There are multiple aspects of the liver in metabolism ranging from glucose formation, bile secretion, protein synthesis, detoxification and formation of urea in our body. The majority of these functions are performed by parenchymal cells of the liver called hepatocytes, these cells have been much used in research mainly to study liver functions and the metabolism of drugs and toxicants in vitro. These various functions of the liver or diseases targeting the organ have been studied using the primary culture of hepatocytes. Examples of studies done using primary cultures include the expression patterns and functioning of cytochromes P450 and other drug-metabolizing enzymes, cytotoxicity and drug-drug interactions. Thus, primary hepatocyte culture is a vital tool to assess liver functions and diseases.
The approach to isolate hepatocytes using two-step collagenase perfusion dates back to 1969 by scientists, Berry, and Friend. This protocol has been subject to several modifications and the 1976 protocol by Seglenin is most followed. This involves dissociation of hepatocytes from anesthetized adult rats using collagenase perfusion through the portal vein. A 100 μm pore size mesh nylon filter is used to filter the cells that are cultured on plates. This is followed by replacing the medium after 4 hours with or without serum-based media for further time.
An article published in the Journal of visualized experiments in 2012 by Shen and team discussed the protocol in detail that allows the isolation of viable hepatocytes in significant numbers. The viability is reported as 88 ~ 96%; while the cell yield is 1.0 x 108 cells per preparation.
Several points suggested by the authors to be kept in mind during the primary hepatocyte culture include:
- The use of strict aseptic techniques is of utmost importance as required for any cell culture; this avoids contamination from bacterial or fungal pathogens.
- The back wall of the portal vein should not be perforated before the perfusion.
- Cell-to-cell adhesion is mediated by a structure called Desmosome, also known as macula adherens. This structure requires calcium, hence, perfusion uses Ca2+ –free medium and EDTA that chelates the metal to break down cell attachments and isolate cells by the action of collagenase in the next step. This is when Ca2+rich medium is used to allow the action of collagenase that is dependent on Ca2+ to allow efficient digestion.
- The preparation of hepatocytes requires correctly done collagenase treatment. The recommended flow rate of Perfusion buffer II plus collagenase is 25 ml/min.
- As the hepatocytes are fragile immediately after isolation, the changing of media after 4 hours should be done carefully. As direct force can damage the cells, do not pipette on top of the cells directly but down the side of the well.
- Points to check if the hepatocyte culture is unsuccessful: 1) poor dissociation of cells if the perfusion speed is low or improper buffer warming. 2) Extra collagenase digestion causes cell death.
Doo-Hoon Lee and Kwang-Woong Lee. Hepatocyte Isolation, Culture, and Its Clinical Applications. Hanyang Medical Reviews, 2014;34:165-172. Shen, L., Hillebrand, A., Wang, D. Q., & Liu, M. (2012). Isolation and primary culture of rat hepatic cells. Journal of visualized experiments: JoVE, (64), 3917. doi:10.3791/3917.