Cell count and viability of T cells can be robustly and accurately determined during adoptive CAR-T cell manufacturing using the NucleoCounter® NC-200™. In the unique Via1-Cassette™, cell sampling, staining, and counting chamber loading are carried out as a single step. For increased robustness, the NucleoCounter® is then used for automatic image acquisition and analysis.
The Via1-cassette™ is pre-loaded with Acridine Orange and DAPI to facilitate accurate T cell detection even when beads, erythrocytes, or formulation reagents are present. In summary, the NucleoCounter® system is ideally suited for adoptive CAR-T cell manufacturing.
Engineered CAR-T cells for adoptive immunotherapies
In cellular therapies, immunotherapies using autologous T cells are one of the most promising methods. The toxicity and specificity of T cells can be enhanced in response to various diseases, like cancer and graft-versus-host disease, by engineering these patient-specific T cells to express chimeric antigen receptors (CAR) or T cell receptors.
In adoptive CAR-T cell therapy, peripheral blood mononuclear cells (PBMC) are collected from the patient and then engineered CAR-T cells are created by enrichment of the T cell subset of interest and genetic modification is carried out by viral transfection.
The next step is to expand the engineered CAR-T cells on a large scale, followed by infusing them back into the patient. The NucleoCounter® NC-200™ allows continuous monitoring in every step of the entire CAR-T cell manufacturing process under GMP conditions (Figure 1), where the NC-200™ is used to monitor the entire process, from leukapheresis to the formulated product.
Figure 1. Monitoring the whole process from leukapheresis to the formulated product. With the NC-200™, it is easy to monitor all different steps of the purification, expansion and formulation of CAR-T cells using the same instrument to ensure precise and reliable results.
Monitoring the whole process using the NC-200™
Thanks to easy and user-adaptable protocols, cell counting can be directly performed from expanded and purified CAR-T cells as well as from leukapheresis using a single instrument.
Acridine Orange and DAPI dyes are pre-loaded into the Via1-Cassette™ to eliminate some of the issues that cause huge inaccuracies when the bright field based counting method is used. When erythrocytes are present in counting samples, the sample is pre-treated with an erythrocytes lysing buffer to eliminate the quenching effect of hemoglobin.
The next step is to use the Via1-Cassette™ to stain the cells with Acridine Orange and DAPI for determination of the total cell count and the dead cell count, respectively. The analysis provides very accurate results due to the exclusion of cell fragments and artifacts such as micelles and undersized events such as platelets.
Isolation of T cells may involve the use of magnetic beads, which do not influence the results of the cell counting process using the NucleoCounter® NC-200™. In summary, the NucleoCounter® NC-200™ can be employed for all steps of the cell manufacturing process.
Literature: Counting of PBMC and T cells using the NucleoCounter® NC‑200™
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About ChemoMetec A/S
Founded in 1997, Chemometec specialises in the design, development and production of high quality instruments using patented technology for a wide range of applications in cell counting and evaluation.
Chemometec work closely with life science companies, research institutes, universities, hospitals, specialist clinics and a wide range of food and beverage manufacturers, matching our technical development expertise to customer needs - with quality results, reliability, cost-efficiency and ease-of-operation as our guiding principles.
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