Isolation and culture of rabbit nucleus pulposus cells in vitro by type Ⅱ collagenase digestion methods plus explant culture method

doi:10.3969/j.issn.2095-4344.2013.39.001

Abstract

Abstract:

BACKGROUND: Intervertebral disc can bear load but lack vessels. Nucleus pulposus cells have the problem of phenotype loss during in vitro culture that can lead to degenerative changes. The mechanism of intervertebral disc degeneration remains unclear.
OBJECTIVE: To explore the approaches of isolation, adherence culture, amplification and identification of the rabbit nucleus pulposus cells in vitro, to observe the growth characteristics of nucleus pulposus cells in different generations.
METHODS: Type Ⅱ collagenase digestion method plus explants culture method was used to isolate and purify nucleus pulposus cells and then amplify in vitro. The morphology and growth of primary and passaged cells was observed under the inverted microscope, the number of cells was counted and the growth curve was draw. The morphology of the cells was observed under light microscope after hematoxylin-eosin staining, and the expressions of collagen type Ⅱ and aggrecan were examined by immunocytochemistry.
RESULTS AND CONCLUSION: Nucleus pulposus cells of rabbit were isolated, cultured and amplified in vitro successfully. Growth activity was observed, and found that the 1-3 generation nucleus pulposus cells proliferated more rapidly and vigorously. The proliferation of nucleus pulposus cells was decreased while the cell passaged more generations. These isolated and cultured nucleus pulposus cells could positively express the collagen type Ⅱ and aggrecan. In vitro combination of type Ⅱ collagenase digestion method and explants culture method could obtain high purity nucleus pulposus cells, and the cultured nucleus pulposus cells were grew in round or polygonal. The 1-3 generation of cells had strong activity.

Key words: collagen type Ⅱ, microbial collagenase, cells, cultured, proteoglycans

CLC Number:

R318

Li Shu-wen, Wu Hai-jun, Yin He-ping, Bai Ming, Du Zhi-cai. Isolation and culture of rabbit nucleus pulposus cells in vitro by type Ⅱ collagenase digestion methods plus explant culture method[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(39): 6861-6866.

Figures/Tables 6

References

[1]Alini M, Li W, Markovic P, et al. The potential and limitations of a cell-seeded collagen/hyaluronan scaffold to engineer an intervertebral disc-like matrix. Spine(Phila Pa 1976).2003; 28(5):446-454.
[2]Urban JP, Roberts S. Degeneration of the intervertebral disc. Arthritis Res Ther. 2003,5(3):120-130.
[3]An HS, Anderson PA, Haughton VM, et al. Introduction: disc degeneration: summary. Spine (Phila Pa 1976).2004;29(23): 2677-2678.
[4]Hiyama A, Mochida J, Iwashina T, et al. Transplantation of mesenchymal stem cells in a canine disc degeneration model. J Orthop Res. 2008;26(5):589-600.
[5]Watanabe T, Sakai D, Yamamoto Y, et al. Human nucleus pulposus cells significantly enhanced biological properties in a coculture system with direct cell-to-cell contact with autologous mesenchymal stem cells. J Orthop Res.2010; 28(5):623-630.
[6]Comes ME, Bossano CM, Johnston CM, et al. In vitro localization of bone growth factors in constructs of biodegradable scaffolds seeded with marrow stromal cells and cultured in a flow perfusion bioreactod. Tissue Eng. 2006; 12(1):177-188.
[7]Smith LJ,Chiaro JA,Nerurkar NL,et al．Nucleus pulposus cells synthesize a functional extracellular matrix and respond to inflammatory cytokine challenge following long-term agarose culture．Eur Cell Mater.2011;22:291-301.
[8]Roberts S,Evans H,Trivedi J,et at．Histology and pathology of the human intervertebral disc. J Bone Joint Surg Am.2006; 88(S2):10-14.
[9]Wang H,Liu H,Zheng ZM,et al．Role of death receptor,mitochondrial and endoplasmic reticutum pathways in different stages of degenerative human lumbar disc. Apoptosis. 2011;16(10): 990-1003.
[10]Zhao CQ,Wang LM,Jiang LS. et al．The cell biology of intervertebral disc aging and degeneration.Ageing Res Rev. 2007; 6(3):247-261.
[11]Yang SH, Wu CC, Shih TT, et al. Three-dimensional culture of human nucleus pulposus cells in fibrin clot: comparisons on cellular proliferation and matrix synthesis with cells in alginate. Artif Organs. 2008;32(1): 70-73.
[12]王锋,吴小涛,王运涛,等.单纯Ⅱ型胶原酶消化法分离、培养人退变椎间盘髓核细胞的形态学观察[J].中国脊柱脊髓杂志,2010, 20(4):300-304.
[13]孙浩林,李淳德.原代培养差速贴壁法分离纯化大鼠腰椎间盘髓核细胞[J]. 中国组织工程研,2011,15(46):8569-8573.
[14]Wang H,Liu H,Zheng ZM,et al.Role of death receptor, mitochondrial and endoplasmic reticutum pathways in different stages of degenerative human lumbar disc. Apoptosis. 2011;16(10):990-1003.
[15]Yang Z, Huang CY, Candiotti KA, et al. Sox-9 facilitates differentiation of adipose tissue-derived stem cells into a chondrocyte-like phenotype in vitro.J Orthop Res. 2011;29(8): 1291-1297.
[16]Hu MH, Hung LW, Yang SH, et al. Lovastatin promotes redifferentiation of human nucleus pulposu s cells during expansion in monolayer culture. Artif Organs.2011;35(4): 411-416.
[17]Kluba T, Niemeyer T, Gaissmaier C, et al. Human anulus fibrosis and nucleus pulposus cells of the intervertebral disc: effect of degeneration and culture system on cell phenotype. Spine (Phila Pa 1976). 2005; 30(24): 2743-2748.