The Trypan Blue exclusion method, currently endorsed by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA), is the standard for total cell number and cell viability assessment in cell therapies. However, as cell therapy progresses with the utilization of increasingly intricate cell samples, such as immune cells, primary cells and stem cells, the limitations of the Trypan Blue method are becoming more pronounced, characterized by overestimation of cell viability, contamination with red blood cells, and subjectivity in interpretation.
In response to these challenges, fluorescence-based cell counting methodologies have been introduced. Leveraging fluorescent dyes that selectively target cell nuclei, these techniques are impervious to the influence of debris or red blood cells (RBCs), thereby ensuring more precise cell counts and viability assessments. So far, the approved methods for fluorescence-based cell counting have been confined to the use of flow cytometers. [1]∙[2]. However, from an industrial standpoint, there is a pressing need to expand the use of fluorescence-based automated cell counting instruments to accelerate both research and development speed and production efficiency.
Recognizing these limitations, the Korean Ministry of Food and Drug Safety (MFDS) has updated its guidelines for cell therapy products to endorse the use of automated cell counters with fluorescence nuclear staining. NanoEntek is at the forefront of this advancement, offering a comprehensive range of advanced automated fluorescent cell counters.
NanoEntek’s line-up includes the single fluorescence channel cell counter ADAM™ MC2/CellT and dual fluorescence cell counter ADAM™ MC Plus/CellT Plus. Furthermore, NanoEntek provides the sophisticated 3 fluorescence channel cell analyzer ADAMII™ LS/CDx. For applications demanding high-throughput and precision, NanoEntek's automated high-throughput dual fluorescence cell counter EVE™ HT FL stands out, making it exceptionally well-suited for both cell therapy research and manufacturing (refer to the image ‘NanoEntek’s LS product line-up’ below). This shift towards fluorescent nuclear staining methods, supported by these advanced automated cell counters, offers a more reliable and efficient option for assessing cell viability in cell therapy processes.
1. Current US FDA and EMA Guidelines
Currently, the U.S. FDA and EMA recognize the Trypan Blue exclusion method, both in manual and automated, as a standard method for quality control in cell therapies. However, this method has inherent limitations that make it less suitable for complexities of modern cell therapies. For example, TB based viability tends to overestimate viability compared to fluorescence-based methods [3]∙[4], the presence of RBCs in samples makes total cell counts unreliable, and TB based manual counting is inherently subjective and more variable [5]. Addressing this issue, the regulatory agencies have partially approved fluorescence-based cell counting methods. Nevertheless, this approval is currently limited to flow cytometers.
From an industrial perspective, there is a growing demand to expand the use of fluorescence-based automated cell counting instruments. Doing so would not only accelerate research and development processes but also enhance production efficiency. Expanding approval to include automated fluorescence cell counters could greatly enhance the quality and efficiency of cell therapy manufacturing.
2. Limitation of traditional cell measuring method: Trypan blue exclusion method
(1) Definition of Trypan Blue Exclusion Method:
The trypan blue exclusion assay is one of the earliest and most commonly used method for determining cell viability. The principle behind the staining mechanism of Trypan blue (azo dye) is based on the differential permeability of cell membranes.
Viable cells possess an intact and functional cell membrane that selectively allows the passage of certain molecules. Trypan blue particles, due to their size and charge, can permeate the compromised or damaged membranes of non-viable cells, entering the intracellular space.
Normally, the intracellular environment, particularly the cytoplasm, is maintained by active processes such as ATP-dependent ion pumps and transporters in case of healthy cells. These cells utilize ATP energy to actively extrude the Trypan blue particles through a process known as exocytosis, effectively removing the dye from the intracellular space and preventing the cells from being stained.
Conversely, non-viable or dead cells lack the ability to generate ATP and, consequently, are unable to perform exocytosis. As a result, they retain the Trypan blue dye within their cytoplasm, causing them to appear blue or stained under a microscope.
By using this staining method, researchers can directly identify and count live cells (unstained) and dead cells (blue) within a given population.
(2) Limitations of Trypan Blue exclusion method:
In the past, cell-based research primarily utilized immortalized cell lines, such as CHO cells. However, as the field of cell therapy advances, more complex sample types are being used, including primary cells, peripheral blood mononuclear cells (PBMCs), stem cells, dissociated tumor cells, and even engineered T cells. These cells vary in size, shape, and aggregation properties, making the traditional Trypan Blue exclusion method less reliable for accurate results, especially with heterogeneous samples like PBMCs, stem cells, and primary cells.
Here are some limitations of the Trypan Blue exclusion method:
Firstly, trypan blue is toxic to mammalian cells. Even viable cells are eventually stained with trypan blue as the toxic dye permeates their cell membrane, leading to cell death about 5 to 30 minutes after exposure. As a result, accurate measurements must be taken within 3 to 5 minutes after dye introduction. Additionally, Trypan Blue is harmful not only to cells but also to humans, being identified as a carcinogen that may cause cancer after prolonged exposure.
Furthermore, trypan blue can alter the morphology of dead cells to a diffuse shape, potentially leading to overestimation of viability. Cells immediately start to rupture upon staining with trypan blue, becoming diffuse objects. Some dead cells may disappear after trypan blue staining, leading to an undercounting of total cells and thus an overestimation of cell viability. [4] In other words, the toxicity of trypan blue can affect measurement accuracy by altering viability over time, resulting in an underestimation of cell population viability. [4]
These inaccuracies could impact the outcomes of cellular therapies, which rely on accurate measurements of immune cells that will be infused back into patients, furthermore, highlighting the need for improved solutions, such as the fluorescence cell nucleus staining method.
3. Necessity of fluorescence nuclear staining methods in the process of manufacturing cell therapies
In the production of cell therapy products, various immune cells are extracted from human blood. However, these cells often come contaminated with RBCs, which can be incorrectly identified as dead cells when using the traditional trypan blue exclusion method. This contamination extends to cellular debris and non-cellular particles, leading to skewed data. Additionally, red blood cells mixed with PBMCs are indistinguishable during immune cell counting, resulting in inaccurate total cell count values. These inaccuracies can significantly impact the research, development, and production of various immune cell therapies like CAR-T and NK cell therapies, ultimately affecting the overall quality of the final cell-based therapies.
Moreover, primary cells extracted from blood, tissues, or organs pose similar issues due to the relatively high presence of debris compared to immortalized cell lines. This can lead to the miscounting of debris as cells, further exacerbating the inaccuracy of total cell count and diminishing the quality of research, development, production, and manufacturing of cell-based therapies.
Furthermore, the limitations of the Trypan Blue based cell counting method also extend to its effect on cell survival rates. The inconsistency and inaccuracy in total cell count values directly influence cell viability assessment. For instance, cells abundant in immune cells such as PBMCs exhibit a higher variability in cell viability coefficient of variation (CV %) due to their smaller size (8μm) compared to conventional cell lines (>10μm) when examined under bright field microscopy.
Unlike conventional cell lines, where Trypan Blue staining enables the distinction between living cells (bright internally) and dead cells (dark blue), determining the viability of smaller cells like PBMCs based on internal brightness poses a significant challenge. This challenge leads to notable discrepancies among users and a poor ability to differentiate viability using conventional cell counting microscopes.
To mitigate these issues, companies are now employing automated cell counters with cell nucleus fluorescent staining techniques. This approach involves staining cell nuclei fluorescently to achieve consistent and accurate values, regardless of cell size. Dead cells are counted based on the presence of stained cell nuclei using reagents like PI or DAPI, ensuring accuracy and minimizing external interference.
This method is effective for both immortalized cell lines and primary cells extracted from blood, tissues, or organs, providing consistent and accurate results despite debris or RBC contamination. Using automated cell counting methods based on cell nucleus fluorescent staining ensures high accuracy and minimal external interference in both total cell count and viability measurement, enabling reliable data acquisition and facilitating research, development, and process management.
4. South Korea: Proactive Changes in the "Guideline on Release Tests for Cell Therapy Products”
The Korea MFDS (Ministry of Food and Drug Safety) has recently made a significant update to its "Guideline on Release Tests for Cell Therapy Products". This update has been made in recognition of the accuracy and reliability of the fluorescence nuclear staining method. As a result, automated cell counters utilizing this method are now officially recognized as a valid testing procedure.
The latest revision of the guideline acknowledges the limitations of the Trypan Blue staining method and has officially embraced the use of the automated cell counter using fluorescence nuclear staining method in parallel. This development is a positive step forward, as it addresses concerns related to the approval process and opens the door to more accurate cell counting using fluorescence-based cell counters.
With the modification of regulation, NanoEntek's range of automated fluorescence cell counters (refer to the figure ‘NanoEntek’s LS Product Line-up’), including single fluorescence channel cell counter ADAM™ MC2/CellT, dual fluorescence cell counter ADAM™ MC Plus/CellT Plus, 3 fluorescence channel cell analyzer ADAMII™ LS/CDx, and high throughput automated dual fluorescence cell counter EVE™ HT FL, can now seamlessly integrate into the development, manufacturing, production, and clinical trials of cell therapy products. This recognition of fluorescence-based cell counting methods as a valid testing procedure ensures that accurate and reliable data can be obtained, contributing to the overall quality and safety of cell therapy products.
5. NanoEntek's Advanced Cell Counters: Ideal Tool for Cell Therapy Development and Manufacturing
As previously mentioned, fluorescent cell nucleus staining method stands out for its high accuracy and reliability in the manufacturing and quality control of cell therapy products. NanoEntek's range of automated fluorescent cell counters, including the ADAM™ MC2/CellT, ADAM™ MC Plus/CellT Plus, and ADAMII™ LS/CDx, are designed to deliver precise and reliable results for both the total cell number and their viability.
Specifically, ADAM II series is capable of accurate cell identification and absolute counting using cell surface marker (CD marker). Moreover , the EVE™ HT FL, a high-throughput fluorescence cell counter, can process up to 48 samples within just 3 minutes. This exceptional precision and speed make the EVE™ HT FL ideal for a wide range of applications, including the analysis of cell lines and primary cells.
Overall, the ADAM™ series, ADAMII™ series, and EVE™ HT FL represent highly suitable equipment for use in the development of cell-based therapies. They offer unparalleled accuracy, reliability, and efficiency, making them indispensable tools for research, development, and production processes in the field of cell therapy.
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2. Council of Europe. Nucleated cell count and viability (2.7.29). European Pharmacopoeia, 10th, 2019: 297-9.
3. Mascotti K, McCullough J, Burger SR. HPC viability measurement: trypan blue versus acridine orange and propidium iodide. Transfusion. 2000 Jun;40(6):693-6. doi: 10.1046/j.1537-2995.2000.40060693.x. PMID: 10864990.
4. Chan LL, Rice WL, Qiu J. Observation and quantification of the morphological effect of trypan blue rupturing dead or dying cells. PLoS One. 2020 Jan 24;15(1):e0227950. doi: 10.1371/journal.pone.0227950. PMID: 31978129; PMCID: PMC6980413.
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