Collagen vitrigel membranes for the in vitro reconstruction of separate corneal epithelial, stromal, and endothelial cell layers

Abstract

The goal of this study was to evaluate the potential suitability of collagen Vitrigel (CV) membrane as a substrate for the separate reconstruction of the three main cellular layers of the cornea. Limbal explants, keratocytes, and endothelial cells were cultured on transparent membranes made of type I collagen. The resulting cell sheets were evaluated using RT-PCR, in addition to light and electron microscopy. Tensile testing was also performed to examine the mechanical properties of CV. Limbal explant cultures resulted in partially stratified epithelial sheets with upregulation of the putative stem cell marker p63. Keratocytes cultured in serum on CV exhibited stellate morphology along with a marked increase in expression of corneal crystallin ALDH and keratocan, (a keratan sulphate proteoglycan: KSPG), compared to identical cultures on tissue culture plastic. Endothelial cells formed dense monolayers with uniform cell size, tight intercellular junctions, and expression of voltage-dependent anion channels VDAC2 and VDAC3, chloride channel protein CLCN2, and sodium bicarbonate transporter NBC1. Epithelial and endothelial cells exhibited adhesive structures (desmosomes and hemidesmosomes) and evidence of apical specialization (microplicae), while endothelial cells also produced a Descemet's membrane-like basal lamina. CV was found to possess ultimate tensile strengths of 6.8 +/- 1.5 MPa when hydrated and 28.6 +/- 7.0 MPa when dry. Taken together, these results indicate that CV holds promise as a substrate for corneal reconstruction.

Transplantation of multiciliated airway cells derived from human iPS cells using an artificial tracheal patch into rat trachea

“…It exhibits excellent transparency and permeability for high‐molecular weight proteins and has, therefore, been successfully used as a cell culture scaffold in various studies. Human corneal models (Ko et al, ; McIntosh Ambrose et al, ; Takezawa, Nishikawa, & Wang, ; Chae et al, ; Yamaguchi et al, 2016; Yoshida et al, ; Yamaguchi & Takezawa, ) and human hepatocytes models (Oshikata‐Miyazaki & Takezawa, ; Watari et al, ) have been successfully reconstructed using the collagen vitrigel membrane. An artificial skin based on collagen vitrigel was shown to promote the epithelialization of regenerative skin and to inhibit scar formation and inflammation, whereas the existing products such as DuoACTIVEV ET of a hydrocolloid dressing and Pelnac of a collagen sponge was not able to sufficiently inhibit scar formation (Aoki et al, ).…”

Extracellular Matrix is an Important Component of Limbal Stem Cell Niche

“…Collagen vitrigel which is made of type I collagen is used to culture epithelial cells, keratocytes and endothelial cells. Vitrigel possesses proper tensile strengths, thus makes it a potential scaffold for corneal stroma reconstruction [17]. …”

In vitro biomimetic platforms featuring a perfusion system and 3D spheroid culture promote the construction of tissue-engineered corneal endothelial layers

“…Collagen sheets were prepared as previously described with modifications 77 . Briefly, a ring-shaped sterilized nylon membrane with an inner and outer diameter of 23 and 33 mm, respectively, was inserted into a polystyrene culture dish with a diameter of 35 mm.…”