Use of cells for drug discovery as well as therapeutic and diagnostic applications has been increasing exponentially. “No matter what the scientific question or downstream application, if tissue culture is involved, it’s a safe bet that someone will count cells somewhere in the process,” explains Chris Laucius, Ph.D., product manager, development at BioTek Instruments.
The hemocytometer, consisting of a thick glass slide with an etched grid and a sample chamber, has traditionally been used to count cells in a defined volume under the microscope, to get a measure of cell concentration. However, this manual method of cell counting takes time, effort, and is prone to error and contamination. Hence, it is no surprise that in recent years, labs have switched to more automated methods for cell counting. Today, cell counting can be done using techniques such as impedance, flow cytometry, or bright field or fluorescence microscopy. The difference between these techniques lies in their speed, sample handling, and customization capabilities.
Improving speed and accuracy
The majority of automated cell counters on the market are low throughput and work well for labs that don’t process a lot of samples. However, in an environment where speed is critical, such as in cell production or in clinical testing, high-throughput cell counters are in use. The Cellaca MX is a fast, high-throughput, fluorescence-based imaging cell counter offered by Nexcelom Bioscience that can count 24 samples in less than 2.5 minutes. “Having a cell counting system that is image-based and quantitative not only helps with accurately timing and planning cell passaging for valuable samples, but also helps with early detection of any problems with the cell culture,” says Leo Chan, Ph.D., technology R&D manager at Nexcelom. “In some applications, time is of the essence as cells may behave differently with time. Cellaca can be used for large amounts of sample allowing researchers to be more efficient and assays to be more robust by not having the samples sit around.”
The CellDrop Automated Cell Counter from DeNovix uses DirectPipette™ technology to count cells. It uses two optical-grade, sapphire surfaces that form a variable-height sample chamber. Analysis typically takes around three seconds for brightfield images and 8.5 seconds for dual-channel fluorescence and brightfield images.
Image: CellDrop Automated Cell Counter: DirectPipette™ Technology replaces plastic slides with a variable-height, wipe clean sample chamber. Image courtesy of DeNovix.
“The advantage of having control over the height of the chamber is primarily the range of cell densities that can be assayed without the need for diluting or concentrating samples,” says Andrew Jones, market development manager at DeNovix. For high-density cell cultures, a lower chamber height can be set, decreasing the sample volume in the field of view. Conversely, a higher chamber height can be used for low-density samples, increasing the volume and therefore the number of cells present. The DirectPipette system also avoids the need for disposable plastic slides, which is a big advantage from a cost, usability, and environmental waste perspective.
BioTek has introduced the Cell Count & Viability App for use with its Cytation™ Cell Imaging Multi-Mode Readers and Lionheart™ automated imagers. The application enables rapid hemocytometer-based counting of suspended mammalian cells. “In addition, the app provides a built-in dilution calculator for downstream cell seeding, which allows you to quickly calculate how much media to mix with your cell suspensions,” says Laucius.
Image: The Cell Count & Viability App from BioTek enables rapid hemocytometer-based counting of suspended mammalian cells. Image courtesy of BioTek Instruments.
Along with speed, it is essential that the cell counts obtained are accurate. The new generation of image-based cell counters offers a more reliable option for specific applications. According to recent studies, cell counters that use trypan blue staining could see some systematic errors in overestimation of viability in certain cell types, due to rupturing of dead cells caused by the dye. “If you are working with cells that are fairly healthy and the cell viability is more than 80% then trypan blue is fine,” says Chan. “However, if the cell viability is below 80% and you are using certain cell types, like immune cells for cell and gene therapy research, then cell counts obtained using trypan blue staining may have detrimental effects, and fluorescence imaging may prove to be more accurate.”
Cell counters integrated with high-resolution cameras can mitigate some of the errors associated with differentiating live cells from dead cells and debris. “Growth rates can be altered by contamination, like mycoplasma,” says Hiroyuki Kimura, product manager of the Incubation Monitoring System at Olympus Corporation. “The visual information and quantitative records enable you to identify these problems early. The data monitoring also lets you check whether lab members are following the correct protocol without the error and subjectivity of manual counting.”
Minimizing cell handling and contamination
The Olympus Provi CM20 incubation monitoring system (currently available only in the U.S. and Japan) periodically scans and counts multiple points on the entire surface of the culture vessel or multiple wells in a microplate to track the health and confluency of cells or colonies. The Incubation Monitoring Head stays inside the incubator, and the data is wirelessly communicated to the computer workstation connected via an optional router. The software calculates the cell count, confluency, growth curve, and doubling time using the acquired images. “This image-based, label-free cell counting does not require any special preparation and can be monitored without entering the clean room,” says Kimura. “The combination of remote monitoring and label-free cell counting is very unique and greatly improves the cell culture process.”
Image:The Olympus CM20 incubation monitoring system provides quantitative data remotely. Image courtesy of Olympus Corporation.
The Flex2 analyzer by Nova Biomedical can be integrated with bench-scale to large production bioreactors for automated sampling and measurements. The system can be optimized to get cell counts for individual cell types, including cells with high density, aggregation, and adherence. “Cell counting has been a challenge when densities go up to 100 million/mL in the production stage,” says Matthew McRae, biotechnology sales product line manager at Nova Biomedical. Taking the sample out of the bioreactor can introduce errors during dilution and can sometimes cause the cells to be shocked, which can also change the viability readings. “With this system we can go up to 160 million/mL without any dilution needed for cell counting.”
Image:BioProfile FLEX2 Comprehensive Cell culture Analyzer with CDV. Image courtesy of Nova Biomedical.
MilliporeSigma’s Scepter 2.0 Handheld Automated Cell Counter is portable and provides reliable cell counts in less than 30 seconds. “It combines the ease of automated instrumentation and the accuracy of the Coulter principle of impedance-based particle detection for accurate and reliable results,” says Michele Halter, global product manager at MilliporeSigma.
Increasing customization and ease-of-use
“I’ve found that many scientists are hesitant to make the switch to an automated counter and when asked why, they say ‘I just don’t trust the counts’ or ‘I don’t save much time anyway’,” says Laucius. “Hence, it’s very important that the cell counter provides an easy-to-use alternative to the manual method and provides settings to tailor the count analysis to specific cell lines.”
The imaging and data analysis software for cell counting should be intuitive and simple enough for people to start using after a few minutes of instruction. Having the software save images for record-keeping and analyzing, without having to save the sample, is also a huge benefit.
CellDrop uses EasyApps software to provide default settings to quickly count and export data, while also providing a platform to optimize some specific parameters. “This is a highly efficient way of creating cell-specific protocols for future use,” says Jones. “To enhance cell identification the software includes HD edge detection to identify cells and particles and accurately report their size using a proprietary decluttering algorithm. In addition, there is a Small Cell Mode enabling detection of even non-fluorescent samples down to 4 µm.”
“Cell counting is important at ATCC since we work with so many different cell types,” says Sheela Jacob, Ph.D., scientist at ATCC. “We don’t want to introduce any variability in cell culture between R&D and manufacturing processes for our cell development.” ATCC’s collection includes more than 4000 human and animal cell lines. Hence, they use the same automated trypan blue-based cell counter, such as the Vi-CELL from Beckman Coulter, to optimize cell counting, whether it’s for population doubling level (PDL) calculations, scaling up, cryopreservation, technology transfer, or other processes in cell production. “The Vi-CELL counter has pre-defined settings for different cell types, and you can use the pull-down menu to select the one you need or we have freedom to set the parameters for new cell types,” says Jacob. “We add the samples, walk away, and come back to get the cell count and other measurements in different output formats such as histograms or in an Excel sheet.”
Countdown to better standardization
As cell and gene therapy gains prominence, cell counting is going to get stringent and will be required to strictly adhere to protocols for meeting Good Manufacturing Practices (GMP) standards. The International Organization for Standardization (ISO) has released two sets of guidance that relate to cell counting. Part 1 lists six key areas to ensure good cell counting practices. “It’s important to investigate and select an appropriate cell counting system—not only the instrument but the reagents, software, and everything that contributes to the results,” says Chan. “It’s important to also understand the cell sample composition, the assay for which its being used, and whether it’s fit-for-purpose. It’s important to see cell counting as a process—from sampling to diluting and staining—to minimize all possible errors and finally, it’s important to provide consistent and continuous training to the user.”
Besides cell counting, there are a number of other parameters such as cell purity, cell morphology, cell size, cell health, apoptosis, and population analysis that can now be measured using some of the newer instruments. All the images and the reports from these measurements can be analyzed before the experiment is done to eliminate problems downstream. “Cell counting is a ubiquitous task in all labs that culture cells, and it will remain essential for the foreseeable future,” says Laucius.
Hero image: Antigen presenting cells dissected from a mouse spleen are analyzed for viability and count on the CellDrop. Image courtesy of DeNovix