Primary Keratocytes Form Spheroid Bodies in vitro

Abstract

Abstract: : Purpose: Keratocytes of the cornea are the source of the transparent stromal extracellular matrix essential for corneal transparency. In culture, keratocytes maintain their unique phenotype and matrix production only under quiescent conditions; after passaging they become irreversibly fibroblastic. Tumor cells, neural progenitor cells, and hepatocytes under appropriate conditions in vitro, aggregate to form attachment–independent spheroid bodies. Primary hepatocytes in spheroids exhibit differentiated hepatocyte functions and can be expanded extensively in vitro without loss of phenotype. This study examined conditions and effects of culturing corneal keratocytes in a spheroid modality. Methods: Primary bovine keratocytes, isolated by collagenase digestion, were cultured on tissue culture plastic in media of varying composition. Keratocyte differentiation was examined by RT–PCR for keratocan, aldehyde dehydrogenase, cellular fibronectin and by immunostaining for keratan sulfate. Results: Monolayer cultures of primary keratocytes in serum–free media containing fibroblast growth factor–2 (FGF2) and insulin or insulin–like growth factor (IGF1) slowly formed multilayered aggregates. After 3 weeks in culture the aggregates floated free as tight spheres with 0.1–1 millimeter diameters. Formation of spheroids was accelerated on non–adhesive surfaces. PDGF and transforming growth factor also accelerated spheroid formation. Addition of serum or lipid–rich albumin blocked spheroid formation. Free floating spheroids expressed about twice as much keratocan mRNA as attached cells in the same culture. Immunostaining for normal corneal matrix showed keratocan protein expressed pericellularly throughout the cluster of cells and keratan sulfate to localize around the peripheral cells. Conclusions: These results show that primary keratocytes form viable attachment–independent spheroids in which the cells exhibit a high level of differentiation. Spheroid culture presents a potential manner of expanding keratocytes without loss of differentiated phenotype. The technique may be useful in tissue engineering approaches to bioengineering of corneal stroma.

Keywords: cornea: stroma and keratocytes • extracellular matrix • proteoglycans/glycosaminoglycans