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

doi:10.3969/j.issn.2095-4344.2013.28.007

Abstract

Abstract:

BACKGROUND: The commonly used culture methods for primary intervertebral disc cells are type Ⅱ collagenase alone digestion method, and type Ⅱ collagenase 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 cell suspension was applied for the determination of cell viability using trypan blue staining. The cell growth was detected with MTT assay. The cell morphology was observed under laser confocal microscopy.

RESULTS AND CONCLUSION: Nucleus pulposus cells were successfully 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 cell viability was 99%, and passage 3 cells had 93%-95% viability. The proliferation of nucleus pulposus cells gradually decreased as the generation increased. Compared with passage1 cells, passage 2 and 3 cells at logarithmic phase trended to be smoother. The cell morphology observed under laser confocal microscopy was similar to the results under phase contrast microscope. The improved simple method can successfully acquire a variety of cells from human degenerative intervertebral disc, and these cells show fine biological properties.

Key words: tissue construction, cartilage tissue construction, nucleus pulposus cells, improved simple method, intervertebral disc, lumbar intervertebral disc protrusion, cell culture, cell proliferation, morphology, biological property, type Ⅱ collagenase, cell growth curve, National Natural Science Foundation of China

CLC Number:

R318

Wang Hai, Huang Bo, Zhou Yue, Wang Jian, Li Chang-qing. Biological characteristics of nucleus pulposus cells from human degenerative intervertebral disc using the improved simple method[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(28): 5139-5144.

Figures/Tables 4

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