Excellence of differential velocity adherent procedure in primary culture of nucleus pulposus cells of intervertebral disc / 中国组织工程研究

ABSTRACT

BACKGROUND:As the main function cel of intervertebral disc, nucleus pulposus cel s are the focus of studying the degenerative mechanism;thereby, it is crucial to maintaining the physiological function of nucleus pulposus cel s in vitro and the stability of the cel phenotype. OBJECTIVE:To study the excel ence of differential velocity adherent procedure in primary culture of nucleus pulposus cel s of rat intervertebral disc through comparison. METHODS:Twenty male Wistar rats aged 4 weeks were enrol ed, and then nucleus pulposus cel s of intervertebral disc were isolated and cultured in vitro;cel passage culture was performed in different groups when the primary cel s were merged to 90%. Differential velocity adherent group cel s adhered for 30 minutes, and non-adherent cel s were aspirated and transferred to new culture dish after readjusting the concentration;the controls received no intervention. Passages 1 and 2 cel s in the differential velocity adherent group were isolated and purified by the same procedure. The morphology of three generations of cel s in the two groups was compared, the purity of the identification was detected by immunohistochemistry, the cel viability was detected by cel counting kit-8 and the cel growth curve was drawn. RESULTS AND CONCLUSION:Inverted phase contrast microscope and hematoxylin-eosin staining revealed that the cel homogeneity of the differential velocity adherent group was significantly higher than that of the control group, and there were more kinds of fibroblast-like cel s in nucleus pulposus cel s in the control group. Identification and purity analysis of col agen type II showed that the cytoplasm of two groups were both stained brown, indicating that they were the nucleus pulposus chrondrocytes. The positive rate of differential velocity adherent group was significantly higher than that of the control group (P<0.05). The cel growth curve of cel counting kit-8 showed cel s in the two groups al passed by the latency phase within 2 days, then to the logarithmic phase of 3 days, and entered the lag phase, while the growth rate of the control group was more rapid during the latency and the early logarithmic phases. These findings suggest that differential velocity adherence method is a practical and effective procedure for the isolation and purification of primary cultured rat nucleus pulposus cel s. Through the primary culture, twice differential velocity adherence procedure, the passage 3 rat nucleus pulposus cel s are metabolic exuberant, consistent with the phenotype, the cel purity is higher, and the logarithmic growth phase can be used as the optimal time for studying the mechanism of intervertebral disc cel s.