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Viral vectors are efficient at transporting desired pieces of DNA into cells, and are used for, among other things, transfecting chimeric antigen receptor (CAR) genes into patient lymphocytes for CAR T cell therapy. But for some gene therapies, vectors come with “a litany of frustrations,” says Masaru Rao, a mechanical engineer at the University of California, Riverside.

Some viral vectors are limited in the size of DNA they can carry, and they integrate that DNA randomly into the genome, risking damaging mutations, Rao explains. The presence of viral particles in the body can, in certain types of gene therapies, induce an innate immune response in patients, he adds. Furthermore, the production of viral vectors, which depends on culturing cell lines, can be difficult to scale up. 

“A non-viral transfection method is critical for the field,” says biomedical engineer Abraham Lee of the University of...

SPIKED CELLS: Cells in suspension are pipetted into a reservoir with an array of cell-size wells each containing a single spike. Microfluidic channels running through the wells generate a suction force that draws cells into individual wells (1), where the spikes pierce the cell membranes (2). The flow is reversed to release the cells (3), which are then mixed with the DNA of interest (4). The DNA diffuses into the cells via the temporary pores, which then close up on their own. WEB | PDF
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Most of the approaches developed so far, however, including electroporation, cell squeezing, and acoustic shearing, indiscriminately disrupt the cells’ membranes to allow the entry of genetic material, says Rao. “The number and size of holes is not well controlled,” he says. As a result, some cells are ripped apart, while others may remain intact but do not take up the DNA. There is often a trade-off between transfection efficiency and cell viability, he explains.

To avoid this problem, Rao and colleagues created a device that generates a single transient pore in each cell, allowing DNA to enter but minimizing the rate of cell death. Using microfluidic manipulations, cells in suspension are guided into individual cell-size wells that are arranged in an array at the bottom of the cell reservoir. Each well houses a single spike, which pierces the cell as it slips into the well. The fluid flow is then reversed to release the perforated cells, which are collected and incubated with the desired DNA before the membrane heals itself. 

The team has optimized flow rates to maximize cell viability and tested the device with various human cell types. The researchers achieved transfection efficiencies of greater than 80 percent for a T cell line as well as T cells isolated from blood. An electroporation protocol optimized for the same T cell line, by contrast, yielded an average efficiency of around 20 percent. 

The device currently pierces 10,000 cells at a time, but could be scaled up to house a larger array and could be automated for high throughput, says Rao. A typical CAR T therapeutic dose is several million to several hundred million cells. “With their fabrication technique, I believe they could [scale up],” says Lee, who was not involved in the project. “This is an elegant technology and . . . a great addition to the field.” (Nano Lett, 20:860–67, 2020)

Non-viral transfection technique
How DNA gets in
Cell viability
Transfection efficiencyNumber of cells transfected at once
ElectroporationA current is passed through a suspension of cells, disrupting the cell membranes.Varies greatly depending on cell type and electrical currentVaries, but in Rao’s report, using a protocol optimized for a human T cell line, 20 percent5 million to 10 million
Deterministic mechanoporationCells are sucked into wells and pierced by a micro-scale spike.
Close to 100 percent
For the same human T cell line, 88 percentCurrently 10,000, but could be scaled up

Ruth Williams is a freelance journalist based in Connecticut. Email her at ruth@wordsbyruth.com or find her on Twitter @rooph.

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