Flow cytometry-based cell sorting is widely used to isolate cells of interest from a heterogeneous population. However, the electrostatic droplet-based cell sorting techniques (jet-in-air and cuvette-hybrid) employed by most conventional cell sorters can be damaging to many cell types, causing a phenomenon known as Sorter Induced Cellular Stress (SICS) (DeLay et al., 2018; Lopez et al., 2020) and limiting the utility of sorted cells for downstream research. One way of mitigating SICS is to sort cells by a gentler method that avoids the use of high pressure and electric charge, and that allows the sheath fluid to be customized to the cell type. In this article, we consider the benefits of switching to modern technologies for sorting cells and explain how replacing the conventional techniques with microfluidic chip-based air-over-liquid cell sorting can significantly reduce SICS.
Most conventional cell sorters rely on a jet-in-air or cuvette-based technique to isolate cells. After being introduced to the cell sorter via a nozzle, immunolabeled cells are injected into a stream of sheath fluid that is forced through the system under high pressure. As the cells move past an interrogation point, the stream is partitioned into droplets (each containing a single cell) and a charge is applied to each droplet containing the target cell of interest. These charged droplets are then directed into a collection reservoir as they pass through an electric field, allowing the cells to be used for further analysis.
The combination of high pressure, electric charge, and the frequency at which high-speed collisions occur within a conventional electrostatic droplet cell sorter can all result in undesirable cellular changes. These include morphological changes, delayed cell growth, a decrease in the production of cellular metabolites, functional changes, and even cell death, and have collectively been termed Sorter Induced Cellular Stress (DeLay et al., 2018; Lopez et al., 2020). Irrespective of how the sorted cells will be used, minimizing SICS is essential for downstream studies to be reliable and reproducible.
Although best practice guidelines have been established to reduce SICS when sorting cells using a conventional cell sorter (e.g. increasing the size of the nozzle and reducing the sheath pressure), these measures are often ineffective, particularly when sorting extremely large or sensitive cell types. Neurons can be especially difficult to work with, often failing to demonstrate neurite outgrowth after conventional sorting, whereas a large proportion of sperm that are sorted via electrostatic droplet-based techniques have been found to be unsuitable for in vitro fertilization (IVF).
Spheroid sorting can also be problematic using conventional methods. Spheroids are easily damaged by the high shear stresses required to isolate them into a uniformly sized population for downstream applications and, in many cases, are too large to pass through the nozzle. Another challenging application is the removal of undifferentiated cells from induced pluripotent stem cell (iPSC) cultures prior to transplantation; achieving a pure population is critical to prevent undifferentiated cells from becoming tumorous following implantation.
A growing awareness of the damage conventional sorting can cause has led many researchers to consider moving away from electrostatic droplet-based techniques to sort cells. Included among the various alternative technologies being developed, air-over-liquid flow shift cell sorting has proven successful at reducing SICS in many sensitive cell types, including neurons and sperm cells, and in large particles such as spheroids; it has also enabled multi-step negative sorting to be used for removing rare, undifferentiated cells from iPSC.
Image: Images of fetal rat brain hippocampus cells without sorting (a), and sorted by electrostatic droplet based-cell sorter (b) and microfluidic chip-based cell sorter, On-chip Sort (c), and cultured for three and seven days.
Instead of applying a charge to isolate cells, air-over-liquid cell sorting uses pressurized air to generate a short liquid pulse that deflects the target cells into the collection reservoir. This approach eliminates all of the damaging steps involved when cells are sorted by conventional methods, and also provides several further advantages. First, because flow shift technology is based around a small, disposable microfluidic chip, all of the fluids required for a cell sorting experiment (sample, sheath fluid, and waste) remain fully contained throughout analysis and sorting. The risk of sample-to-sample contamination or contamination to the instrument itself is therefore removed, contributing to improved data quality.
Image: Air-over-liquid flow shift cell sorting mechanism on disposable microfluidic chip of microfluidic chip-based cell sorter, On-chip Sort.
Additionally, since both the sample material and the sheath fluid are loaded on to the chip during air-over-liquid cell sorting—rather than being introduced directly into the cell sorter—the choice of sheath fluid is no longer dictated by the instrumentation. As such, the sheath fluid can readily be customized to suit the cell type under investigation, providing a consistent environment between culture and sorting to help prevent any unwanted cellular change.
The On-chip Sort is a microfluidic chip-based cell sorter that provides damage-free cell sorting and significantly reduces SICS for a diverse range of cell types. To learn more, visit on-chipbio.com/onchip_sort
DeLay, M., Lopez, P., Schiemann, M. Cell sorting for function and viability. CYTO 2018, Prague, Czech Republic.
Lopez, P., Rose, R.E., Ryan, K., Jones, D.R. A review of Sorter Induced Cellular Stress (SICS). CYTO Virtual 2020.