Morphologic characteristics and proliferation of rabbit corneal stromal cells onto complexes of collagen-chitosan-sodium hyaluronate under simulated microgravity.

PURPOSE

We investigated the morphologic characteristics and proliferation of rabbit corneal stromal cells (CSCs) onto scaffolds under simulated microgravity.

METHODS

Rabbit CSCs were cultured under simulated microgravity (SMG) and static condition. Complexes of collagen-chitosan-sodium hyaluronate with pores were used as scaffolds. Rotational speed was set at 15, 20, and 30 rpm in the first, second, and third weeks of culture, respectively. Histology, immunofluorescence staining, atomic force microscope (AFM), and scanning electron microscope (SEM) examinations were performed. The cell proliferation was analyzed by cell counting kit-8 (CCK-8) assay.

RESULTS

In the SMG group, more CSCs adhered to the carriers in 24 hours. Confocal microscopic evaluation showed aggregated cells positively immunostained with vimentin. The SEM displayed the complex network of triangular or polygonal dendritic morphology of the cell bodies with many fine and long processes, which adhered to the scaffolds tightly. After 18 days of SMG culture, keratocyte-like CSCs with rich cell interconnections not only grew on the surface, but also into the interior of scaffolds. There were degradation phenomena in scaffolds in the SMG condition. Under static condition, cells just grew on scaffolds forming a monolayer. Cells showed elongating spindle shape and developed less processes. The absorbance values of the CCK-8 assay in the SMG group were significantly higher (P < 0.01) than in the conventional group.

CONCLUSIONS

The condition of SMG and porous collagen-chitosan-sodium hyaluronate scaffolds facilitate the proliferation of CSCs. Cells showed robust growing characteristics and morphologic properties of keratocytes. The techniques for microgravity culture of keratocyte-like CSCs on scaffolds can yield cell aggregates or cell sheets that are favorable to the reconstruction of tissue engineering of the corneal stromal layer.