Abstract:

BACKGROUND: Obtaining corneal stromal fibroblasts with high purity, activity and biological characteristics closer to in vivo state is the foundation of corneal restoration research.
OBJECTIVE: To develop a new culture method for bovine corneal stromal fibroblasts in vitro.
METHODS: The bovine corneal stroma was digested by type Ⅰcollagenase using two-step digestion method to prepare suspension. The cell suspension was transferred into culture bottles for cultivation. Cells in good growth status were subcultured. Cell survival rate was measured by trypan blue staining immediately after digestion. The growth status of bovine corneal stromal fibroblasts was dynamically observed using inverted phase-contrast microscope. Immunohistochemical staining with vimentin was used to identify the bovine corneal fibroblasts. Cell proliferation was detected using MTT assay. Bovine corneal stromal fibroblasts in logarithmic growth phase were obtained for growth curves and doubling time.
RESULTS AND CONCLUSION: Bovine corneal stromal fibroblasts were successfully isolated and cultured in vitro. Microscopic examination and immunohistochemical staining with vimentin confirmed that the cultured cells were bovine corneal stromal fibroblasts. According to trypan blue staining, the immediate survival rate of bovine corneal stromal fibroblasts was 93.5%. Cell growth curve approximated the "S" shape, and cell population doubling time was 38.70 hours. These findings demonstrate that the cell culture method of two-step digestion is a simple, economic and efficient method for the primary culture of bovine corneal stromal fibroblasts.