Fish Scale-Derived Scaffolds for Culturing Human Corneal Endothelial Cells.

Bert Van den Bogerd,Diego Ponzin,Nadia Zakaria,Mohit Parekh,Stefano Ferrari

doi:10.1155/2018/8146834

Abstract

Purpose To investigate the biocompatibility of fish scale-derived scaffolds (FSS) with primary human corneal endothelial cells (HCEnCs). Methods HCEnCs were isolated from 30 donor corneas in a donor-matched study and plated in precoated Lab-Tek slides (n = 15) and FSS (n = 15). Cell morphology, proliferation/migration, and glucose uptake were studied (n = 30). Hoechst, ethidium homodimer, and calcein AM (HEC) staining was performed to determine viability and toxicity (n = 6). The cell surface area was calculated based on calcein AM staining. HCEnCs were stained for ZO-1 (n = 6) to detect tight junctions and to measure cell morphology; Ki-67 (n = 6) to measure proliferating cells; and vinculin to quantify focal adhesions (n = 6). The formation of de novo extracellular matrix was analyzed using histology (n = 6). Results HCEnCs attach and grow faster on Lab-Tek slides compared to the undulating topography of the FSS. At day 11, HCEnCs on Lab-Tek slide grew 100% confluent, while FSS was only 65% confluent (p = 0.0883), with no significant difference in glucose uptake between the two (p = 0.5181) (2.2 μg/mL in Lab-Tek versus 2.05 μg/mL in FSS). HEC staining showed no toxicity. The surface area of the cells in Lab-Tek was 409.1 μm2 compared to 452.2 μm2 on FSS, which was not significant (p = 0.5325). ZO-1 showed the presence of tight junctions in both conditions; however, hexagonality was higher (74% in Lab-Tek versus 45% in FSS; p = 0.0006) with significantly less polymorphic cells on Lab-Tek slides (8% in Lab-Tek versus 16% in FSS; p = 0.0041). Proliferative cells were detected in both conditions (4.6% in Lab-Tek versus 4.2% in FSS; p = 0.5922). Vinculin expression was marginally higher in HCEnCs cultured on Lab-Tek (234 versus 199 focal adhesions; p = 0.0507). Histological analysis did not show the formation of a basement membrane. Conclusions HCEnCs cultured on precoated FSS form a monolayer, displaying correct morphology, cytocompatibility, and absence of toxicity. FSS needs further modification in terms of structure and surface chemistry before considering it as a potential carrier for cultured HCEnCs.

Highlights

The human cornea is the outermost, transparent tissue of the eye
Human corneal endothelial cells (HCEnCs) are responsible for maintaining this transparency through a pump-and-leak mechanism [1]. This leaky barrier of hexagonally shaped cells allows passive diffusion of nutrients flowing from the anterior chamber to the corneal stroma and epithelium but simultaneously averts corneal edema by pumping excessive fluid back to the anterior chamber
If the overall number of HCEnCs drops below a certain threshold of less than 500 cells/mm2, irreversible edema eventually arises, leading to an opaque cornea

Summary

Introduction

The human cornea is the outermost, transparent tissue of the eye. It is the principal refractive element of the visual system, and its function depends mainly on its optical clarity. Human corneal endothelial cells (HCEnCs) are responsible for maintaining this transparency through a pump-and-leak mechanism [1]. To do so, this leaky barrier of hexagonally shaped cells allows passive diffusion of nutrients flowing from the anterior chamber to the corneal stroma and epithelium but simultaneously averts corneal edema by pumping excessive fluid back to the anterior chamber. Due to a mitotic arrest in vivo after birth, the number of endothelial cells decreases throughout life [2]. This decay can dramatically be accelerated by trauma or several diseases. If the overall number of HCEnCs drops below a certain threshold of less than 500 cells/mm, irreversible edema eventually arises, leading to an opaque cornea

Methods

Results

Discussion

Conclusion