Development of a hemicornea from human primary cell cultures for pharmacotoxicology testing.
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.
Builles
N
N
Banque de Cornées des Hospices Civils de Lyon, Lyon, France.
Bechetoille
N
N
Justin
V
V
André
V
V
Barbaro
V
V
Di Iorio
E
E
Auxenfans
C
C
Hulmes
D J S
DJ
Damour
O
O
D000826:Animal Testing Alternatives; D015415:Biomarkers / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000378:metabolism / Q000648:ultrastructure; D003315:Cornea / Q000166:cytology* / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D022002:Hemidesmosomes / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology* / Q000378:metabolism; D000097794:Kalinin
D015415:Biomarkers; D015815:Cell Adhesion Molecules; D024141:Collagen Type IV; D008565:Membrane Proteins; D000097794:Kalinin; C081876:CKAP4 protein, human