Characterization of human conjunctival cells grown in a 3D-model

Abstract

Purpose: To characterize primary conjunctival cells grown in a bioengineered 3D-model of human conjunctiva.

Methods: Human conjunctival epithelial cells and fibroblasts were obtained from cadaveric donors, and grown in biocompatible matrices. Fibrin matrices were made using human plasma or cryoprecipitate, with a final fibrinogen concentration of 1.5 mg/ml. Fibroblasts were grown inside the matrices and epithelial cells above them. Complete constructs were allowed to grow up to 14 days, and several parameters were evaluated at different times. Cell proliferation in the matrices was evaluated with AlamarBlue assay at days 3 and 7 (n=5). Construct processing for histology study and sectioning (6 μm) was done. Hematoxylin/eosin was used to characterize the constructs at 3, 7, and 14 days. At the same days, immunofluorescence staining was performed against different markers: cytokeratin (CK) 19 was used to determine if epithelial cells maintains their phenotype in the matrices; HPA lectin was used to identify secretory products from goblet cells; Ki67 was used as an indicator of proliferative capacity of cells.

Results: Fibrin matrices made from either human plasma or cryoprecipitate allowed conjunctival cell growth. 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. The epithelial cells were able to stratify, and they maintained the expression of CK19. Some cells in the upper layers secreted high molecular weight glycoconjugates identified by lectin HPA. A small percentage of epithelial cells grown in the matrices maintained proliferative capacity measured by Ki67.

Conclusions: Epithelial cells grown in our 3D-model retained their phenotype for at least 14 days. Some of these cells secreted glycoconjugates, and some others maintained proliferative capacity. These results suggest that this 3D-model of human conjunctiva could be used for in vitro assays involving proliferation and/or secretion.

Keywords: 474 conjunctiva • 608 nanomedicine • 607 nanotechnology