For decades, biologists have used flow cytometry-based cell sorting to isolate cells from complex samples like blood or serum. This involves labeling the cells of interest with antibodies before introducing them—along with any unlabeled cells—into a cell sorter via a nozzle. As the cells enter the sorter, they are injected into a stream of sheath fluid that is forced through the system under high pressure. They then move past an interrogation point, and the stream is partitioned into droplets each containing a single cell.
By applying a charge to the droplets containing the cell type of interest, the cell sorter directs the target cells into a collection tube as they pass through an electric field, allowing them to be used for further analysis. This technique, known as electrostatic droplet-based cell sorting, can broadly be divided into two categories—jet-in-air cell sorting or cuvette-based cell sorting—depending on the nature of the flow cell.
Although both approaches offer distinct advantages, a major problem with electrostatic droplet-based cell sorting in general is that it can be damaging to many cell types. This is largely due to the combination of high pressure, electric charge, and the frequency at which high-speed collisions occur within a conventional cell sorter.
As well as causing morphological changes, these factors can lead to delayed cell growth, decreased production of cellular metabolites, functional changes, and even cell death—a set of highly undesirable cellular events that have collectively been termed Sorter Induced Cellular Stress, or SICS (Delay, M., Lopez., Schiemann, M. CYTO 2018).
Irrespective of how the sorted cells will be used, minimizing SICS is essential for downstream studies to be reliable and reproducible. While best practice guidelines have been established to reduce SICS during conventional sorting, they are often ineffective, particularly when sorting extremely large or sensitive cell types. This has restricted cell sorting mainly to the study of immune cells, despite its vast potential to advance many other fields of research.
Neuroscience is one such field that could benefit from gentler cell sorting. Although conventional sorting techniques often have little impact on the viability of neurons, they can prevent neurite outgrowth, even after sorted cells have spent several days in culture.
The field of reproductive research is another example. For instance, a large proportion of sperm sorted via electrostatic droplet-based techniques have poor mobility and have been found to be unsuitable for in vitro fertilization.
Spheroid sorting is also problematic. Not only are spheroids frequently too large to pass through the nozzle but, when they do make it into the cell sorter, they are easily damaged by the high shear stresses required to isolate them.
Yet another challenging application is the removal of rare, undifferentiated cells from induced pluripotent stem cell cultures prior to transplantation. This is essential to prevent tumors from developing, yet undifferentiated cells can often slip through the net using established cell sorting techniques.
Various alternative technologies have been developed to overcome the limitations of conventional cell sorting.
One promising approach is air-over-liquid flow shift cell sorting, which differs mainly from conventional techniques in that it uses pressurized air to generate a short liquid pulse for directing the target cells into the collection reservoir. By eliminating all of the damaging steps involved in conventional cell sorting, flow shift cell sorting has been proven to reduce SICS in many different cell types.
These include sensitive cell types such as neurons and sperm cells, and large particles such as spheroids. Flow shift cell sorting has also been successfully used to remove rare, undifferentiated cells from iPSC.
As well as providing better quality cells, flow shift cell sorting offers several other advantages over conventional techniques. First, because the technology is based around a small, disposable microfluidic chip, the sample, sheath fluid and waste are contained throughout analysis and sorting. This removes the risk of sample-to-sample contamination, or contamination to the instrument itself, enhancing data quality.
Second, since the sample and the sheath fluid are both loaded on to the chip during flow shift cell sorting—rather than being introduced directly into the cell sorter— the choice of sheath fluid is no longer dictated by the instrumentation. This means the sheath fluid can be customized to suit the target cell type, providing a consistent environment between culture and sorting to further help prevent SICS.
Flow shift cell sorting reduces SICS for better quality cells that suit a broader range of research applications. Why not give it a try with the On-chip Sort? To learn more, visit onchipbio.com
More Info