The interactions between corneal nerve, epithelium, and stroma are essential for maintaining a healthy cornea. Thus, corneal tissue models that more fully mimic the anatomy, mechanical properties and cellular components of corneal tissue would provide useful systems to study cellular interactions, corneal diseases and provide options for improved drug screening. Here a corneal tissue model was constructed to include the stroma, epithelium, and innervation. Thin silk protein film stacks served as the scaffolding to support the corneal epithelial and stromal layers, while a surrounding silk porous sponge supported neuronal growth. The neurons innervated the stromal and epithelial layers and improved function and viability of the tissues. An air-liquid interface environment of the corneal tissue was also mimicked in vitro, resulting in a positive impact on epithelial maturity. The inclusion of three cell types in co-culture at an air-liquid interface provides an important advance for the field of in vitro corneal tissue engineering, to permit improvements in the study of innervation and corneal tissue development, corneal disease, and tissue responses to environmental factors.

With insufficient options to meet the clinical demand for cornea transplants, one emerging area of emphasis is on cornea tissue engineering. In the present study, the goal was to combine the corneal stroma and epithelium into one coculture system, to monitor both human corneal stromal stem cell (hCSSC) and human corneal epithelial cell (hCE) growth and differentiation into keratocytes and differentiated epithelium in these three-dimensional tissue systems in vitro.Coculture conditions were first optimized, including the medium, air-liquid interface culture, and surface topography and chemistry of biomaterial scaffold films based on silk protein. The silk was used as scaffolding for both stromal and epithelial tissue layers because it is cell compatible, can be surface patterned, and is optically clear. Next, the effects of proliferating and differentiating hCEs and hCSSCs were studied in this in vitro system, including the effects on cell proliferation, matrix formation by immunochemistry, and gene expression by quantitative reverse transcription-polymerase chain reaction. The incorporation of both cell types into the coculture system demonstrated more complete differentiation and growth for both cell types compared to the corneal stromal cells and corneal epithelial cells alone. Silk films for corneal epithelial culture were optimized to combine a 4.0-μm-scale surface pattern with bulk-loaded collagen type IV. Differentiation of each cell type was in evidence based on increased expression of corneal stroma and epithelial proteins and transcript levels after 6 weeks in coculture on the optimized silk scaffolds.

New in vitro tissue models to mimic in vivo conditions are needed to provide insight into mechanisms involved in peripheral pain responses, potential therapeutic strategies to address these responses, and to replace animal models for such indications. For example, the rabbit cornea Draize test has become the standard method used for decades to screen ophthalmic drug and consumer product toxicity. In vitro tissue models with functional innervation have the potential to replace in vivo animal testing and provide sophisticated bench tools to study ocular nociception and its amelioration. Herein, full thickness, innervated, 3D human corneal tissues are grown under physiologically relevant culture conditions to study nociceptive-related responses, by mimicking ocular environmental cues, including intraocular pressure (IOP) and tear flow (TF). Capsaicin, a chili pepper-derived irritant known to cause a burning sensation in mammalian tissues is utilized as a nociceptive stimulant to induce pain, while subsequent serum treatment is used to mimic healing. Pain mediators released upon capsaicin stimulation and cell regrowth after serum treatment are characterized to assess ocular responses in this new, innervated, human corneal tissue system for comparison of outcomes to established animal and related responses.