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Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro
Published on: December 20, 2019
Primary hepatocyte culture in collagen gel mixture and collagen sandwich
Ying-Jie Wang1, Hong-Ling Liu, Hai-Tao Guo
1Institute of Infectious Diseases, Southwest Hospital, Third Military Medical University, Chongqing 400038, China. wangyj103@263.net
Aim:
To explore the methods of hepatocytes culture in a collagen gel mixture or between double layers of collagen sandwich configuration and to examine the functional and cytomorphological characteristics of cultured hepatocytes.
Methods:
A two-step collagenase perfusion technique was used to isolate the hepatocytes from Wistar rats or newborn Chinese experimental piglets. The isolated hepatocytes were cultured in a collagen gel mixture or between double layers of collagen sandwich configuration respectively. The former was that rat hepatocytes were mixed with type I rat tail collagen solution till gelled, and the medium was added onto the gel. The latter was that swine hepatocytes were seeded on a plate precoated with collagen gel for 24 h, then another layer of collagen gel was overlaid, resulting in a sandwich configuration. The cytomorphological characteristics, albumin secretion, and LDH-release of the hepatocytes cultured in these two models were examined.
Results:
Freshly isolated rat hepatocytes were successfully mixed and fixed in collagen gel, and cultured in the gel condition. During the culture period, the urea synthesized and secreted by rat hepatocytes was detected throughout the period. Likewise, newborn experimental piglet hepatocytes were successfully fixed between the double layers of collagen gel, forming a sandwich configuration. Within a week of culture, the albumin secreted by swine hepatocytes was detected by SDS/PAGE analysis. The typical cytomorphological characteristics of the hepatocytes cultured by the above two culture models were found under a phase-contrast microscope. There was little LDH-release during the culture period.
Conclusion:
Both collagen gel mixture and double layers of collagen sandwich configuration can provide cultural conditions much closer to in vivo environment, and are helpful for maintaining specific hepatic functions and cytomorphological characteristics. A collagen gel mixture culture may be more eligible for the study of bioartificial livers.
