Establishment and significance of separation and culture of human nucleus pulposus cells

LI Dapeng; WU Yan; HUANG Yonghui; JIANG Lu; SUN Jifu; YUE Jiawei

doi:10.3969/j.issn.1002-266X.2017.32.001

Abstract：Objective To establish a new method for isolation and culture of human nucleus pulposus cells (NPCs) in vitro and to explore its significance.Methods Human normal NPCs were isolated by type Ⅱ collagenase digestion, then were cultured and expanded in monolayer.Cell morphology was observed by inverted microscope;the proliferation ability (expressed as absorbance value) of passage 1, passage 2 and passage 3 NPCs were detected by CCK-8 assay for consecutive 12 days;the positive expression rates of type Ⅱ collagen (Col-2), cytokeratin 18 (KRT-18), KRT-19, hypoxia-inducible factor-1α(HIF-1α), glucose transporter 1 (GLUT-1), Sox-9, aggrecan (ACAN), and CD24 in passage 2 NPCs were detected by immunohistochemistry.Results The isolate human NPCs were polygonal or short spindle, similar to chondrocytes.However, the nucleus pulposus cells after passage 4 grew slowly, the protrusion of cells extended, and the cells were long spindle shape, all of these indicated the cells were aging.The growth curve of NPCs was S-shaped, cells grew slowly in 1-2 days, then grew into the logarithmic growth phase in 3-7 days, and after 8 days the cells proliferated slowly and entered the plateau.After consecutive 12-day culture, the difference of absorbance values at 450 nm among passage 1, passage 2 and passage 3 NPCs was not statistically significant at the same time point (all P>0.05).Immunohistochemical staining showed: the positive rates of Col-2, KRT-18, KRT-19, GLUT-1, HIF-1α, Sox-9, ACAN, and CD24 in passage 2 NPCs were no less than 85.6% (85.6%-91.2%).Conclusions Human NPCs can be effectively isolated by 0.2% type Ⅱ collagenase digestion method, and can be cultured by monolayer adherence method.The cells we cultured were identified as nucleus pulposus cells by immunohistochemistry.There are no significant changes in the morphological and proliferation abilities of NPCs within 3 generations, and could be used as seed cells for study of intervertebral disc degeneration.