Separation and culture of high-purity primary rat hepatocyte
- VernacularTitle:高纯度原代大鼠肝细胞的分离与培养
- Author:
Xinying WANG
;
Weiqin LI
;
Ning LI
;
Jieshou LI
- Publication Type:Journal Article
- Keywords:
Hepatocyte;
Separation;
Culture;
Rat
- From:
Journal of Medical Postgraduates
2003;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
Objectives: To establish a method of separation and culture of high-purity primary rat hepatocyte. Methods: The perfusion solutions used in the first and the second step were D-Hank's solution containing EGTA, and the digestive solution contained DNase I and collagenase respectively. Purified hepatocytes were separated from the dissociative cells by low-speed centrifugation (50 g, 5 min) 3 times. The survival rate was measured by typan blue exclusion and the purity was measured by routine hematoxylin and-eosin cytochemistry. Separated hepatocytes were cultured in RPMI 1640 culture medium with some special nutrient elements. Throughout the in vitro culture, we kept continuous observation of the shape changes of the hepatocyte and measured the survival rate within the first 5 days. Results:(1.0～1.5)?10 8 cells were prepared from a 180-200 g rat on average, the survival rate was higher than 50% and the hepatocyte purity was higher than 98%.Microscopically, the hepatocytes were normal in shape and viability. Conclusions: The modified in situ perfusion digestion method and the culture in RPMI 1640 and special nutrients are advantageous to get high viability and purity hepatocyte with normal shape.
