Primary cell culture and morphological observation of bulbar conjunctival fibroblasts in conjunctivochalasis / 国际眼科杂志(Guoji Yanke Zazhi)

ABSTRACT

AIM: To observe the growth status and morphological changes of primary cultured bulbar conjunctival fibroblasts in different stages of conjunctivochalasis(CCH), and to determine the best passage time, so as to obtain stable and consistent CCH bulbar conjunctival fibroblasts.METHODS: CCH primary bulbar conjunctival fibroblasts were obtained by tissue block adhesion method. The fibroblasts were purified by trypsin differential digestion method. The growth status and morphological changes of fibroblasts in different periods were observed and recorded under inverted microscope. The fibroblasts were identified by immunofluorescence cytochemical staining.RESULTS: After 24h of CCH conjunctival tissue adherent to the wall, a small number of cells would be seen crawling out around the tissue blocks. The logarithmic phase of cell growth was from the 2-7d. The cells grew fast, with vigorously proliferation, clear outline, uniform distribution, increas in numbers and clear nuclei. From the 9-15d, the cell growth entered the plateau stage, the tissue blocks gradually aged and lost activity. The cells grew slowly, arranged loosely, the volume became larger, the shape became flat, and a large number of granular substances and vesicles were produced in the cytoplasm. Some cells fell off from the bottom of the culture bottle, and large gaps appeared between the cells. After subculture and purification, the size and morphology of fibroblasts were basically the same. Through cell identification, fibroblasts were long spindle, flat star or multi-process spindle, wide in the middle, oval nucleus, relatively small at both ends, with 2-3 slender processes of different lengths extending outward.CONCLUSION: Primary CCH bulbar conjunctival fibroblasts can be successfully obtained by tissue block adhesion method. When the cells grow to the 8d, they can be digested and passaged to obtain stable and consistent CCH conjunctival fibroblasts.