Excellence of differential velocity adherent procedure in primary culture of nucleus pulposus cells of intervertebral disc

doi:10.3969/j.issn.2095-4344.2016.42.008

Abstract

Abstract:

BACKGROUND: As the main function cell of intervertebral disc, nucleus pulposus cells are the focus of studying the degenerative mechanism; thereby, it is crucial to maintaining the physiological function of nucleus pulposus cells in vitro and the stability of the cell phenotype.
OBJECTIVE: To study the excellence of differential velocity adherent procedure in primary culture of nucleus pulposus cells of rat intervertebral disc through comparison.
METHODS: Twenty male Wistar rats aged 4 weeks were enrolled, and then nucleus pulposus cells of intervertebral disc were isolated and cultured in vitro; cell passage culture was performed in different groups when the primary cells were merged to 90%. Differential velocity adherent group cells adhered for 30 minutes, and non-adherent cells were aspirated and transferred to new culture dish after readjusting the concentration; the controls received no intervention. Passages 1 and 2 cells in the differential velocity adherent group were isolated and purified by the same procedure. The morphology of three generations of cells in the two groups was compared, the purity of the identification was detected by immunohistochemistry, the cell viability was detected by cell counting kit-8 and the cell growth curve was drawn.
RESULTS AND CONCLUSION: Inverted phase contrast microscope and hematoxylin-eosin staining revealed that the cell homogeneity of the differential velocity adherent group was significantly higher than that of the control group, and there were more kinds of fibroblast-like cells in nucleus pulposus cells in the control group. Identification and purity analysis of collagen type II showed that the cytoplasm of two groups were both stained brown, indicating that they were the nucleus pulposus chrondrocytes. The positive rate of differential velocity adherent group was significantly higher than that of the control group (P < 0.05). The cell growth curve of cell counting kit-8 showed cells in the two groups all passed by the latency phase within 2 days, then to the logarithmic phase of 3 days, and entered the lag phase, while the growth rate of the control group was more rapid during the latency and the early logarithmic phases.
These findings suggest that differential velocity adherence method is a practical and effective procedure for the isolation and purification of primary cultured rat nucleus pulposus cells. Through the primary culture, twice differential velocity adherence procedure, the passage 3 rat nucleus pulposus cells are metabolic exuberant, consistent with the phenotype, the cell purity is higher, and the logarithmic growth phase can be used as the optimal time for studying the mechanism of intervertebral disc cells.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Intervetebral Disk, Cells, Cultured, Cell Aging, Tissue Engineering

CLC Number:

R318

Hu Yong-kai, Sun Hao-lin, Qi Long-tao, Li Chun-de. Excellence of differential velocity adherent procedure in primary culture of nucleus pulposus cells of intervertebral disc[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(42): 6284-6289.

Figures/Tables 2

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