Culture of human nucleus pulposus cells from degenerated intervertebral discs by explants adherent method after enzymatic pre-digestion

Abstract

Objective To compare the efficiency of explants adherent after enzymatic pre-digestion and traditional typeⅡ?collagenase digestion methods for culturing human nucleus pulposus cells from degenerated intervertebral discs.Methods Human nucleus pulposus tissues collected from patients'degenerated intervertebral discs were divided into Groups A and B.In Group A,the nucleus pulposus tissues were cultured using explants adherent method directly after 0.025 ％ typeⅡ?collagenase digestion for 30 minutes.In Group B,the tissues were firstly digested with 0.025 ％ typeⅡ?collagenase for five hours,and then underwent filtration,centrifugation and inoculation successively Success rate and primary cell fusion time of the two culture methods were compared.Cell morphology and expressions of sex determining region Y-box 9(Sox-9),collagenⅡ?and aggrecan were observed by HE staining,toluidine blue staining and immunofluorescence cell staining.Results There was no significant difference in the successful culture rate between the two groups(P＞0.05).However,the average fusion time of primary passage in Group A was significantly shorter than that in Group B(P＜0.01).In Group A,the nucleus pulposus cells could be stained as azure by toluidine blue and immunofluorescence cell staining showed positive expressions of Sox-9,collagenⅡ?and aggrecan.Conclusions Compared with traditional typeⅡ?collagenase digestion,explants adherent method after typeⅡ?collagenase pre-digestion for the culture of human nucleus pulposus cells from degenerative intervertebral discs has a high success rate and obtains a large number of cells in a short time.Meanwhile,the cells have strong expressions of Sox-9,collagenⅡ?and aggrecan. Key words: Intervertebral disc; Nucleus pulposus cells; Cell culture; Immunofluorescence technique; Type Ⅱ collagenase