Natural Corneal Cell-Based Microenvironment as Prerequisite for Balanced 3D Corneal Epithelial Morphogenesis: A Promising Animal Experiment-Abandoning Tool in Ophthalmology

Abstract

To achieve durable recognition as a promising animal experiment-abandoning tool in ophthalmology, in vitro engineered tissue equivalents of the human cornea should exhibit proper morphogenesis. Regarding this issue, we were seeking for the natural cell microenvironment fulfilling the minimum requirements to allow human corneal keratinocytes to develop a balanced epithelial morphology with regular spatial appearance of tissue homeostatic biomarkers. Hence, we established cocultures of 3D cell-based collagen scaffolds comprising immortalized corneal keratinocytes combined with a gradual cornea-derived in vivo-like cell microenvironment, together with immortalized stromal fibroblasts alone (nonholistic) or fibroblasts and immortalized endothelial cells (holistic). With matched non-holistic microenvironments revealing mostly flattened cells and putative apical cell ablation foci at day 6, and 9 in HE stains, holistic counterparts yielded proper epithelial stratification with cell flattening restricted to apical layers. Concordantly, RT 2 -PCR showed a tremendous increase in gene expression for progressive and terminal biomarkers of corneal keratinocyte differentiation, cytokeratin (CK) 12, and filaggrin (FIL), in response to nonholistic environments, while involucrin (INV) was moderately but significantly upregulated. Although visible, this increase was moderate in corneal keratinocytes with a holistic environment. On the protein level, indirect immunofluorescence revealed that only epithelia of holistic environments showed diminishment in CK19, counteracted by CK12 rising over time. This time-dependent progression in differentiation coincided with declined proliferation and tissue-regular focus of differentiation biomarkers inv and fil to suprabasal and apical cell layers. Our novel findings suggest the interplay of native tissue forming cell entities, important for balanced corneal epithelial morphogenesis. In addition, they provide evidence for a holistic cell microenvironment as a prerequisite for development of an in vitro engineered corneal epithelial tissue equivalent, exhibiting a regular appearance of tissue homeostatic biomarkers. Such equivalents will be promising tools in ophthalmology, for example, for mechanistic studies in basic research and/or testing of generics or preclinical validation of innovative cornea-tailored biomaterials, desired for regenerative strategies.

Modulation of focal adhesion constituents and their down-stream events by EGF: On the cross-talk of integrins and growth factor receptors

“…target biomarker and thereby its level of localization and/or its activation stage, as it was the case with EGFR. This approach, on the following termed semiquantitative fluorescence imaging (SQFI), has been successfully used in our former studies to quantitatively estimate the frequency of occurrence of various tissue cell-specific biomolecules [28,29,37,38]. Moreover, to veritably show that increase in specific antigen fluorescence coincides with increased frequency of occurrence of an antigen under study, inlays in Figs.…”

Corneal Tissue Engineering: An <em>In Vitro</em> Model of the Stromal-nerve Interactions of the Human Cornea

“…Corneal tissue engineering applications have garnered great interest across various fields of science 10,11,12,13,14 , but there are still major limitations during actual application, including corneal graft rejections, infections, and scarring 10,11,12,13,14 . There are several studies that have successfully developed and established various in vitro models 3 , 15,16,17,18,19,20,21,22,23,24,25,26 . The 3D in vitro models are the most promising and of great scientific interest.3D models are known to better mirror the in vivo cellular and physiological events that are critical during fibrosis and wound healing 15,27,28,29 .These in vitro models play an integral role in finding new therapeutic approaches for treating different disease conditions including corneal complications.…”