Abstract
Purpose:
The goal of this study was to isolate three cell types from the choroid (fibroblasts, endothelial cells and melanocytes) in order to reconstruct a choroid in vitro using the self-assembly approach of tissue engineering.
Methods:
Human choroids were incubated in dispase to remove retinal pigment epithelial cells, then in collagenase to dissociate the choroidal cells. Vascular endothelial cells were isolated using CD31-coated magnetic beads, then cultured in EBM-2 growth medium. The remaining cells were plated in growth media optimized for the culture of melanocytes or fibroblasts. Culture purity was assessed using immunostainings (CD31, HMB45, vimentin, keratins 8/18). To reconstruct the choroidal stroma, fibroblasts were cultured in the presence of serum and ascorbic acid to promote extracellular matrix (ECM) assembly. Melanocytes and endothelial cells (primary cultures or GFP-HUVEC) were separately seeded on top of the stromal substitutes. Sheets were then stacked in order to sandwich the endothelial cells between the ECM sheets. Stromal substitutes were analysed by mass spectrometry and by immunostaining (collagens, proteoglycans). Development of vascular networks was observed throughout time using the GFP-HUVEC cell line. Choroidal substitutes were further characterized by histology.
Results:
The technique used to isolate choroidal cells yielded pure cultures of fibroblasts, melanocytes and vascular endothelial cells. The stromal substitutes engineered using the self-assembly approach were composed of collagen (types I, VI, XII and XIV), proteoglycans (such as decorin, lumican) and other ECM proteins. Protein expression was confirmed using immunostaining. Endothelial cells spontaneously assembled into tubular structures and vascular networks when cocultured within the fibroblast-containing ECM sheets. The seeded melanocytes adhered and survived on the stromal substitutes as confirmed by the presence of pigmented HMB45-positive cells with a dendritic morphology.
Conclusions:
This study shows that the self-assembly approach of tissue engineering can be used to reconstruct a choroid using native cells. This model represents a unique tool to better understand the crosstalk between the different choroidal cell types and cell-ECM interactions.
Keywords: 452 choroid •
519 extracellular matrix