Sponsored content brought to you by
In Paris, at the 2023 annual meeting of the International Society for Cell and Gene Therapy (ISCT), scientists from Charles River showcased a poster that demonstrated a method of dramatically improving the production of adoptive cell therapies, which can be used to treat a range of cancers and other diseases. Making these therapies safe and effective, however, depends on the genetic engineering of cells, and that can be challenging.
The challenge begins with loading cells with specific molecules—such as mRNA, DNA, endonucleases, or transposon systems—that orchestrate the production of a therapy. In many cases, electroporation (EP) is used because a pulse of high-frequency voltage creates transient pores in a cell membrane, thereby allowing the molecules to enter. Although EP often works well for gene editing and transfection, it can also produce high levels of cell death.
In a search to improve the survival and viability of genetically engineered cells, Charles River scientists tested a new approach to the process. EP was applied to introduce a tagged transgene to T cells or hematopoietic stem cells (HSCs), and then the cells were cultured in standard media or media plus a pro-survival, defined supplement. Controls were created by loading T cells or HSCs in an electroporator, not exposing them to EP, and then culturing them in media with or without supplementation. Then, at prescribed time points, Charles River scientists analyzed the cultures for cell numbers, cell viability, and transfection.
Success of supplementation
T cells were cultured for 16 hours with or without supplementation, and then they were stained with the fluorescent 7-aminoactinomycin D (7-AAD) and analyzed for cell death with flow cytometry. About 50% of the T cells died after EP and culturing in unsupplemented media, but less than 20% of the T cells died when they were cultured in supplemented media after the EP process.
After 24 hours, the scientists analyzed T cells, HSCs, and controls for cell recovery and viability. By comparison to the controls, adding the supplement to culture media improved cell recovery by at least 50% and as much as 80–100%. An analysis of cell viability showed that nearly 100% of the control cells survived, but approximately 50% of the T cells and HSCs died in the unsupplemented condition. Conversely, nearly 100% of the T cells and HSCs cultured with supplemented media remained alive after 24 hours.
Four days after EP, Charles River scientists analyzed the cells for transfection efficiency. For the T cells, transgene expression reached about 25% for cells cultured in unsupplemented media, but it surpassed 50% for the cells in supplemented media. For the HSCs, unsupplemented media produced transgene expression above 30%, and that number increased beyond 75% for cells cultured in supplemented media.
Overall, this case study showed that adding the pro-survival supplement to culture media after EP increased cell viability by 30–40%. Moreover, the data indicated similar improvements in cell recovery and viability in T cells and HSCs—revealing that the results were not cell-type specific. The improvements in cell survival and consequent expansion increased transgene expression as much as twofold in both T cells and HSCs.
Despite some of the challenges of using EP to produce adoptive cell therapies, these results showed that supplementing culture media can dramatically improve cell recovery, viability, and transfection efficiency. As a result, genetically engineered adoptive cell therapies could be produced much more efficiently. Consequently, these therapies could be made available to patients more affordably, more quickly, and perhaps able to treat a wider range of diseases.
Learn more https://bit.ly/3NdSOPw