Trypsin plus type II collagenase digestion for isolation of nucleus pulposus cells:the optimal glucose concentration in complete medium / 中国组织工程研究

ABSTRACT

BACKGROUND:Intervertebral disc degeneration is the pathological basis of degenerative spinal diseases. Studies on the influentialfactors of intervertebral disc degeneration contribute to the prevention and treatment of degenerative spinal disease. OBJECTIVE:To observe the growth and proliferation of nucleus pulposus cels isolated by trypsin plus type II colagenase digestion in complete medium with different glucose concentrations, exploring the optimal glucose concentration for growth of nucleus pulposus cels. METHODS:Nucleus pulposus cels isolated and cultured by trypsin plus type II colagenase digestion method were observed under an inverted microscope, and thecelnumber was counted. Morphology of nucleus pulposus cels was observed afterhematoxylin-eosinstaining and toluidine blue staining. Colagen type II immunoreactivity was detected by immunohistochemical staining combined with immunofluorescent staining.Nucleus pulposuscels were incubated in complete medium containing various glucose concentrations (0, 6.25, 12.5, 17.5, and 25 mmol/L) for 24 hours, and then cel proliferation and apoptosis were determined. RESULTS AND CONCLUSION:The stained nucleus pulposus cels showed polygonal and short spindle, with one or two nuclei. Celularpseudopod appeared gradualy and then became slim with increased passage numbers. The isolated and cultured nucleus pulposus cels positively expressed colagen type II and aggrecanProliferative activity of nucleus pulposus cels cultured in medium with 17.5 mmol/L glucose was significantly higher than that in medium with 0 and 25 mmol/L glucose (P< 0.05 orP< 0.01). There wasno significant differencein cel apoptosis between these groups except for 0 mmol/L glucose (P<0.05). These results confirm that a large number of nucleus pulposus celscan beharvested by trypsin plustype II colagenase digestion and the optimal glucose concentration is 17.5 mmol/L.