Impact of cell source on human cornea reconstructed by tissue engineering.
OBJECTIVE
To investigate the effect of the tissue origin of stromal fibroblasts and epithelial cells on reconstructed corneas in vitro.


METHODS
Four types of constructs were produced by the self-assembly approach using the following combinations of human cells: corneal fibroblasts/corneal epithelial cells, corneal fibroblasts/skin epithelial cells, skin fibroblasts/corneal epithelial cells, skin fibroblasts/skin epithelial cells. Fibroblasts were cultured with ascorbic acid to produce stromal sheets on which epithelial cells were cultured. After 2 weeks at the air-liquid interface, the reconstructed tissues were photographed, absorption spectra were measured, and tissues were fixed for histologic analysis. Cytokine expression in corneal- or skin-fibroblast-conditioned media was determined with the use of protein array membranes. The effect of culturing reconstructed tissues with conditioned media, or media supplemented with a cytokine secreted mainly by corneal fibroblasts, was determined.


RESULTS
The tissue source from which epithelial and mesenchymal cells were isolated had a great impact on the macroscopic and histologic features (epithelium thickness and differentiation) and the functional properties (transparency) of the reconstructed tissues. The reconstructed cornea had ultraviolet-absorption characteristics resembling those of native human cornea. The regulation of epithelial differentiation and thickness was mesenchyme-dependent and mediated by diffusible factors. IL-6, which is secreted in greater amounts by corneal fibroblasts than skin fibroblasts, decreased the expression of the differentiation marker DLK in the reconstructed epidermis.


CONCLUSIONS
The tissue origin of fibroblasts and epithelial cells plays a significant role in the properties of the reconstructed tissues. These human models are promising tools for gaining a thorough understanding of epithelial-stromal interactions and regulation of epithelia homeostasis.
Carrier
Patrick
P
Department of Oto-Rhino-Laryngology and Ophthalmology, Laboratoire d'Organogénèse Expérimentale, Centre de Recherche (FRSQ) du CHA, Laval University, Quebec, Canada.
Deschambeault
Alexandre
A
Audet
Caroline
C
Talbot
Mariève
M
Gauvin
Robert
R
Giasson
Claude J
CJ
Auger
François A
FA
Guérin
Sylvain L
SL
Germain
Lucie
L
D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques; D003319:Corneal Stroma / Q000166:cytology* / Q000378:metabolism; D017077:Culture Media, Conditioned; D016207:Cytokines / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D008027:Light; D008856:Microscopy, Fluorescence; D012542:Scattering, Radiation; D012867:Skin / Q000166:cytology* / Q000378:metabolism; D023822:Tissue Engineering; D054457:Tissue Scaffolds
D017077:Culture Media, Conditioned; D016207:Cytokines