Overview of Hepatocytes
Hepatocytes are the predominant cell in the liver, comprising about 60% of liver cells and 80% of liver mass. One of the main hepatocyte functions is the production of bile. Hepatocytes are also responsible for the regulation of metabolism, detoxification, and the manufacture of important circulating proteins, making them a critical component of studies involving liver disease, drug metabolism, and toxicology.
Primary hepatocytes are very difficult to maintain in culture, displaying a rapid decline in viability and liver-specific functions. Optimization of culture conditions, including extracellular matrices (ECM), culture media / supplements, and co-culture with other cell types required for the normal cell-cell interactions displayed in vivo (micro-patterned co-cultures, MPCC) have enabled researchers to maintain hepatocyte viability and function for several weeks in culture. Recent studies have also proven cryopreservation (for up to 4 years) as a viable method of maintaining access to viable, functional primary hepatocytes.
Ploss, A., et al. (2010). Persistent Hepatitis C Virus Infection in Microscale Primary Human Hepatocyte Cultures, PNAS. 107(7), 3141-3145. Doi: 10.1073/pnas.0915130107.
Malarkey, D. et al. (2005). New Insight into Functional Aspects of Liver Morphology, Toxicologic Pathology.33:27-34. Doi: 10.1080/01926230590881826.
Malhi, H. et al. (2010). Hepatocyte Death: A Clear and Present Danger, Physiol. Rev. 90:1165-1194. Doi: 10.1152/physrev.00061.2009.
Lu, J.-N., et al. (2011). The Behaviors of Long-term Cryopreserved Human Hepatocytes on Different Biomaterials, Artificial Organs. 35:E65-E72. Doi: 10.1111/j.1525-1594.2010.01191.x.
Importance of Accurate Hepatocyte Counts
Primary hepatocytes are regularly used to measure toxicity of drug candidates during the drug screening process. Most cytotoxicity assays are plate-based assays involving the plating of hepatocytes in each well. Precise and accurate cell concentration and viability measurement are crucial to obtaining reproducible, high quality data.
- Multi-well plates are used to screen drug candidates at varying concentrations with repeated measurements, generating a large number of data points for analysis and comparison
- Multi-well plates need to be seeded with identical numbers of cells with known viability for correct interpretation of data
Hepatocyte Challenges & Cellometer Solutions
Due to hepatocytes' variable morphology, fragile nature, and tendency to clump, traditional manual counting methods can be time-consuming and inaccurate. Because hepatocytes lose viability over time, extended or variable counting times can generate inaccurate and inconsistent viability determinations. Hepatocytes are too fragile to evaluate using flow cytometry due to flow-induced shear stress.
Cellometer image cytometry is an ideal method for determination of hepatocyte concentration and viability, overcoming all of the difficult hepatocyte characteristics:
|Fragile||Non-fluidic Imaging System|
|Irregular in Shape||Specialized algorithm for counting of irregular (non-round) shapes|
|Variable in Size||User-adjustable size range for counting|
|Form Clusters or Clumps||Proprietary Cellometer Software identifies and counts individual cells within clusters|
|Lose Viability Over Time||Analysis in < 60 seconds|
Cellometer Hepatocyte Viability Method
Due to hepatocytes' variable morphology, fragile nature, and tendency to clump, traditional manual counting methods can be time-consuming and inaccurate. Because hepatocytes lose viability over time, extended or variable counting times can generate inaccurate and inconsistent viability determinations. Hepatocytes are also too fragile to evaluate using flow cytometry due to flow-induced shear stress. Cellometer image cytometry is the most reliable method for determination of hepatocyte viability.
Dual-fluorescence Staining Procedure
For viability determination, 20µl of hepatocyte sample is mixed with 20µl of Cellometer AO/PI Staining Solution. The acridine orange (AO) dye stains DNA in all nucleated cells, generating green fluorescence and easily differentiating hepatocytes from debris. Propidium iodide (PI) stains DNA in all cells with compromised cell membranes, generating red fluorescence. In cells stained with both AO and PI, the green fluorescence is absorbed by the red fluorescence via FRET (fluorescence resonance energy transfer), so all dead hepatocytes fluoresce red and can be easily counted. The procedure is fast, gentle, and accurate.
Immediately after mixing, 20µl of stained sample is loaded into the Cellometer Counting Chamber and inserted into the Cellometer Vision instrument. The sample is imaged directly from the counting chamber. Because the counting chamber is disposable, no washing is required between samples and there is no risk of cross-contamination. Samples are imaged and analyzed using pre-set parameters for primary hepatocytes.
Automated Viability Results
The Cellometer Vision software reports:
- Cell images and data can be instantly saved to a secure network or printed directly from the Vision software.
- Images and tables can be exported for further analysis, presentation, or publication.
- Automated analysis and reporting eliminates inter-operator variability and potential user errors in counting or recording.
Hepatocyte Species Analyzed
- Rainbow Trout