Hepatocytes are essential for liver function and critical in drug testing and biological research. But their unique traits—irregular shapes, multiple nuclei, and clumping—make accurate automated and manual hepatocyte counting challenging.
Yet, accurate counts are key, especially in fields like preclinical drug development, where fairly minor errors can derail research and inflate costs.
This article explains the challenges of hepatocyte counting, provides practical tips for preparing high-quality hepatocyte samples, and showcases the latest machine learning-based automated hepatocyte counting tools.
What Are Hepatocytes?
Hepatocytes are the predominant cell type in the liver and constitute approximately 80% of the liver’s mass.
They are responsible for many of the liver’s essential processes, such as detoxification, metabolism, and secretion of albumin and clotting factors. One of the many responsibilities of hepatocytes includes drug metabolism. [1]
Primary hepatocytes retain their metabolic behavior and function following extraction from liver tissue and are considered the gold standard for in vitro liver models.
As such, they are used extensively in preclinical drug safety assessments. However, hepatocytes are an unusual cell type due to their large size, irregular shape, and complex internal structure.
Why Accurate Hepatocyte Counts Matter
Many assays require a precise quantification of cell numbers for cell seeding purposes and to allow the normalization of data between different assays.
An accurate hepatocyte count is imperative because of their various important biological applications, including:
- Drug testing and development: Accurate hepatocyte counts ensure the correct dosage in drug testing, affecting the efficacy and toxicity measures. [2]
- Biological research: Studying liver diseases, liver regeneration, and drug metabolism research necessitates precise cell counts for reliable results. [3]
- Clinical diagnostics: Hepatocyte counts are used for liver functions and understanding diseases like hepatitis and cirrhosis.
Inaccuracies in hepatocyte counting compromise downstream analysis and lead to erroneous conclusions with potentially costly ramifications in drug development and clinical treatments.
How to Prepare High-quality Hepatocyte Suspensions for Your Counts
Preparing a high-quality sample of hepatocytes is the critical first step for producing reliable, accurate, and reproducible data, including the final count.
An optimized extraction protocol will increase cell viability and yield, maintain cell function, minimize contamination, and reduce the need for troubleshooting and repeating experiments.
To ensure your final data is valid, always aim for consistency in your counting technique, such as a cell counter specifically validated on hepatocytes.
Investing time and effort in optimizing your preparation process will help you get accurate counts, so below are some practical tips for optimizing hepatocyte extraction via a perfusion protocol.
Use Fresh Tissue or Freshly Thawed Cryo Samples and Optimize Perfusion Conditions
Freshly isolated liver tissue or freshly thawed cryo-frozen hepatocytes will have higher cell viability. [4] This is important because your hepatocytes will start to degrade once you have isolated them from their native tissue.
If doing an isolation, adjust the perfusion buffer flow rate and temperature. A gentle flow rate and keeping the tissue at an optimal temperature (usually around 37 °C) will improve cell viability.
Minimize Handling Time
Working efficiently will reduce the time the hepatocytes are exposed to unfavorable conditions. If using a cryopreserved hepatocyte sample, thaw the samples so the vial has only just defrosted, and some ice is still present.
Maintain a Sterile Setup
Contamination may affect cell viability and introduce additional challenges and variability in later steps. So, keep all your buffers and equipment sterile.
Optimize Your Centrifugation Steps
High centrifugation speeds may reduce cell viability by damaging them. Consider testing whether certain speeds are better for your sample by measuring cell viability over a range of centrifugation speeds.
Take Advantage of Dedicated Kits
Hepatocyte extraction kits such as the rat and mouse Liver Perfusion Kit available from Miltenyi will help streamline the extraction process.
The pre-formulated reagents are already optimized to achieve a maximum cell yield and viability, and the standardization of the materials provided has the added benefit of increasing consistency and reproducibility between each extraction procedure.
Use an Automated Perfuser
Automated perfusers, such as the gentleMACS Perfuser, are particularly advantageous for high-quality, viable, and reproducible cell preparations.
They provide precise control of overflow rate and pressure, helping to minimize cell damage.
Perfusers also reduce the handling time of the hepatocytes. This in turn reduces the likelihood of error and contamination and enables scalability for high-throughput studies.
Which Cell Counting Method Should You Use?
After preparing your hepatocytes, the next step is to count and assess their quality. Here’s a summary of common cell counting methods and their key features:
Hemocytometer
A manual, cost-effective tool requiring a microscope and basic lab skills, where cells are counted on a grid etched into a glass slide. This method is prone to human error and variability, making it less reliable for complex samples like hepatocytes.
Viability Stains (e.g., Trypan Blue)
Viability stains like trypan blue differentiate live cells from dead ones based on membrane integrity; dead cells absorb the stain and appear blue under a microscope, making this method useful for basic viability assessments but limited in specificity. [5]
Nuclei Extraction
Nuclei extraction is ideal for samples where intact cells are difficult to count, as stains like DAPI highlight nuclei for counting under a microscope. However, it is less accurate for multinucleated cells, such as hepatocytes.
Fluorescence-based Cell Counting
Fluorescence-based methods use dyes or proteins for high sensitivity and specificity, allowing for differentiation between live and dead cells or high-throughput counting with flow cytometry. These methods require specialized equipment and expertise, but they deliver fast, consistent results and are amenable to manual and automated counting.
The Benefits of Automated Cell Counting
Manual cell counting is often labor-intensive and prone to user variability, making your results unreliable.
Even the more accurate cell counting methods such as fluorescent labeling require additional experimental steps.
If you want accurate counts with as few steps as possible, automation is the way forward.
Early automated cell counters, primarily developed for tissue culture applications, employed simplistic algorithms tailored to detect bright objects against a dark background.
While traditional automated cell counters are effective at accurately counting some cell types, they can struggle when the cell morphology is irregular, and the cells clump.
Hepatocytes are classic examples of a cell that is challenging to count with conventional methods (Figure 1) due to the following reasons:
- They are irregularly shaped
- They contain multiple nuclei
- High levels of intracellular vitamin A, which causes autofluorescence
- The cells tend to clump
Better automation of this fundamental laboratory technique addresses several needs:
- Standardization of cell counts: Automated cell counting helps to standardize cell counts between users within a lab and across different labs, ensuring consistent and reliable results.
- Increased accuracy and precision: Automated cell counters can use specific algorithms developed using machine learning to achieve the optimal cell count for specific cell types. This can greatly improve the accuracy for challenging cell types like hepatocytes, where conventional software and techniques cannot reliably achieve > 95 % counting accuracy.
- Data management: Automated workflows typically facilitate consistent, comprehensive documentation as counting data is logged by the cell counter.
- Regulatory compliance: Automating techniques such as cell counting enables laboratories to meet the requirements for Good Manufacturing Practices (GMP) and Good Laboratory Practices (GLP).
- Automatic calculations: Cell concentration and dilution calculations are automated, which helps streamline workflows.
The CellDrop™ FLi Automated Cell Counter and Hepatocyte App from DeNovix addresses the demands of counting hepatocytes accurately while offering all the benefits listed above.
For the Most Accurate Counts in the Shortest Time—Use the CellDrop™ FLi Automated Cell Counter
The CellDrop FLi uses a hepatocyte-specific machine learning algorithm created by experts within the DeNovix Team. Trained on an extensive hepatocyte image dataset from a range of species, extraction methods, and sample types, the application is the first of its kind to provide fast, accurate, automated hepatocyte counts.
Figure 2 below demonstrates the counting accuracy of the DeNovix machine learning cell count algorithm.
If you routinely require consistent, accurate hepatocyte counts, you should choose the CellDrop FLi. Here’s why:
Get Your Counts Faster
The CellDrop FLi will count your hepatocytes within 30–40 seconds, faster than any other counting method!
During this time, it will also automatically perform:
- Size gating
- Autofluorescence thresholding
- Debris detection and quantification
- Lymphocyte counts
- Free nuclei counts
The algorithm provides rich information about your sample that other methods cannot offer while removing user subjectivity.
It also comes with other powerful features designed to simplify your experiments.
The autofocus feature automatically gets your sample into sharp focus and provides focal plane consistency between experiments.
Its powerful thresholding technology automatically distinguishes live cells from dead ones.
Figure 3 below shows the objects in your samples that are automatically categorized and quantified.
Live hepatocytes are shaded green, lymphocytes are shaded blue, nuclei are shaded orange, dead hepatocytes are shaded red, and cell debris is unstained and appears gray.
Eliminate Plastic Waste with DirectPipette™ Technology
Load your sample into the chamber without using any slides and cut down the plastic waste your lab produces.
The CellDrop’s unique DirectPipette™ technology takes advantage of surface tension and capillary action to draw samples into a fixed chamber, ensuring precise measurements and eliminating carryover between samples.
The sample chamber volume is adjusted using a precision screw mechanism, ensuring you achieve accurate hepatocyte counts from sample volumes as low as 2.5 µL.
Fluorescence-based Methods for Sensitive Sample Detection
The CellDrop FLi uses AO/PI, a combination of two fluorescent dyes: acridine orange (AO) and propidium iodide (PI).
Preparing your sample is as easy as mixing your hepatocyte suspension with AO/PI at a 1:1 ratio.
AO is a live cell fluorescent stain and will pass through the cell membrane of both live and dead cells, staining the nucleic acid green.
In contrast, PI will only pass through damaged cell membranes and will stain the nucleic acid of those cells red.
The PI also acts as a quencher of AO, so live cells will be stained green, and dead cells will be stained red.
The CellDrop FLi automatically distinguishes live cells (stained green) from dead cells (stained red).
Important Calculations and Sample Parameters Are Calculated for You
As shown in Figure 4 below, along with your hepatocyte count, the CellDrop FLi will also display:
- Live hepatocytes per mL
- Dead hepatocytes per mL
- Percentage hepatocyte viability
- Percentage of live hepatocytes
- The number of lymphocytes, free nuclei, and debris per mL and in total
Plus, diluting your sample down to the desired number of hepatocytes per mL is as easy as entering your target value. The CellDrop FLi will use the calculated hepatocyte concentration in your sample and display the corresponding dilution factor you need to use.
Versatile Data Management and Compliance Ready Software
The CellDrop FLi is equipped with a 7-inch HD touchscreen and a powerful processor, so it doesn’t require a separate PC to operate. It also features Wi-Fi, ethernet, and USB connectivity.
For every measurement you make, your data is documented and saved to the 128 GB SSD, simplifying your data management and ensuring your experimental results are always traceable.
Thanks to the inclusion of 21 CFR Part 11 Compliance Ready Software, you can easily prepare GxP compliance-ready results.
Plus, you can export these results in whatever format suits your workflow, whether email, network folders, or USB sticks.
Click here to learn more about the benefits of the CellDrop FLi.
Automated Hepatocyte Counting Summarized
The CellDrop FLi is setting new standards for cell counting, significantly improving the accuracy of your counts and the time taken to get them.
Whilst traditional counting methods like hemocytometry can work, they have major drawbacks when you are trying to count complex cell types like hepatocytes.
If your lab work involves routinely counting hepatocytes, consider making the switch to automation today to save time and consumable waste, and bring a new level of standardization to your research.
To see a demonstration of how easy hepatocyte counting with the CellDrop FLi is and for more information on automation in cell counting, watch our webinar with Application Scientist Dan Schieffer.
FAQs
Q. Do I need to train the machine learning software on my own samples?
A. No.
During the development of the software, machine learning-based optimization was used extensively to create an app that can accurately count hepatocytes automatically.
Now the software is fully developed and validated, no further machine learning is required and no machine learning occurs during end-user hepatocyte counts.
In other words, the software is finished, and there is no need to train it on your samples.
Q. Is the software affected by the species my hepatocytes are from or the extraction method I use?
A. No.
Many species were tested during software development, including human, mouse, and rat tissue, and other common species from which hepatocytes are extracted.
Similarly, different extraction methods were also tested, including thawed cryo-preserved hepatocytes, and the DeNovix scientists recreated excellent and poor sample preparations to ensure the software could handle a wide spectrum of sample quality.
This extensive testing means the software is fit for purpose in real-life labs and unaffected by the species used.
Q. What are the minimum and maximum cell concentrations the CellDrop FLi can measure?
A. With DirectPipette™ technology, the top of the sample chamber is attached to a motorized screw and can be adjusted to three different sample chamber heights—50 µm, 100 µm (standard), and 400 µm. This allows the sample chamber to accommodate 5 µL, 10 µL, and 40 µL of sample respectively.
These chamber height options enable a wide dynamic range, allowing the CellDrop FLi to measure samples with cell densities of 7 x 10^2 – 2.5 x 10^7 cells per mL.
References
- Gong J, Tu W, Liu J, Tian D. (2023). Hepatocytes: A key role in liver inflammation. Front Immunol. 13:1083780
- Soars MG, McGinnity D, Grime K, Riley. (2007). The pivotal role of hepatocytes in drug discovery. Chemico-Biological Interactions. 168:2–15
- Hora S, Wuestefeld T. (2023). Liver Injury and Regeneration: Current Understanding, New Approaches, and Future Perspectives. Cells. 12(17):212
- Terry C, Hughes R. (2009) An optimised method for cryopreservation of human hepatocytes. Methods Mol Biol. 481:25-34.
- Strober W. (2015). Trypan Blue Exclusion Test of Cell Viability. Curr Protoc Immunol. 111:A3.B.1–A3.B.
