Cell therapies for cancer: CAR-Ts in development
Cancer with 18.1 million new cases and 9.6 million cancer-related deaths observed in 2018 is still one of the most prevalent threats to human health and well-being. Therefore, there is a strong need for better cancer treatment. Cancer immunotherapy makes use of components of the immune system like antibodies that bind to, and inhibit the function of, proteins expressed by cancer cells. More promising novel immunotherapies rely on patient-derived, genetically modified cells like T-Cells or Natural Killer Cells that express chimeric antigen receptors (CAR).
Primary Human T-Cells are difficult to modify genetically using chemical transfection reagents, just as virtually all non-dividing primary cell. Viral transduction methods depend on the cumbersome production of the viral vectors. Classical electroporation methods are often limited in throughput and can result in impaired cell viability and functionality. Therefore, we optimized the transfection and culture procedure for primary human T-Cells using the 4D-Nucleofector™ System and 96-well Shuttle™ Device allowing the high-throughput transfection of up to 96 independent transfection samples in parallel.
Human T-Cells enriched from buffy coats were transfected with pmaxGFP™ Vector through a high viability or high efficiency Nucleofector™ program in 20 μl volume. Donor-dependent transfection efficiencies of up to 70% with high cell viability were achieved 48 hours after transfection. Transfection of eGFP mRNA resulted in up to 60% transfection efficiency with more than 90% cell viability 24 hours after transfection.
In a second step, we stimulated isolated human T-Cells for 2–3 days prior to transfection via CD3 and CD28. 1.0 x 106 cells were transfected with the high viability program using pmaxGFP™ Vector in 20 μl volume. Cells were analyzed 24 hours post transfection revealing transfection efficiency and cell viability comparable to the results of unstimulated T-Cells.
In a last evaluation step, using unstimulated human T-Cells, we could show very low intra- and interplate variability of the 96-well Shuttle™ System. Transfection efficiencies varied between 62% and 77%, while a cell viability of more than 80% compared to non-program control was observed.
In summary, we present an efficient and reliable transfection system for primary human T-Cells that allows the parallel processing of up to 96 independent samples. The showcased method will support cell-engineering approaches including screening of siRNA libraries, CRISPR-based genome editing and rapid evaluation of different CAR constructs to advance novel biomedical treatments including immunotherapy approaches.