Purpose:
Primary hepatocytes provide scientists with a valuable tool for evaluating metabolic, biochemical, and molecular functions in a physiologically relevant, readily controlled in vitro experimental system. However, as is the case for all primary cells, there are unique considerations that must be taken into account to minimize batch-to-batch variability and ensure the quality, reliability, and reproducibility of data. The following protocol outlines the materials and methods for the (relatively) time- and cost-effective isolation and 2D-culture of high-quality, responsive, and functionally-consistent primary mouse hepatocytes. These techniques were developed using the C57 BL/6 strain of mice(6-20 weeks of age; 20-40g total body weight), have been scaled successfully in rats, and may, with minimal adjustments, be applicable to related species.
Background:
Historically, primary hepatocytes have been utilized for the study of drug metabolism, xenobiotic transformation, metabolite processing, hormonal function, and numerous other purposes. When properly isolated, hepatocytes strongly adhere to a variety of surfaces, including standard vacuum-plasma-treated polypropylene cell culture dishes, gelatin, collagen, and various ECM-based coatings, and remain functionally viable for at least 48 hours post-plating. The majority of hepatocyte-based studies are completed within this time frame, and while many authors have published data using > 3 day-old cells, longer-term (> 2 days) preservation of hepatocyte morphology and function appears to have more to do with the culture and/or substrate conditions, and less on the actual isolation procedure.
Our experience is limited to the isolation and short-term culture of hepatocytes, and towards this end, our goal is to simply provide the information needed for the isolation of healthy, fully functional cells. Instructions for the performance of select functional assays will be added in the near future, as these will serve as benchmarks to assess the quality and consistency of isolated hepatocytes.
We have successfully and consistently performed a number of assays in isolated hepatocytes, including:
| Readout | Tested agonists* | Tested antagonists* | Tested metabolic substrates* |
|---|---|---|---|
| ATP (intracellular) | Rotenone | - | Glucose |
| Beta-oxidation | Carnitine | Etomoxir | Palmitate (3-H) |
| EROD | PCBs, BNF | Multiple | - |
| Glucose output | Glucagon, forskolin | Insulin, AICAR, Metformin | Lactate, pyruvate, glycerol |
| Glycogen synthesis | Insulin | Glucagon, forskolin | Glucose, lactate, pyruvate, dihydroxyacetone, glycerol |
| Glycogenolysis | Glucagon, forskolin | Insulin | - |
| Immunoblotting | Insulin, Adenoviruses, Lipofectamine | Kinase inhibitors | Free fatty acids, glucose |
| Lactate output | - | - | Glucose |
| LDH release | Rotenone, hypotonic lysis | - | - |
| Lipogenesis | Insulin | - | Glucose (14-C) |
| Oil Red O staining | - | - | BSA-coupled palmitate, oleate, stearate, and linoleate |
| qRT-PCR | Multiple | Multiple | Multiple |
*All tested agonists/antagonists/substrates listed are those which we have observed to perform in a manner consistent with the known mechanism of action as well as the published literature.