In the rapidly evolving field of immunotherapy, Chimeric Antigen Receptor T-cell therapy is changing the way we look at cancer treatments. CAR-T therapy is an innovative approach that involves genetically modifying a patient's own T-cells to express a chimeric antigen receptor (CAR) that can recognize and target specific cancer cells.
Cell counting and sizing play a vital role in ensuring the success of this therapy.
Before the infusion of CAR-T cells, it is important to determine the number of cells that will be administered to the patient. This is typically done through cell counting methods, which involve using various techniques to count the number of cells in a given sample. Accurate cell counting ensures that the patient receives the correct dose of CAR-T cells.
Cell sizing is equally important. The size distribution of the CAR-T cells helps determine the quality and consistency of the CAR-T cell product. Various techniques are used by researchers and clinicians for effective cell sizing, but choosing the most effective and affordable technique is critical for laboratories.
Steps requiring cell counting and sizing in the CAR-T workflow
Take a look at the steps that involve cell counting and sizing, and learn why they are crucial for success.
Collection of Peripheral Blood Mononuclear Cells (PBMCs)
The need for accurate cell analysis begins at step one. Most often, PBMCs are used as the starting material for CAR-T cell manufacturing. During the collection process, cell counting is essential to determine the cell concentration and to ensure an adequate number of cells for subsequent steps.
T-cell Isolation
The next step involves isolating T-cells from the collected PBMCs, which is done through centrifugation. Cell counting is crucial here to determine the number of T-cells obtained, as this affects subsequent calculations and adjustments during the manufacturing process.
T-cell Activation and Expansion
T-cells are then activated and expanded in vitro to generate a sufficient number of CAR-T cells for therapeutic purposes. CAR-T activation window is small, so knowing when the cells are ready is vital. Cell counting is performed at regular intervals during the expansion process to monitor cell growth, proliferation, and viability. Cell sizing is critical to monitoring the activation state of T-cells. This is the step in which Coulter-Principle-based systems, like ORFLO’s Moxi systems, excel with their precise volumetric measuring capabilities. Moreover, they give results in seconds, allowing you to monitor the progress of the activation state during the course of expansion.
Transduction with CAR Gene
CAR-T cells are engineered by introducing a chimeric antigen receptor (CAR) gene into the T-cells to enable them to bind to tumor antigens. Before transduction, cell counting is necessary to determine the number of T-cells to be transduced and to optimize the transduction efficiency. After the cells are engineered, they are allowed to grow and multiply to generate enough cells for suitable use. Here again, accurate cell counts are required.
CAR-T Cell Harvesting
Once the CAR gene has been successfully introduced, the CAR-T cells are harvested for subsequent processing or infusion. Accurate cell counting is crucial at this stage to determine the yield and viability of the CAR-T cell product, which in turn is important for ensuring consistent and effective cell therapy outcomes.
Quality Control and Characterization
After harvesting, the CAR-T cell product undergoes comprehensive quality control and characterization. This involves assessing cell viability, cell concentration, and cell size distribution. Cell counting and sizing are integral to evaluating the final product's potency, purity, and overall quality. Deviations in cell size can affect their function and potency. Only the most viable and potent cells can be infused back into patients.
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