CAR-T cell therapy is a type of immunotherapy that uses T cells to kill cancer. If you work with these cells, you know that proper quality control is essential to maintaining their effectiveness.
For that, you can count on The Inish Analyser, an automatic cell counter capable of performing cell counting, viability, and transfection analysis with precision.
This article will look at the basics of CAR-T cells and how to analyze these cells to get the most out of your experiments.
CAR-T Cells Basics
As a life science researcher, you've undoubtably heard about CAR-T cells. Although research in this area has been going on for a long time, it was only recently that the first therapies using CAR-T were approved. Here, we'll review some concepts about this revolutionary technology.
Chimeric antigen receptor (CAR)-T cell therapy is a revolutionary technique that can produce effective and durable clinical responses in cancer treatment, [1].
CARs are synthetic receptors containing, [1]:
An extracellular antigen-binding domain – this portion is responsible for the target antigen specificity.
A hinge region – this portion provides flexibility and allows the antigen-binding domain to access the targeted epitope.
A transmembrane domain – this portion anchors the CAR to the T cell membrane and may influence the CAR-T cell's function.
One or more intracellular signaling domains – co-stimulatory domains have different functions. Overall, they help improve the CAR-T cell durability, persistence, and T cell response.
CAR's function is to redirect lymphocytes (mainly T cells) to recognize and eliminate cells expressing a particular target antigen. CARs binding to these target antigens is independent of the MCH receptor, resulting in a potent T cell activation, [1].
How Does CAR-T Therapy Works?
The first step in CAR-T therapy is to collect the patient's blood. Then, a technician isolates the T cells from the peripheral blood sample; this technique is called leukapheresis. After that, researchers insert the CAR gene into the cells using a vector. These CAR-T cells expand in the laboratory before they are reintroduced into the patient, [2].
Potential Applications
In August 2017, the FDA approved a CAR-T cell-based therapy for the first time. The drug (Tisagenlecleucel) is used to treat patients with acute lymphoblastic leukemia (LLA), [3].
CAR-T cell therapy is currently used to treat blood cancers like multiple myeloma, lymphoma, and leukemia, [4].
But CAR-T cells are not restricted to cancer treatment. They could also be used to treat autoimmune diseases (e.g. Lupus Erythematosus), allergies, asthma, HIV, hepatitis, COVID-19, and many more, [5].
Once the patient receives the treatment with CAR-T cells, they can survive in the body for many years, keeping the ability to find and destroy cancer cells. This treatment reduces the chances of the disease getting back and avoids more invasive therapies like stem cell transplantation, [2].
Quality Control of CAR-T Cells
Proper quality control of CAR-T cells is essential for treatment effectiveness and reproducibility. CAR-T cells manufacturing quality control involves inspecting the materials used in production, process control, and finished product, [6].
The production materials include the cells, vectors, and reagents (i.e. culture medium). These substances should be assessed for pathogen contamination, sterility, purity, and biological activity, [6].
Testing the quality of cells during the preparation process is critical for ensuring the batch-to-batch consistency of the final products, [6]. Also, before the product release, researchers run tests to ensure that the final product meets the specified release criteria. These tests include product identification, biological efficacy, purity, impurities, live cell count, and the number of functional cells, [6].
Transfection efficiency is another crucial metric in the quality control of CAR-T cells since it can affect their efficacy. So, transfection efficiency assays allow researchers to verify how many T cells were efficiently transfected, [6].
How Can The Inish Analyser Help You?
Cell count and viability assays often require several steps due to the use of dyes. But did you know that you can do these analyses stain-free?
Cellix’s Inish Analyser is an automated method for cell counting and viability analysis based on impedance spectroscopy. Here are some advantages of this method:
Time-saving
Unlike manual cell counting with hemocytometers, the Inish Analyser requires no cell staining, reducing the steps in your workflow.
Accuracy and Reproducibility
Automated cell counters like the Inish Analyser help to avoid user-to-user variability for more reliable results.
Cost-effective
The Inish Analyser doesn't require staining or expensive slides/chambers.
Transfection Efficiency Prediction
The Inish Analyser predicts transfection efficiency unlike traditional cell analysis assays. Using the Inish Analyser in tandem with your transfection experiments, you can easily optimize transfection settings and quickly determine the useful population, saving inserts, media, and time.
How Does it Work?
The process is very simple. After isolating your primary T-cells from whole blood, pipette 40 uL of your sample into a tube, add 360 uL of the Inish Analyser buffer, run the sample via the Inish Analyser's touchscreen. The results are available to you in a few seconds.
For a Cell Counting & Viability assay, you´ll see 3 populations displayed in your results:
Live cells, represented as green dots.
Dead cells, represented as red dots.
Debris population
For a Transfection Efficiency Prediction assay, you’ll see 4 populations displayed in your results:
Live cells with closed membranes represented as green dots.
Live cells with open membranes represented as blue dots – this is the useful population; as these cells are the only ones capable of undergoing transfection.
Dead cells, represented as red dots.
Debris population
After that, you can select the population of interest for analysis, simple as that.
If you'd like to learn more about Cellix's Inish Analyser, contact Cellix to book an online demo or request a quote.
References
1. Sterner, R.C., Sterner, R.M. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 11, 69 (2021). https://doi.org/10.1038/s41408-021-00459-7
2. Miliotou AN, Papadopoulou LC. CAR T-cell Therapy: A New Era in Cancer Immunotherapy. 2018;5–18. DOI: 10.2174/1389201019666180418095526
3. National Cancer Institute. CAR T-Cell Therapy Approved for Some Children and Young Adults with Leukemia [Internet]. 2017 [cited 2021 Nov 3]. Available from: https://www.cancer.gov/news-events/cancer-currents-blog/2017/tisagenlecleucel-fda-childhood-leukemia
4. Seimetz, D., Heller, K., & Richter, J. (2019). Approval of First CAR-Ts: Have we Solved all Hurdles for ATMPs?. Cell medicine, 11, 2155179018822781. https://doi.org/10.1177/2155179018822781
5. Zmievskaya E, Valiullina A, Ganeeva I, Petukhov A, Rizvanov A, Bulatov E. Application of CAR-T Cell Therapy beyond Oncology: Autoimmune Diseases and Viral Infections. Vol. 9, Biomedicines . 2021. https://www.mdpi.com/2227-9059/9/1/59
6. Li Y, Huo Y, Yu L, Wang J. Quality control and nonclinical research on CAR-T cell products: general principles and key issues. Engineering. 2019;5(1):122–31. https://www.sciencedirect.com/science/article/pii/S2095809918308786