Development of a hemicornea from human primary cell cultures for pharmacotoxicology testing

Abstract

We report the reconstruction and characterization of a hemicornea (epithelialized stroma), using primary human cells, for use in research and as an alternative to the use of animals in pharmacotoxicology testing. To create a stromal equivalent, keratocytes from human corneas were cultured in collagen-glycosaminoglycan-chitosan foams. Limbal stem cell-derived epithelial cells were seeded on top of these, giving rise to hemi-corneas. The epithelium appeared morphologically similar to its physiological counterpart, as shown by the basal cell expression of p63 isoforms including, in some cases, the stem cell marker p63DeltaNalpha, and the expression of keratin 3 and 14-3-3sigma in the upper cell layers. In addition, the cuboidal basal epithelial cells were anchored to a basement membrane containing collagen IV, laminin 5, and hemidesmosomes. In the stromal part, the keratocytes colonized the porous scaffold, formed a network of interconnecting cells, and synthesized an ultrastructurally organized extracellular matrix (ECM) containing collagen types I, V, and VI. Electron microscopy showed the newly synthesized collagen fibrils to have characteristic periodic striations, with diameters and interfibril spacings similar to those found in natural corneas. Compared to existing models for corneal pharmacotoxicology testing, this new model more closely approaches physiological conditions by including the inducing effects of mesenchyme and cell-matrix interactions on epithelial cell morphogenesis.

Cell culture models of the human cornea — a comparative evaluation of their usefulness to determine ocular drug absorption in-vitro

“…Therefore, most cell culture models of corneal tissue comprise only epithelial cells (Hornof et al 2005). However, to reflect the in-vivo properties of the cornea more accurately, epithelium-stroma-equivalents (Parnigotto et al 1998;Builles et al 2007), as well as cell culture models of the whole cornea, consisting of epithelial, stromal and endothelial cells, have been developed (Griffith et al 1999;Tegtmeyer et al 2001;Reichl & Müller-Goymann 2003). An ideal corneal cell culture model for in-vitro permeation studies should exhibit a similar multilayered architecture to that observed in-vivo, form a tight epithelium and have barrier properties similar to excised cornea for a wide spectrum of substances and drugs with different physico-chemical characteristics.…”

Development of a Reconstructed Cornea from Collagen–Chondroitin Sulfate Foams and Human Cell Cultures

“…41,42 Previous studies by the authors have shown that neosynthesized collagen deposition in collagen foams is a slow process. An initial study 13 showed that 35-day culture provides incomplete filling of the pores of the foam. In another study, 43 we tried to increase collagen deposition and to observe the effect of tetracycline hydrochloride treatment on a 74-day culture with some success.…”

Orthogonal scaffold of magnetically aligned collagen lamellae for corneal stroma reconstruction

“…Scaffolds can be used as stand-alone implants, or be seeded with cells to create cell-scaffold composites, which might be allowed to mature in vitro until tissue-like properties develop. In addition to their potential clinical uses as 3D tissue mimics, tissueengineered corneas should facilitate the in vitro study of the complex physiology of living tissue and would also serve as alternatives to animal models for pharmacotoxicity testing [3,4,13]. While the two in vitro human corneal models currently commercially available for the latter purpose, from SkinEthic Laboratories (Nice, France) and…”