Abstract: Objective To study pieces-stick primary culture of conjunctival tissue and identification of bulbar conjunctiva fibroblast from patient of conjunctivochalasis，for conjunctivochalasis provide a large cells in vitro study. Design Experimental study. Participants Cultures of conjunctiva fibroblasts from conjunctivochalasis. Methods The bulbar conjunctivafrom patient who accepted operation of conjunctivochalasis removal was the experimental material. Fibroblasts were cultured with tissue pieces-stick method,  trypsin-digest and transferred in vitro. Then, morphological observation was carried out, and expression of fibroblast cytoplasm-specific protein was identified by immunofluorescence and detected by Flow Cytometry. Main Outcome Measures Cellular morphology, structure and purity. Results After culture with primary culture method for 2-5 days, tissue pieces were closely attached with six well plates. After 10-15 days, lots of cluster of cell proliferation were observed under inverted microscope, cells had a mosaic shape with indistinct borders. When cells were subcultured into secondary generation, cells under microscope presented with a shuttle shape and radical arrangement. Size and morphology were uniform. There was an oval-shaped nucleus in cytoplasm, and crosslinking was formed between cells by cellular processes. Expression of Vimentin and Keratin (C11) in bulbar conjunctiva fibroblasts from patients with conjunctivochalasis were detected as positive and negative respectively by fluorescence microscope and flow cytometry. Conclusions Stable conjunctivochalasis conjunctiva fibroblast can be obtained by cell culture with tissue pieces-stick method, and can also be further identified by morphologic observation, flow cytometry and immunofluorescence. (Ophthalmol CHN, 2013, 22: 105-108)

Key words:  , bulbar conjunctiva； fibroblasts； cells, cultured