Downsize Your Cell Sorting with Microfluidics

Since the 1960s, cell sorters have enabled researchers to tease apart differences in cells' immunological makeup and gene and protein expression. More recently, fluorescence activated cell sorting (FACS, often called “flow sorting”) has been used to create distinct cell populations for research and therapeutic purposes.

FACS traditionally is performed in core laboratories on large, expensive, operator-dedicated instruments that subject cells to high pressure for high-throughput sorting. These must be cleaned and disinfected between runs to avoid cross contamination, and they may expose operators to toxic or biohazardous aerosols. But a new generation of smaller, microfluidics-driven instruments with disposable cartridges at their core promises to dramatically alter the flow-sorting landscape.

Jet-in-air

A traditional (“jet-in-air”) cell sorter hydrodynamically focuses a stream of cells in a column of fast-moving sheath fluid, where they are interrogated by one or more lasers. As they break off from the stream into droplets, cells that meet the sorting criteria are given an electrostatic charge with which they can be deflected and hence collected. Those of a given size, expressing green fluorescent protein (GFP) and the CD4 antigen bound by a cherry red-labeled antibody, for example, may be diverted into one well, while GFP+ CD4- cells are sent into another and the rest go to waste.

Vendors boast sorting speeds upwards of 100,000 cells per second, “but users say that the rate to get good sorting is 10 to 20,000 per second,” says David Weitz, Mallinckrodt professor of physics and applied physics at Harvard University. “Really precise sorting is really much slower than the maximum.”

Still, fluids moving through air at such speeds can create aerosols. The cytometric community recommends taking the precaution—at least—of wearing a respirator and housing the cytometer in a containment hood when sorting infectious or pathogenic cells and those of primate origin [1].

There’s also an issue of cell health. Sorted cells are subjected to “about 20 [to] 30 psi [pounds per square inch], which is like the pressure in your tires,” notes Isaiah Luke Hankel, global product manager for Miltenyi Biotec. This can affect viability—only about 70% to 80% of cells come through the sorter alive—and behavior. “The phenotype can change at those high pressures as cells are flattened against the fluidic walls.”

Blu-ray

By constraining cells to tight, narrow, well-defined microfluidic pathways in which they can be manipulated, flow sorters have been made that circumvent some of these pitfalls while still offering capabilities seen in larger, more conventional sorters.

Sony Biotechnology, for example, made use of its parent company’s expertise in Blu-ray technology to design a plastic, disposable microfluidic chip where the sample gets hydrodynamically focused and the cells can be precisely interrogated by laser. “You just put in that chip like a DVD, it auto-aligns the lasers and then sets up the instrument for sorting in an automated fashion,” explains global product manager Deena Soni.

The Sony Biotechnology SH800 is ready to sort within 15 minutes of being turned on and can be operated by a user with only minimal training—this instead of the typical 45 minutes for a skilled operator to manually align and tweak the system before running samples. The system boasts six fluorescent channels plus forward and side scatter, and it can accommodate up to four lasers.

Peter Lopez, director of the flow cytometry and cell sorting center at the NYU Langone Medical Center, sees the SH800 as occupying a grey zone between traditional and microfluidic sorting. It focuses by using a chip, he says, “but the sorting is still done in a conventional way”—with droplets leaving the chip to be diverted into tubes or microplates. “So there’s an aerosol generated there.”

Microfluidics

Several more wholly microfluidic sorters are in various stages of beta testing, and others are in earlier stages of design.

For example, Miltenyi Biotec plans in the next few months to roll out its closed-system, walk-away MACSQuant Tyto. “Here, every experiment is done in a separate, one-use cartridge, which is completely sterile, with no possibility of contamination and no aerosols given off,” says Hankel.

The MACSQuant Tyto requires no sheath fluid, using instead the media the cells are already in. And cells run through at 4 psi—close to normal atmospheric pressure—leaving at least 95% of them viable, Hankel reports. But the instrument is no slouch: Featuring eight color channels plus side and two backscatter channels, it can sort up to 50 million cells per hour (about 14,000 per second).

The current iteration of Cytonome’s GigaSort™—available to collaborators for early access—uses 24 microfluidic chips in parallel to sort up to 48,000 cells per second, says Jack Lapidas, vice president of business development. The high-end, single-laser, four-color, two scatter-channel instrument will be used “mostly by the translational clinical market,” as well as for high-throughput screening, where speed, safety and gentleness are of primary concern, Lapidas says. Subsequent instruments will include up to 72 parallel microfluidic channels.

At the other end of the spectrum, NanoCellect Biomedical president and CEO Jose Morachis wants to “tap into the 50% to 80% of the market that wants to sort GFP+ cells and the very common stains” in a safer, gentler way. He envisions the company’s initial offering—to be launched “ideally in 2015”—as a single-laser, two- to three-color, “nice compact system that can be affordable and accessible to most researchers … [for] under $100,000,” Morachis says. Cells emerge from the microfluidic channels in one of three sterile pools: sorted A, sorted B and unsorted, which can be sorted again, if desired.

In the works

Academic labs and start-ups are also in the microfluidic sorting game. Tony Huang, professor of engineering science and mechanics at The Pennsylvania State University (Penn State), for instance, founded Ascent Bio-Nano Technologies, a company that is using sound to sort cells. This “very early stage—more like a research-lab/proof-of-concept” company, develops devices based on standing surface acoustic waves (SSAW) that use the same frequency as sonic (ultrasound) imaging, Huang says, adding that “SSAW uses sound to do both [cell] focusing and sorting.”

Weitz’s lab uses a different acoustic-wave technology to sort cells within picoliter droplets after first hydrodynamically focusing them inside a microfluidic chip. This enables throughput comparable to traditional jet-in-air sorters, but in a gentler way and with no aerosols—and without the need for “gallons of sheath fluid,” Weitz says.

“Everything would be flowing through a chip, and you could just mount it in a way that would be automatically aligned,” Weitz explains. He adds, “Furthermore, the optics would be much easier, because you have flat faces from the chip and you wouldn’t have to try to match some sort of semi-fluidical flow. And the cleanup and the preparation would be much easier, because you just replace the chip.”

The resulting microfluidic FACS machines could possibly be built for $40,000 to $50,000, he says—“the sort of cutoff line where people can afford to buy them individually.”

Reference

[1] Holmes, KL, et al., “International Society for the Advancement of Cytometry cell sorter biosafety standards,” Cytometry A, 85:434–53, 2014. [PubMed ID: 24634405]