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Are you suffering from poor cell viabilities after cell sorting? Or have you observed low replating efficiencies and expansion rates of your cells after sending them through a traditional droplet-based sorter? Don`t worry, you are not necessarily doing anything wrong. It`s not you, it`s the instrument!
During cell sorting on a conventional droplet sorter, cells are hydrodynamically focused and linearized before they get packed into little droplets for the actual sorting process. Hydrodynamic focusing requires high pressures that typically range from ~2,0–4,8 bar (~30–70 psi). It is well accepted that even relatively low increases in hydrostatic pressure of up to ~0.2 bar can cause significant alterations in cell behavior, including changes in morphology, cell proliferation, and migration, increased apoptosis, and even changes in gene expression. While these changes in cell behavior can already be observed at 0.2 bar, it is not hard to imagine that exposure to 10–24 times that pressure on conventional droplet sorters may cause severe cell damage
It is widely accepted that decompression and resulting shear forces during conventional droplet sorting are main contributors to cell damage. When cells exit the nozzle, they move from a high-pressure environment (~2–4.8 bar) to atmospheric pressure within milliseconds. Only few studies have investigated the exact effects on cell function and viability, but when comparing this drastic change in pressure to a situation well known to man, the harmful consequences become obvious. Take a deep-sea diver for example. A pressure of 4.8 bar is similar to what you`d experience when diving 48 m deep. A diver that would ascend to normal atmospheric pressure within milliseconds would experience severe decompression sickness, which can even result in permanent paralysis or death. It´s probably not a good idea to put your cells through that!
Cells spend a great deal of their metabolic energy maintaining and actively regulating their various membrane potentials, particularly those of the plasma membrane and mitochondria. Na+, K+, Ca++ and H+ are all key drivers of cells’ ability to sense and respond to their environment, and their appropriate local concentrations across membranes are broadly recognized as key diagnostics of a cell’s “health.” Micro currents, tiny in magnitude, location and time, are perhaps the most proximate measure of responsiveness we have.
The distribution of the electric field in a charged droplet containing an electrostatic cell has not been characterized, but the concern among experimental scientists is long standing. Sorting with charged droplets has been a successful technical strategy to understand purified cell populations for many years now, but it has remained difficult to confidently isolate and understand the effects of applying a high voltage in a small volume, even briefly. New sorting strategies that avoid such issues will help us understand any untoward effects.
In traditional droplet sorters, the high pressures that are typically applied are not applied homogeneously, in time or space. As the cells enter and exit the nozzle, pressure is applied radially and instantaneously, with the sheath fluid squeezing the cells into extended rods far from their natural shape. The sheath fluid moves much faster at its external boundary, setting up a velocity gradient that keeps cells pinned to a narrow channel in the center of the stream; great for optics, maybe not great for cells. In addition, cells are dispensed into collection vessels at high speed, causing potentially harmful shear forces upon hitting the collection liquid.
We know that mechanical distortion is a powerful operator on cell differentiation. Of course cells can distort extensively when they choose diapedesis, wherein blood cells squeeze through micrometer-scale gaps in endothelia, but this nearly fluid behavior is slow and requires extensive remodeling of the cytoskeleton and organelles to achieve it. We can’t be surprised, if violent distortion causes damage.
Learn more about gentle, microchip-based cell sorting on our MACSQuant® Tyto® Cell Sorter.
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