Isolation and culture of rabbit nucleus pulposus cells in vitro by type Ⅱ collagenase digestion methods plus explant culture method
10.3969/j.issn.2095-4344.2013.39.001
- VernacularTitle:Ⅱ型胶原酶消化结合组织块贴壁法分离培养兔髓核细胞
- Author:
Shuwen LI
;
Haijun WU
;
Heping YIN
;
Ming BAI
;
Zhicai DU
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2013;(39):6861-6866
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND:Intervertebral disc can bear load but lack vessels. Nucleus pulposus cel s 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 cel s in vitro, to observe the growth characteristics of nucleus pulposus cel s in different generations.
METHODS:Type Ⅱ col agenase digestion method plus explants culture method was used to isolate and purify nucleus pulposus cel s and then amplify in vitro. The morphology and growth of primary and passaged cel s was observed under the inverted microscope, the number of cel s was counted and the growth curve was draw. The morphology of the cel s was observed under light microscope after hematoxylin-eosin staining, and the expressions of col agen type Ⅱ and aggrecan were examined by immunocytochemistry.
RESULTS AND CONCLUSION:Nucleus pulposus cel s of rabbit were isolated, cultured and amplified in vitro successful y. Growth activity was observed, and found that the 1-3 generation nucleus pulposus cel s proliferated more rapidly and vigorously. The proliferation of nucleus pulposus cel s was decreased while the cel passaged more generations. These isolated and cultured nucleus pulposus cel s could positively express the col agen type Ⅱ and aggrecan. In vitro combination of type Ⅱ col agenase digestion method and explants culture method could obtain high purity nucleus pulposus cel s, and the cultured nucleus pulposus cel s were grew in round or polygonal. The 1-3 generation of cel s had strong activity.
