharti. “For both diseases or stages of AMD, the dry form or the wet form, there's no good model system to study these hypotheses that people have come up with. That's what our model tried to do — recreate and study both processes in a dish.”

Bioprinting

The motivation for Bharti’s study was to understand how the disease starts and progresses better. To do that, they created human RPE in a dish. “We started using bioprinting as an approach because for blood vessels to form the three cell types, endothelial cells, pericytes, and fibroblasts, that make the blood vessels and the matrix around them, they had to be together for a long time — a few days — and bioprinting allowed us to achieve that and create a specific pattern of blood vessel network,” noted Bharti.

He explained that the bioprinter could be programmed to move in any way in the X, Y, and Z orientation. The needle that prints the material is filled with bio-ink. “Bio-ink is essentially three cell types, endothelial cells, fibroblasts, and pericytes, mixed in a very high concentration with a hydrogel that allows it to be printed,” said Bharti.

“The three cells stay together and start making small vessels or capillaries. From where they're printed, the capillaries are formed in that area and then spread. They undergo angiogenesis as capillaries spread and then become confluent throughout the tissue. That's a whole bioprinting approach,” he added.

Complications

In theory, this concept is excellent; however, Bharti described the challenges associated with the bio-printing process. He explained that optimizing the hydrogel was a significant challenge he and his team had to overcome before the project could progress. The team needed to determine what hydrogel would work best for the cells, the proper ratio of hydrogel, and the correct ratio of the three cell types.

After solving these problems, they also had to ensure that the cell types were available on the day of bioprinting. After optimizing the methods to make the cell types, the team expanded and cryopreserved them to be available on the day of bioprinting.

“There are still some factors to consider after that. Even the humidity and the temperature of the room affect the bioprinting process. If it's too hot or too dry, the consistency of the bio-ink, a toothpaste-like material, changes, and that may completely lead to failure of the bioprinting process,” cautioned Bharti.

Applications

One of the team’s goals was to determine whether the printed tissue could simulate the physiological process of AMD. To do so, they recreated the conditions associated with dry and wet AMD, finding that the tissue reacted the way they had hypothesized.

Bharti and his collaborators took it one step further, saying, “if this is a true wet AMD phenotype, it should be treatable with how we treat wet AMD patients nowadays.” They treated the wet AMD phenotype with anti-vascular endothelial growth factor (anti-VEGF) antibodies.

“VEGF is known to induce capillary hyperproliferation in tumors and wet MD patients. They are usually given antibodies that sequester VEGF; hence the capillaries don't proliferate anymore,” explained Bharti. “We added those antibodies in our tissue and could suppress capillary reformation in our tissue, clinically validating that our tissue is a wet MD model and can be used to discover and test new drugs in this space.”

After developing these tissues, Bharti and other researchers at the National Eye Institute hope to use this technology to create more effective drugs for wet AMD and new medications for dry AMD.

Considering the FDA’s latest announcement, noting that it will allow certain researchers to forgo animal trials, this technology will be critical for future research. Rather than testing AMD medications on animal models before beginning human trials, researchers can test the medications — even multiple medications — in a dish on actual human tissue.

“The advantage is that we are creating human tissue in a dish to study human biology, human diseases, and test drugs,” continued Bharti. “We are miniaturizing it to do large-scale drug screens, tying directly into the FDA’s concept that we can use these tissues for drug discovery pipelines.”