How do automated cell counters work?

Why automated cell counters are important

The pharmaceutical industry is known for taking a deliberate approach to new technologies. However, with the arrival of Pharma 4.0, industry leaders are rapidly testing and deploying new automated solutions.

The keystone to this paradigm shift is Process Analytical Technology (PAT).

PAT entails understanding and controlling the manufacturing process in real time to ensure optimal quality. This approach enables quicker and more efficient analysis of manufacturing processes.

The result of PAT is to support the Quality by Design (QbD) approach to drug manufacturing. QbD aims for a “right first time” approach so that quality can be ensured in the finished pharmaceutical product. Benefits of this approach are many, including faster timelines and safer medicines.

Results of automated, PAT approaches to biopharmaceutical manufacturing include greater:

• drug product quality and yield
• process understanding and control
• reproducibility.

There are currently over three thousand cell and gene therapies in development. For these and other cell-based medicines (e.g., vaccines and biologics), variations in cell count and viability during the stages of the manufacturing process can result in not-so-good product quality and yields.

Goods such as beer and wine also require precise concentrations of yeast to optimize taste and quality.

Automated cell counters help in keeping track of these attributes in a way that is reliable. Furthermore, they may help provide an edge in the rush to get products to the clinic or market as fast as possible.

Reducing downtime and avoiding bottlenecks is goal shared by pharmaceutical companies. Improving quality and process understanding also benefits patients, by helping create safer products.

Automated cell counters improve precision in cell counting. Yet, they do have some common sources of variability. The most common? Variation between instruments, variation in sample preparation, and variations between users.

Let’s now explore what sets automated cell counters apart from semi-automated and manual methods.

What are automated cell counters?

Automated cell counters are analytical instruments that quantify the number of cells in a sample— with minimal human interaction.

In addition to cell count, they often measure cell size and calculate cell viability.

One common goal among automated cell counters is to reduce human bias in the process.

This has the potential to make research more reproducible, manufacturing better controlled, and products safer.

Automated cell counters rely on software to analyze the cells. The data they provide is also easily exported to further analysis.

Some automated cell counters have added functionality that ensures compliance with 21 CFR part 11 and current Good Manufacturing Practices (cGMP).

Despite being ‘automated,’ many automated cell counters will require some manual steps.

The purpose of an automated cell counter however should be to eliminate as many manual steps as possible. These considerations will be discussed further below.

Types of automated cell counters

There are three categories of automated cell counters:

1. Electrical impedance-based cell counters
2. Flow cytometers
3. Image cytometers

All three types are available for use today, produced by a range of manufacturers. Some instruments may be a better fit for academia, whereas others may be designed for cGMP manufacturing of human medicines.

Others may be a better fit as an on-line measurement tool, whereas others may be great at-line or off-line tools. The right cell counter for one process may not be the preferred cell counter for another.

Considerations on which type of automated cell counter is best for you depend on total costs, performance and convenience.

Principles of automated cell counters

Electrical impedance

Electrical impedance-based cell counters use the principle of electric current to count cells.

The idea is that particles will have a different conductivity than a surrounding electrolyte solution.

These instruments pass suspended cells in an electrolyte solution through a narrow aperture. This aperture is surrounded by two electrodes. Cells pass through the aperture one by one, and as they do so, electrodes sense a change (or impedance) in electric conductivity.

Each instance this occurs is counted, thus determining the cell count.

This technology has been used to develop a wide range of cell counters and analyzers since the 1950’s. One main advantage is that they do not require cell sample staining.

But there are some common causes of error inherent to these devices. The major one is that particles are counted when they pass through the aperture. Sometimes, more than one particle can pass through, termed a “coincidence event.” Similarly, particles may flow backwards through the aperture and be double counted.

Changing the aperture size or diluting the cell sample are how these issues are typically resolved.

Electrical impedance based instruments are not specific to cells and are sometimes referred to as particle analyzers. As a result, the machines require suspension cells free from contamination to provide accurate counts.

These instruments are excellent at measuring cell size, as the size of the cell is directly proportional to the electrical impedance observed.

Flow cytometers

Flow cytometers measure the optical and fluorescent characteristics of cells via the principle of light scatter. These machines consist of a fluidics system, optical system and electronics.

Suspended cells flow one-by-one down a fluid stream before meeting laser light. From there, the light scatters in different directions based on the cell’s intrinsic and extrinsic characteristics.

The light is collected, converted into data by the computer and visualized.

Light may scatter either forward or sideways. Forward-scattering light is used to determine cell size and viability and distinguish between debris and living cells.

Light that scatters sideways informs us of internal characteristics of the cell.

Flow cytometry allows for cell counting as well as analysis of organelles, cell surface markers and nucleic acid or protein content.

Flow cytometry forms the basis of fluorescence-activated cell sorting (FACS), which can sort cells based on fluorescent markers.

When counting cells, flow cytometers are accurate and can analyze heterogenous cell samples and rare cells at a high rate.

One downside of flow cytometers is that they require daily cleaning and regular maintenance of their fluidics and optics systems.

Even when following cleaning protocols correctly, flow cytometers get clogged, which requires specialized skills and may add to operational costs. Flow cytometers provide invaluable data, helping scientists understand how drugs and disease affects cell health.

Image cytometers

Image-based cell counters, or image cytometers, use advanced digital cameras, microscopes and machine learning algorithms to count cells. The cell sample is loaded onto a slide or a cassette and then placed into the image cytometer.

The automated cell counter then takes several pictures of the cells and the image is analyzed by the algorithm. After the image is processed, the machine provides cell count as well as viability and cell size.

In order for cells to be visualized under the microscope, they must be stained. Some use trypan blue, like hemocytometers, whereas others rely on fluorescent staining. Some image cytometers offer both brightfield and fluorescence channels.

Trypan blue stains dead cells by penetrating their porous cell membrane. As a result of the stain, live cells and dead cells look different under the microscope. This staining method works well on many cell types.

However, it does not work well with complex cell types. Some cell samples for instance may contain (mammalian) red blood cells, and these look very similar to other mammalian cells under the microscope.

Fluorescence microscopy offers a solution in this situation. Fluorescent dyes such as acridine orange (AO) and 4′,6-diamidino-2-phenylindole (DAPI) are often used together to distinguish viable cells from dead cells respectively, by staining their nuclei.

Human red blood cells do not contain nuclei, and would not be visible when analyzed by the image-based automated cell counter.

There are several advantages to image-based cell counters. One is that they generally do not have extensive fluidics systems, which lowers the need for maintenance. Additionally, they have a small footprint.

The algorithm used to count cells is especially important in image cytometry. Most image cytometers are configured with multiple protocols, each containing minor adjustments to the algorithm.

This flexibility allows the user to choose the best approach for the cell sample.

This is one advantage of image-based cell counters. Image cytometers can be programmed to count aggregated cells for instance. Another advantage is that you can see the cells yourself in the instrument’s software (either on a PC or on the cell counter’s screen if it has one).

This provides more confidence that the algorithm is working as intended. One disadvantage of this approach is that too many algorithms may complicate the process for the user.

Overall, image based counters have a small footprint and improve the efficiency of cell counting.

Conclusion

Automated cell counters solve the challenges involved with manually counting cells using a hemocytometer. They help manufacture safer medicines and monitor cell-based processes.

However, operating them may require manual sample handling.

In cGMP manufacturing and bioprocessing, automated cell counters play a vital role in monitoring cell viability, cell growth and cell concentration, ideal for processes such as cell and gene therapy manufacture.

Numerous applications and industries benefit from reliable, automated cell counters and their need is expected to grow.

When considering an automated cell counter, think about its ease of use and performance.

Test it out on a range of cell samples and do your research, such as by reading posts like these and sharing with your colleagues. Happy cell counting!

Further reading

• Mini-review: Manual vs. Automated Cell Counting
• Blog post: The Ultimate Guide to Cell Counters
• Blog post: How to Count Cells with a Hemocytometer
• The NucleoCounter® range: Automated Cell Counters & Analyzers

References

1. Office of Pharmaceutical Science in the Center for Drug Evaluation and Research (CDER). PAT — A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance. Available at: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/pat-framework-innovative-pharmaceutical-development-manufacturing-and-quality-assurance
2. Lohr, A. (2023). 2023’s market outlook for cell and gene therapies. Available at: https://www.cellandgene.com/doc/s-market-outlook-for-cell-and-gene-therapies-0001
3. Simson, E. (2013). Wallace Coulter’s life and his impact on the world. International Journal of Laboratory Hematology – Wiley Online Library. Available at: https://onlinelibrary.wiley.com/doi/full/10.1111/ijlh.12069
4. Wynn, E. J. W., & Hounslow, M. J. (1997). Coincidence correction for electrical-zone (Coulter-counter) particle size analysers. Powder Technology, 93(2), 163–175. https://doi.org/10.1016/S0032-5910(97)03267-1