Optimized method for isolating and culturing human nucleus pulposus cells

doi:10.3969/j.issn.2095-4344.2013.28.009

Abstract

Abstract:

BACKGROUND: There are different methods to isolate and culture human nucleus pulposus cells, and the differences in digestive enzymes components and digestion time quite are significant. So how to rapidly and efficiently harvest human nucleus pulposus cells has become a research hotspot.

OBJECTIVE: To optimize the digestive enzymes components and digestion methods for the preparation of human nucleus pulposus cells.

METHODS: Nucleus pulposus tissue specimens were selected from three adult discs in the Department of Orthopedics, China-Japan Union Hospital of Jilin University. The acute traumatic disc tissues that outstanding to the spinal canal were taken under aseptic conditions, and then the peripheral white annulus and jelly-like nucleus pulposus in the center could be seen. According to different mixed enzyme concentration ratio, the samples were divided into two groups. The enzyme Ⅰ group was treated with 0.2% Ⅱ collagenase; and the mixed enzyme Ⅱ group was digested with 0.25% trypsin for 30 minutes, and then treated with 0.2% Ⅱ collagenase. According to digestion time, each group was divided into three subgroups: 2 hours group, 4 hours group, and overnight group. Finally, suspended cell volume was decided as 2 mL to count cells. Dulbecco’s modified Eagle’s medium containing fetal bovine serum was used for cell culture in vitro. Trypan blue staining was performed to count total cell number and ratio of living cells. Methylthiazolyldiphenyl-tetrazolium bromide assay was used to detect the growth curve of nucleus pulposus cells.

RESULTS AND CONCLUSION: Based on the two digestion enzyme concentration, the number of digested cells in the enzyme Ⅰ group was larger than that in the enzyme Ⅱ group after digested for 2 and 4 hours, but the difference was not significant (P > 0.05). Overnight, cell survival rate was decreased in the enzyme Ⅰ group after digested for 2 and 4 hours when compared with the enzyme Ⅱ group, and the difference was significant (P < 0.05). After digested for 4 hours, tissue blocks disappeared, and the number of cells reached maximum. The results indicate that enzyme Ⅰ group composite with Ⅱ collagenase is benefit for the separation of nucleus pulposus cells, and the digestion time is appropriate to 4 hours. This condition has the advantages of simple operation, high efficiency and low cost, and it considered that digestion of nucleus pulposus tissues with 0.2% Ⅱ collagenase for 4 hours is the best condition to obtain nucleus pulposus cells.

Key words: tissue construction, cartilage tissue construction, nucleus pulposus cells, human, Ⅱ collagenase, trypsin, digestion, isolation and culture, methylthiazolyldiphenyl-tetrazolium bromide, National Natural Science Foundation of China

CLC Number:

R318

Zhang Zi-yan, Tong Shen, Yan Hua-dong, Jiang Rui, Wu Han. Optimized method for isolating and culturing human nucleus pulposus cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(28): 5151-5156.

Figures/Tables 3

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