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Jiansu Chen, Qiaozhi Lu, Qiongyu Guo, Xiomara Calderón-Colón, Morgana M. Trexler, Oliver Schein, Jennifer H. Elisseeff; Keratocytes and Corneal Endothelial Cells Cultured on Collagen Vitrigel Membrane Preserve Morphology and Gene Expression. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5155.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the biological activity of collagen vitrigel (CV) membranes and their application for expanding keratocytes and corneal endothelial cells (CEC) while preserving morphology and gene expression.
Primary (P0) bovine keratocytes were cultured on CV in 0% (group A) or in 10% serum (group B). Control P0 keratocytes were cultured on tissue culture plastic in 0% (group C) or in 10% serum (group D) or after two passages (P2) in 10% serum on culture plastic (group E). Keratocyte expressions of ALDH 3A1, keratocan and biglycan were evaluated by real time RT-PCR (n = 3). Cell morphology was visualized with cytoplasmic staining using a Live/Dead cell viability assay. A co-culture of rabbit keratocytes and CEC was also performed by application to opposite sides of a CV. Histological cross-sections were stained with H&E. CECs were stained with 0.2% alizarin red.
Keratocytes cultured in DMEM/F-12 containing 0% or 10% serum on CV had a dendritic morphology and formed extensive intercellular networks. For the expression of ALDH mRNA, real-time PCR analyses revealed that keratocytes cultured on CV for 7 days (Group B, used as reference) expressed greater level compared to Group D. The highest ALDH and keratocan mRNA levels were found in Group A, and there were significant differences when compared with comparable cells in Group C. The lowest mRNA levels of ALDH, keratocan and biglycan were found in the passaged cells. CEC on CV cultured for 9 days stained with alizarin red produced more cell-cell borders than that on culture plastic and revealed hexagonal and homogeneous cell morphology. A corneal stromal-endothelial model was constructed by co-culturing keratocytes and CECs on opposite sides of a CV.
Bovine keratocytes expanded on CV expressed greater levels of ALDH and keratocan mRNA in 0% or 10% serum culture medium compared to plastic controls. Significantly increased mRNA levels of ALDH and keratocan will ultimately favor corneal stromal transparency when reconstructing the cornea. Keratocytes and CEC cultured on CV also preserved their native morphology compared to culture plastic. Thus, collagen vitrigel membrane is a potentially useful scaffold for expanding or delivering cells for corneal reconstruction and may be used to engineer new corneal stromal and endothelial layers.
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