Formation of keratocyte spheroids on chitosan-coated surface can maintain keratocyte phenotypes

Corneal keratocytes have been reported to be able to form spheroids that can preserve their phenotypes after being seeded back onto tissue culture plate in specific culture media. In this study, we found that keratocytes could also form spheroids on a bioengineered material, chitosan-coated surface, with 10% horse serum and Dulbecco's modified Eagle's medium. Under scanning electron microscopy observation, the cells in the spheroids were found to adhere each other tightly, and the cellular boundary could not be distinguished. They could return to a dendritic (keratocyte) morphology and proliferate after they were seeded back onto tissue culture plate. Immunocytochemistry was used to characterize these cells. Reverse transcription-polymerase chain reaction revealed that keratocytes in the spheroids were not from the PAX-6-positive progenitor cells. Further, the results of the seeding density and the number of spheroids formation, cell viability (MTT) assays, negative staining of Ki-67, and Live/Dead assay suggested that the spheroids were from cell aggregation instead of cell proliferation. Cells in the spheroids maintained phenotypes and functions characteristic of keratocytes, as seen by reverse transcription-polymerase chain reaction, collagen gel contraction assay, and challenges of keratocytes with transforming growth factor-beta1. Our results showed that corneal keratocytes could form spheroids on a chitosan-coated surface and maintain a keratocyte phenotype. However, such keratocyte spheroids do not proliferate and cannot withstand transforming growth factor-beta from myofibroblast differentiation.