Enhancing cell counting for cell and gene therapy

Blood cancers — including lymphoma, leukemia, and myeloma — are often challenging to treat. Traditional chemotherapy and radiation therapy can have limited efficacy and cause severe side effects, especially in cases of relapse or where there is resistance to therapy.

The recent emergence of novel therapies, notably chimeric antigen receptor (CAR) T-cell therapy, represents a promising advance in addressing these complex conditions. CAR T-cell therapy involves harvesting a patient’s own immune T cells, genetically engineering them to target cancer cells, and then reintroducing them into the patient’s body.

This approach has shown remarkable success in treating blood cancers. Yet for CAR T-cell therapy to realize its full potential, clinicians need to know the precise quantity and quality of immune cells in each sample.

Automating counting

A company called NanoEntek in Seoul, South Korea, embarked on a journey to provide a solution to this problem in the early 2000s. Excited by the promise of microfluidics — a technology that uses small devices with microchannels to analyse fluids — researchers at the company explored its potential to improve cell counting for blood samples.

The conventional approach uses trypan blue dye, which stains only dead cells. A pathologist determines the cell viability by manually tallying the total number of cells and then subtracting the number of stained dead cells. (see ‘Two staining mechanisms for cell counting in cell therapy’).

However, this method is labour intensive and prone to variability among operators. Recognizing the need for better solutions, NanoEntek has developed a way to automate cell counting. By introducing automated cell counters, the company has made the process more consistent, accurate and user-friendly. Furthermore, with the addition of an automated high-throughput cell counter, they have significantly enhanced efficiency.

Cell discrimination

The poor reliability of traditional methods is becoming more evident with the advance of cell therapies that use a wider range of cell types, including primary cells, peripheral blood mononuclear cells (PBMCs) and stem cells.

For example, a significant limitation of trypan blue is its inability to differentiate between red blood cells and PBMCs. This is especially critical when screening red blood cell products for transfusion, since the presence of white blood cells could provoke an immune response in the recipient.

Fluorescent dyes, such as acridine orange (AO) and 4',6-diamidino-2-phenylindole (DAPI), overcome this limitation by selectively staining cell nuclei. These can therefore allow researchers and clinicians to readily distinguish between white blood cells, which possess nuclei, and red blood cells, which lack them.

Additionally, small sizes of blood cells pose additional challenges for manual counting. Fluorescent dyes can overcome this problem since they selectively stain cell nuclei and emit strong fluorescence when observed under a microscope. They thus enable more precise measurement of even the smallest cells, such as PBMCs, T cells and natural killer cells, which are crucial for cell therapy.

In addition, NanoEntek’s multi-frame imaging technique can further enhance measurement accuracy. “An automated imaging system allows multiple images to be taken per sample,” explains Chan Park from NanoEntek. “As all measurements are averaged out, the resulting accuracy is significantly improved.”

Solving complications

Fluorescence-based methods are invaluable when handling samples containing contamination or samples from patients who might have conditions such as severe inflammation.

An accurate cell count is critical for cell and gene therapy, says Beom Choi from InnoBation Bio, a company based in Seoul that uses NanoEntek’s fluorescence cell counter. “The exact number of live lymphocytes, a type of white blood cell, is a simple yet essential criterion for quality control of the starting material,” he explains. “Everything can fall apart if this initial seeding density is incorrectly measured.”

One challenge of conventional techniques arises when the immune system has been activated, leading to inflammation. This can cause lymphocyte samples to be contaminated with a significant number of red blood cells, which conventional methods may incorrectly count as lymphocytes. “However, since red blood cells lack nuclei, they don’t pick up the fluorescent dye,” Choi explains. “This allows us to accurately determine the number of living cells in the starting material."

Freezing, a common practice in pathology laboratories, presents another challenge for conventional cell-counting methods, as trypan blue is known to underestimate the number of dead cells after thawing.

Moonsoo Jin, a professor of biomedical engineering at the Houston Methodist Research Institute in Texas, United States, is currently evaluating one of NanoEntek’s instruments for immunophenotyping. He is enthusiastic about the ability of fluorescence-based methods to enhance precision, particularly in cell-therapy products subjected to multiple freeze–thaw cycles.

“Automated, fluorescence-based cell counting meets the requirement for accurate and fast cell counting and viability of cell-therapy products, which typically require multiple freezing and thawing in the course of manufacturing and product release,” Jin notes. “NanoEntek’s instruments only need small sample volumes and provide accurate and fast results on cell number and viability."

This combination of accuracy, efficiency and small sample volumes underscores the significant benefits of fluorescence-based cell-counting technology for cell therapy.

Looking ahead

Expanding on this fluorescence-based technology, NanoEntek aims not only to differentiate between live and dead cells but also to distinguish between various types of immune cells. This advance holds particular relevance for CAR T-cell therapy for treating blood cancers, where both T cells and natural killer cells are used. As Park notes, the objective is to extend the capabilities of cell-counting instruments to encompass immunophenotyping, aligning with the evolving demands of cell-based therapies.

Building on these innovations, NanoEntek has developed an automated, high-throughput cell counter capable of processing up to 48 samples simultaneously. Moreover, it requires considerably less sample compared to conventional methods. This is especially advantageous for scenarios such as CAR-T cell therapy, where only a small volume is available for various evaluations.

“NanoEntek has combined the speed and accuracy of an automated system with the specificity and selectivity of fluorescent dyes in a single benchtop solution,” says Park. This promises to improve cell analysis, paving the way for greater precision in biomedical research and therapy, Park believes.

“As cell and gene therapy continues to expand, going beyond blood cancers to solid tumours, the application of fluorescence-based, cell-nucleus staining methods is poised to further flourish, promising enhanced utility and efficacy in a broader range of therapeutic contexts,” he adds.