“We have been always very interested in the conjunctiva,” says co-investigator Laura Garcia-Posadas, PhD, a post-doctoral fellow at Massachusetts Eye and Ear, Harvard Medical School. “It is affected in almost every disease of the ocular surface. However, it is much less studied than the cornea and there are not good in vivo models.”
It’s a problem the eye-care community would like to see rectified.
“I agree,” says DED researcher and thought leader Alice T. Epitropoulos, MD, FACS, “that there is a significant need to develop new technologies that reduce reliance on animals and use a 3D model that is structurally similar to conjunctiva that can simulate the in vivo physiology of conjunctival tissue.”
The researchers from Spain and Harvard may well be on the way to determining that structure. In March they published on PLoS One their work detailing an engineered 3-D human conjunctival-like tissue. It is the second time in three years that Dr. Garcia-Posadas et al have published work on a model meant to study inflammation, but this time the team has mastered an important step: the culturing of conjunctival goblet cells. 1
The true magic of this new model is the opportunity to study the immunologic symphony with all of its complexities in response to desiccating stress,” says Laura M. Periman MD, a dry eye thought leader based in Seattle. “What a leap forward from cell culture alone—which lacks ability to study the cellular interactions with the extracellular matrix and scaffolding. This new model takes 2D cell culture to a 4D level.”
“Keep it simple” — but not always
The problem with available models for conjunctiva is that they are oversimplified, according to Dr. Garcia-Posadas. Most of the in vitro research is done with a cell line called the IOBA-NHC cell line. “Usually the models consist only of cell monolayers formed by transformed cell lines that are no longer similar to the actual progenitor tissue,” she says. “In our group, we have done a lot of research using [IOBA-NHC], but although it is very useful, we were aware that the results should be taken with caution.”
In 2013, the team, including then doctoral student Garcia-Posadas, developed a human, primary epithelial cell culture model of the conjunctiva to study conjunctival inflammation. 2 But that new protocol could only be used to grow epithelial cells from conjunctiva on cadaveric tissue.
The road to a better model
Soon after that, Dr. Garcia-Posadas had the opportunity to conduct research at the Darlene Dartt lab at Massachusetts’ Schepens Eye Research Institute — which led to overcoming a major hurdle in conjunctival models, for here she learned to culture conjunctival goblet cells, a subpopulation in conjunctival epithelium. Goblet cells in turn express mucin, an immunomodulator that plays an active role in inflammation. The goblet cells were a crucial missing ingredient from conjunctival models derived from cell lines.
Then a final condition for the team’s most recent improved conjunctival model fell into place: “My supervisor [Dr. Yolanda Diebold, Institute of Applied Ophthalmobiology, University of Valladolid, Spain] had a collaboration with other groups that were studying different scaffolds,” says Dr. Garcia-Posadas. “So we started to combine the cells with the scaffolds, until we found the best conditions.”
A giant step forward?
The result is a part of Dr. Garcia-Posadas’s PhD thesis, “A three-dimensional conjunctival model with human primary cells derived from conjunctival tissues and fibrin matrices made from plasma and cryoprecipitates.”
Says Dr. Garcia-Posadas: “The fibrin-based matrices supported conjunctival cell growth. Epithelial cells grew on the surface of the scaffolds and underwent stratification that increased over time. These cells had microvilli, which suggests cell polarization and functionality. Fibroblasts were integrated into the scaffold and they showed elongated shape. The three-dimensional model was exposed to different conditions to mimic inflammatory diseases, such as dry eye or allergic conjunctivitis. Compared to controls, the increased MUC5AC secretion occurred in partially desiccated and IL-13-treated cultures. The inflammatory status of cells was evaluated by IL-6 levels which were increased in air-lifted and partially desiccated cultures, but not in IL-13-treated ones.”
This model, she concludes, reacts differentially to the analyzed stimuli, and the responses “were in accordance to what occurs in other studies with patients.”
Further, she and her co-investigators point out in their paper that the model could lead to fewer animal experiments, resulting in reduced ethical concerns of in vivo experiments and research costs.
The ophthalmic community weighs in
“This is an impressive model,” says Dr. Epitropoulos, of The Eye Center of Columbus, and clinical assistant professor at Ohio State University Wexner Medical Center. “This would allow us to further study the pathophysiology and etiology of inflammatory ocular surface diseases and potentially new treatments for diseases such as dry eye disease, allergic conjunctivitis and even conditions such as pemphigoid.”
Perry Rosenthal, MD, assistant professor of Ophthalmology, Harvard Medical School and founder of the Boston EyePain Foundation, agrees that the model holds promise for ocular surface disease research. “This work is a significant contribution to our ability to further advance our understanding of an underlying pathogenetic process responsible for causing disabling chronic eye pain, photophobia and even blindness in some of these patients.”
- Garcia-Posadas L, Soriano-Romani L, Lopez-Garcia A, Diebold Y. An engineered human conjunctival-like tissue to study ocular surface inflammatory diseases. PLoS One. 2017 Mar 1;12(3).
Accessed April 26, 2017. Invest Ophthalmol Vis Sci. 2013 Oct 29;54:7143-52. https://www.ncbi.nlm.nih.gov/pubmed/24106119. Accessed May 2, 2017.