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Isabel Arranz-Valsero, Laura Garcia-Posadas, Ana Fernández, Antonio López-García, F. Javier Iglesias, Yolanda Diebold; Development of a bioengineered 3D-model of human conjunctiva. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4698.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a 3D-model of human conjunctiva by tissue engineering, using fibrin-based matrices.
A biocompatible matrix, human fibroblasts and epithelial cells from bulbar conjunctiva from cadaveric donors were used to construct this model. Fibrin matrices were prepared using human plasma or cryoprecipitate, following a method described by our group. Both matrices had the same fibrinogen concentration (1.5 mg/ml). We have previously optimized epithelial cell culture from that source testing different culture procedures and media. Immunocytochemistry was done against E-cadherin, cytokeratins (CK) 7 and 19, MUC5AC, vimentin and ki67 antigens in order to characterize cell phenotypes (n=3). Fibroblasts were grown inside the fibrin matrices and epithelial cells above them. Hematoxilin/eosin was used to characterize the constructs and AlamarBlueTM assay was used to measure cell proliferation at 3 and 7 days (n=5).
Fibrin matrices made from either human plasma or cryoprecipitate allowed cell growth (fibroblasts inside the matrix and epithelial cells above it). Human serum-supplemented medium was the best for epithelial cell culture, and it was chosen to maintain cell-seeded matrices up to 14 days. Fibroblasts showed positive reactivity to the stromal marker vimentin, but negative to epithelial markers. Epithelial cells exhibited positive reactivity to E-cadherin, CK7 and CK19, but negative reactivity to vimentin. Some cells expressed MUC5AC. Fibroblasts inside plasma matrices showed better proliferation rates than that of fibroblasts inside cryoprecipitate matrices (1.95 fold increase at 3 days; p = 0.004). On the contrary, epithelial cells showed higher proliferation rates when grown over cryoprecipitate-matrices (1.28 fold increase at 3 days; p = 0.022). Constructs seeded with both fibroblasts and epithelial cells, and made from plasma or cryoprecipitate, showed a 3.66 and 2.55 fold increase, respectively, in proliferation rates from day 3 to 7. 3D complete constructs displayed similar consistency to that of human conjunctival tissue.
We have optimized a method to culture human conjunctival cells from cadaveric tissue samples. In addition, we demonstrated that fibrin-based matrices supported human conjunctival cells growth when using them either as a scaffold (fibroblasts) or as a substrate (epithelial). It is then possible to bioengineer a fibrin-based 3D model of human conjunctiva.
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