Biological characteristics of nucleus pulposus cells from human degenerative intervertebral disc using the improved simple method

Wang Hai; Huang Bo; Zhou Yue; Wang Jian; Li Chang-qing

doi:10.3969/j.issn.2095-4344.2013.28.007

Abstract： BACKGROUND: The commonly used culture methods for primary intervertebral disc cells are type Ⅱcol agenase alone digestion method, and type Ⅱ col agenase combined trypin digestion method. However, the acquired cells are few. OBJECTIVE: To acquire nucleus pulposus cells from human degenerative intervertebral disc using a systemic and simplified method. METHODS: Nucleus pulposus cells were isolated and cultured. The morphology of nucleus pulposus cells was observed under inverted phase contrast microscope every day. Primary and subcultured cellsuspension was applied for the determination of cel viability using trypan blue staining. The cel growth was detected with MTT assay. The cel morphology was observed under laser confocal microscopy. RESULTS AND CONCLUSION: Nucleus pulposus cells were successful y isolated from from human degenerative intervertebral disc using the improved method, and cells were subcultured to passage 3. Primary cells were fusiform shaped, while cells at passage 1 and 2 were triangle or polygonal, which were similar to fibroblasts. When cells almost reached the confluence, the cells showed slabstone-like appearance. Trypan blue staining showed that, the primary cel viability was 99%, and passage 3 cells had 93%-95% viability. The proliferation of nucleus pulposus cells gradual y decreased as the generation increased. Compared with passage 1 cells, passage 2 and 3 cells at logarithmic phase trended to be smoother. The cel morphology observed under laser confocal microscopy was similar to the results under phase contrast microscope. The improved simple method can successful y acquire a variety of cells from human degenerative intervertebral disc, and these cells show fine biological properties.