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Cell counters are everywhere – from the brewery to the biology lab.

Yes, that’s right. Cell counters are an essential tool when producing a wide range of products (and when doing the scientific research on how to make them).

In the pharmaceutical industry, cell counters are needed to manufacture cell therapies, vaccines and biologics.

Breweries need cell counters to determine the right amount of yeast to pitch before brewing the perfect batch of beer.

Cell counters are used by dairy farmers and animal breeders to optimize milk and meat quality.

In all these processes, it is important to work with a precise number of cells at one or more stages of production. This is where an accurate and dependable cell counter becomes a staple in an efficient workflow.

There are a plethora of cell counters on the market and they all work in different ways. In this post, we will provide an overview of how cell counters work, the different kinds on the market and the pros and cons of each.

Grab your favorite cup of bubble tea and get ready to learn something new!

[table of contents]

• What Is a Cell Counter?
• Types of Cell Counter?
• How Do Cell Counters Count Cells?
• What Is a Cell Counter?
• What Are Cell Counters Used For?

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What Is a Cell Counter?

If you are reading this post, you are most likely familiar with the hemocytometer, the most common method to count cells.

You also likely understand that this manual process requires meticulous pipetting, great eyesight, and dexterous thumbs.

We wouldn’t call the hemocytometer a cell counter, but rather, a tool that is used to count cells.

Cell counters are instruments that enumerate the number, concentration and viability of cells in a cell sample. These machines are often referred to as “automated cell counters.” Cell types that are most counted include blood, mammalian, insect, and yeast.

Cell counters not only count cells, but also perform the following:

• Calculate cell viability
• Present data visually
• Store data securely
• Allow for data export

The information provided by the modern benchtop cell counter is used for production, manufacturing and quality control related to bioprocesses.

How Do Cell Counters Count Cells?

Although performing a cell count may seem relatively straightforward, cell counters perform quite a complex task. Cell counters must be able to identify cells, distinguish dead cells from live cells and exclude non-cell particles from the count.

Advancements in computing power, optics and machine learning are allowing all of this to be done automatically.

Cell Counter Tech

There are three primary principles by which cell counters today operate (see Table 1). They include:

• Imaging
• Laser light scatter
• Electrical impedance

Table 1. Summary of automated cell counters and their principles

Type of cell counter	Principle
Image cytometer	Imaging. Digital images are taken of the cell sample, located within a small chamber inside of a slide or cassette. Algorithms process the images and identify cells.
Flow cytometer	Laser light scatter and fluorescence[JlC2] . As cells are passed through a single tube one-by-one, laser light beams shine on the cell. The way the light scatters reveals information about cell size and internal characteristics.
Impedance-based cell counter	Electrical impedance. Cells in an electrolyte solution are passed through one-by-one a small aperture surrounded by two electrodes. As they pass through, changes in voltage are recorded, indicating cell count and size.

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How do we know that cell counters are counting cells?

To ensure that a cell counter is correctly counting cells rather than debris or other particles, they must be calibrated and validated against a range of cell samples at varying concentrations. These statistical tests will demonstrate how reliable the cell counter is.

Often, the cell counter is compared to the hemocytometer, which continues to this day serve as a reference method.

Types of Cell Counters

There are several types of instruments that count cells. The main types include:

• Hematology analyzer
• Impedance counter
• Flow cytometer
• Image cytometer

Let’s discuss each one in a little more detail. But first, we must talk about the hemocytometer, the oldest cell counting method in existence.

Hemocytometer

The hemocytometer is a glass slide with a centralized chamber (also called the counting chamber) that contains your cell sample. Etched onto this chamber is a grid, used to count cells when viewed under a microscope.

The most contemporary iteration of this is called the Neubauer Improved cell counting chamber, although there are a variety of counting chambers produced.

The cells are identified in the hemocytometer by using brightfield microscopy.

Viability is determined via the principle of trypan blue exclusion; whereby, trypan blue stains non-viable, or dead, cells, yet excludes viable cells. Both viable and non-viable cells are then counted under a microscope.

The hemocytometer has been the dominant method to count cells for quite some time, and remains popular today due to its low cost, but has some common problems.

Human Error and Bias

The hemocytometer works adequately for cells that are easy to visualize and count, such as Jurkat or Chinese hamster ovary (CHO) cells.

But not all cells are so easy. Aggregated cells or cell samples with low cell viability and significant debris are more challenging to visualize and count with the naked eye.

The manual nature of this method implies that subjectivity and bias are introduced during various stages of the process. For example, differences in pipetting technique when preparing a cell sample may introduce human error.

Differences in counting technique and the determination of what a cell looks like under the microscope introduce even more error.

The result: wide variations in cell counts between users and crippled scientific data.

To test this yourself, ask some colleagues to count cells with you. Calculate the standard deviation and the coefficient of variation of your counts, as we describe in this post. Compare the results. How concordant were your results?

Another problem with hemocytometers (as well as that of some other cell counters) is the use of trypan blue, which damages cells, commonly leading to an overestimation of cell viability.^1,2 The stain is also toxic and consequently being phased out in the European Union.

Below are the pros and cons of using a hemocytometer to count cells.

Advantages:

✅ Low cost

✅ Good training for those new to cell counting

✅ Considered a reference method

Disadvantages:

❌ Imprecise and subjective

❌ Laborious and time-consuming

❌ High user-to-user variation

❌ No electronic record

But don’t panic! There are many other ways to count cells that are reviewed below.

Hematology Analyzer

Hematology analyzers are automated cell counters that measure blood cell, platelet and hemoglobin concentration.

They rely on some of the same principles used by automated cell counters for mammalian cells, such as light scatter and electrical impedance.

However, they are used primarily in healthcare settings, such as at physician offices, hospitals, emergency rooms, clinics and research labs.

One of the most common measurements obtained by these machines is the complete blood count (CBC), which includes enumeration of both red blood cells (RBC) and white blood cells (WBC).

In addition, a three- or five-part WBC differential is also a standard measurement.

This data provides clinicians important information about a patient’s health and aids diagnosis.

One would not choose between a hematology analyzer and an automated cell counter for mammalian cells, as hematology analyzers only count blood cells.

Impedance-based Cell Counters

Electrical impedance is the oldest automated method to count cells, developed by Wallace Coulter and patented in 1953.³ They don’t just count cells however – they are best characterized in fact as “particle counters,” and they cannot distinguish between particle types. They have been used to study cells as well as minerals, clay, metals and food.

Electrical impedance is often interchanged with the “Coulter principle,” and refers to the same concept. Instruments using this principle are often referred to as Coulter counters.

Here is how it works. In principle, the cells, along with liquid and an electric current, are pulsed through a very small opening, called the aperture. Surrounding the aperture on both sides are electrodes, which create a “sensing zone.”

When a cell passes through the aperture, the machine detects a change in voltage and registers it as a voltage pulse.

The instrument then counts the number of voltage pulses to determine the total number of cells in the sample.

This information is also used to calculate cell size, which is directly proportional to the change in voltage.

Potential Errors

The most common error with impedance-based cell counters involves more than one cell entering the sensing zone and being falsely counted as one particle. This is termed coincidence error and requires the cell sample to be diluted further before being counted again.

Keep in mind…

When using an impedance-based cell counter to count cells, suspension cells are usually required. Because these instruments count cells one-by-one, aggregated and complex cell types cannot be counted by these instruments. Additionally, the instrument may have trouble with primary cell samples, which are often contaminated with red blood cells.

As long as you have prepared your cell sample per the right protocol, the impedance-based cell counter will provide an accurate absolute count of your cell sample.

They can additionally count both blood cells and non-nucleated cells, and this is why this technology is used by many hematology analyzers.

Electrical impedance also does not require any sample staining, saving you one step in your workflow, but the lack of a viability marker makes the viability dependent only on morphological features picked up from the impedance

Below are the pros and cons of this technology.

Advantages:

✅ Precise particle count and size

✅ Quick cell count

✅ No need to stain samples

Disadvantages:

❌ Coincidence errors

❌ Cannot count aggregated cells

❌ Low accuracy for viability

Flow Cytometers

Flow cytometers are high-throughput cell analyzers providing data on multiple cell parameters. They work via the principle of scattering laser light. After the machine acquires the sample, cells flow one at a time through [JlC5] a thin tube, where they meet laser light.

Light is then scattered in different directions, providing information on intrinsic and extrinsic cellular characteristics.

Flow cytometers are beneficial for single cell analysis, in vitro diagnostics, and medical research.

Similar to impedance-based counters, flow cytometers have cells move in single file, which allows them to count the absolute number of cells in your sample.

However, cells are analyzed as a function of time not volume, which means mixing with reference beads may be required.

Keep in mind…

In order to prepare a cell sample for flow cytometry, you will need to count cells through other means to obtain the right concentration.

This is why a bulky flow cytometer isn’t a popular choice when quick and easy cell counts are needed.

Like impedance-based counters, flow cytometers require suspension cells, as the cells flow through the fluid stream one-by-one.

This requirement means a tedious sample preparation process proceeds the cell count. Sample preparation can take hours with a flow cytometer.

Automated flow cytometers remove some of this burden, but the process is still time-consuming and generally much slower for measuring cell count compared to that of other methods.

Additionally, flow cytometers need to be calibrated before each use and cleaned afterwards. Due to the extensive fluidics system, they also clog from time to time, which may be costly to repair, and requires special care.

The good news is that dedicated cell counters and flow cytometers are often part of the same workflow. For example, when studying the activity of anti-cancer drugs, researchers may investigate how a drug triggers apoptosis.

Obtaining the cell count informs us of the extent of apoptosis and apoptosis assays on a flow cytometer reveal how the drug triggers apoptosis.

Here are some pros and cons of using a flow cytometer to count cells.

Advantages:

✅ Reproducible

✅ Automated, walk-away technology available

✅ Counts and analyzes heterogenous samples

Disadvantages:

❌ Daily calibration and cleaning needed

❌ Start up and shut down time

❌ Bulky and expensive

❌ Not volume based

Image Cytometers

Image-based cell counters, or image cytometers, work similarly to the hemocytometer – the difference is that the microscope is inside the instrument and an algorithm counts the cells instead of a human.

Within the image cytometer are microscopes, cameras and lights used to visualize a cell sample. The cell sample is spread out onto a thin chamber within a slide or cassette, which is placed inside the instrument.

The image cytometer then takes multiple images of the dispersed cell sample. Its software identifies live and dead cells, providing a calculation of cell count, viability and concentration.

Sample Preparation

Unlike impedance-based cell counters and flow cytometers, automated image cytometers typically require minimal to no sample preparation. Some can count aggregated cells and distinguish non-cell particles from cells in your sample as well. This is a challenge that not all image cytometers successfully pass.

Keep in mind…

Some image-based cell counters also perform cell analysis. These instruments include a range of assays and may provide a cheaper and more efficient alternative to flow cytometry. However, flow cytometers have greater capabilities.

Keep in mind the type of stains an image cytometer uses. Some machines are trypan-blue-based, whereas others rely on fluorescent stains such as acridine orange (AO), propidium iodide (PI) or 4′,6-diamidino-2-phenylindole (DAPI).

If your mammalian cell sample is contaminated with red blood cells, a cell counter using fluorescent dyes is needed to differentiate nucleated from non-nucleated cells.

Purchasing stains and reagents may add to the overall cost of using an image-based cell counter in the long-term.

Another important consideration is the microscope’s focusing technique. Some image-based cell counters require the user to manually focus the microscope before visualizing cells, whereas others provide auto-focus or fixed focus.

Manual focusing introduces subjectivity to your cell counts, resulting in greater user-to-user variation.

The differences between image cytometers on the market mainly center around staining methods, sample preparation, the dynamic range of the machine (the concentration of cells it can count) and the precision of the cell count.

Overall, image-based cell counters are smaller, portable and more cost-effective than flow cytometers.

Here are some pros and cons of this type of instrument.

Advantages:

✅ No daily calibration

✅ Low maintenance

✅ Portable and small footprint

Disadvantages:

❌ May require manual staining or dilution steps

❌ Focusing may be subjective

❌ May require trypan blue – a toxic dye

What Are Cell Counters Used For?

Cell counters are used in scientific research and in industry for a wide variety of applications.

Let’s look at some of the most interesting and relevant areas where precise and accurate cell counters are regularly used.

Cell and Gene Therapy Manufacture

Cell and gene therapies (CGT) have been a breakthrough in medicine, allowing the possibility of personalized medicines with curative potential.

The cell and gene therapy market is expected to grow continually over the next decade, with the US Food and Drug Administration (FDA) expecting to approve between 10-20 CGT products annually by 2025.³

These products include chimeric antigen receptor T-cell (CAR T) and natural killer cell (CAR NK) therapies, stem cell therapies and gene therapies.

Throughout the stages of manufacture of these products, cells need to be counted. In the manufacture of autologous CAR T cell therapy for instance, peripheral blood mononuclear cells (PMBCs), T cells and CAR T cell concentration is measured with the help of a reliable cell counter.

Before a CAR T therapy may be released, release testing is performed. Part of these tests include measuring cell count and viability among other key quality attributes. Process validation, in-process testing and research and development to improve the manufacturing process also require cell counting.

Bioprocessing

Many proteins are dependent on correct modifications during their production – that goes for therapeutic proteins as well. Cells are the only manufacturing tool that can be used to produce antigens for vaccine use as well as therapeutic proteins such as antibodies often used for cancer treatments.

For this purpose, different cell lines have been developed for specific biological products. One of the most common cell lines used to manufacture biologics are CHO cells.

Cell counters play an important role in the manufacturing process of these therapies, whether traditional cell culture models are used, or the cells are grown in a bioreactor.

The number of viable cells used in upstream processes may influence product quality, yield and characteristics. Cell count and viability must be measured regularly to track cell health and growth.

Monitoring cell concentration and viability is also crucial when developing recombinant and cell-based vaccines. In any bioprocess requiring cells, cell concentration may become a critical process parameter. By using a reliable cell counting methodology, manufacturers can ensure process efficiency, minimize downtime and optimize yields.

Drug Screening

An important application of cell counters in traditional pharma is in vitro drug screening. For this purpose, 2D and 3D cell cultures are used to explore a drug’s safety and its effect on disease. The purpose of using cell cultures is to create a model of cells that mimics how cells behave in the human body.

Before an investigational drug may be tested in humans, it is tested in vitro first for its toxicity and to understand more about its mechanism of action.

In cancer drug screening for instance, tumor cells are exposed to varying doses of an anticancer drug. Cell count and viability are measured using cell counters to understand the drug’s effect on cell proliferation and death.

Drug toxicity may be determined by exposing human cell lines, such as HeLa cells, to different concentrations of the drug.⁴

Cell culture models are also useful in personalizing cancer treatment. Tumor cells from individual patients can be cultured to test different cancer drugs or drug combinations to determine which treatment may be the most effective.^5,6 These methods are complex and continually evolving, but determining cell count and viability reliably will remain important.

Cultured Meat Production

Also known as cultivated meat or lab-grown meat, the need for cell counters in cultured meat production mirrors that of cell culture systems. Stem cells are obtained from an ideal part of a cow via tissue biopsy and are grown in bioreactors.⁷

Ultimately, the stem cells will differentiate into muscle and fat tissue, resulting in a product that is similar in taste, consistency and nutritional value to meat.

Cell counts are obtained before seeding the cells and determining cell density of the final product. During the stem cell proliferation phase, cell counters are regularly used to track the process.

After Singapore became the first country to approve cultured meat in 2020, anticipation that the European Union and the United States may follow suit began simmering.

Cultured meat is promoted as a sustainable alternative to conventional meat, requiring fewer resources and without animal harvesting.

Numerous hurdles remain before cultured meat may be commercially manufactured in Europe and the US, but the prospect of eating a lab-grown hamburger has many talking.

Animal Breeding

Cell counters are widely used in the selective breeding of both production animals and pets via artificial insemination. For production animals such as bulls and boars, breeding genetically superior animals helps farmers optimize production, increases the longevity of the animals and results in a more favorable impact on the environment.

Stallions and canines on the other hand are bred mainly for desirability and leisure.

To maximize fertility, semen samples must have optimal sperm concentration and viability, as measured by a cell counter.

Conclusion

Cell counters find themselves in a wide variety of applications. With a call towards automated processes in biomanufacturing, the need for automated cell counters is expected to continue growing.

Cell counting does not have to be tedious – it should be easy and reliable, so you can be confident in your process and results.

Happy cell counting!

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References

1. Mascotti K, McCullough J, Burger SR. HPC viability measurement: trypan blue versus acridine orange and propidium iodide. Transfusion (Paris). 2000;40(6):693-696. doi:10.1046/J.1537-2995.2000.40060693.X

2. Tsaousis KT, Kopsachilis N, Tsinopoulos IT, Dimitrakos SA, Kruse FE, Welge-Luessen U. Time-dependent morphological alterations and viability of cultured human trabecular cells after exposure to Trypan blue. Clin Exp Ophthalmol. 2013;41(5):484-490. doi:10.1111/CEO.12018

3. Gottlieb S. Statement from FDA Commissioner Scott Gottlieb, M.D. and Peter Marks, M.D., Ph.D., Director of the Center for Biologics Evaluation and Research on new policies to advance development of safe and effective cell and gene therapies. https://www.fda.gov/news-events/press-announcements/statement-fda-commissioner-scott-gottlieb-md-and-peter-marks-md-phd-director-center-biologics

4. Cai L, Qin X, Xu Z, et al. Comparison of Cytotoxicity Evaluation of Anticancer Drugs between Real-Time Cell Analysis and CCK-8 Method. ACS Omega. 2019;4(7):12036-12042. doi:10.1021/acsomega.9b01142

5. Gorshkov K, Chen CZ, Marshall RE, et al. Advancing precision medicine with personalized drug screening. Drug Discov Today. 2019;24(1):272. doi:10.1016/J.DRUDIS.2018.08.010

6. Mitra A, Mishra L, Li S. Technologies for deriving primary tumor cells for use in personalized cancer therapy. Trends Biotechnol. 2013;31(6):347. doi:10.1016/J.TIBTECH.2013.03.006

7. Melzener L, Verzijden KE, Buijs AJ, Post MJ, Flack JE. Cultured beef: from small biopsy to substantial quantity. J Sci Food Agric. 2021;101(1):7-14. doi:10.1002/jsfa.10663

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