Innovation in Cell Sorting: When FACS and MACS just won’t cut it

Cell sorting is generally a must-do when it comes to biomedical research. Cell sorting may be used to enrich, identify, or evaluate a certain cell type of interest. The most common and highly effective methods of cell sorting are fluorescence- or magnetic-activated cell sorting (FACS or MACS, respectively). FACS and MACS are universally used in fields such as oncology, immunology, and regenerative medicine, just to name a few. Considered the gold standard, FACS and MACS offer high sensitivity and/or high throughput marker-based cell sorting. FACS and MACS, however, may not always meet the needs that innovative science requires. Here, we explore these common cell sorting techniques, discuss how they work, their advantages and limitations, and how CytoRecovery offers a unique solution to a problem not yet resolved by other cell sorting techniques.

So, what are FACS and MACS and how do they work?

To get an idea of the capabilities of FACS and MACS, let’s discuss how they work.

Fluorescence-activated cell sorting (FACS) uses a fluorescent antibody to tag or label the cells of interest in a mixed suspension. The sample is then siphoned into the instrument where single cells encapsulated in a single droplet of buffer pass through a laser and detectors and, subsequently, the deflector. As the laser shines on each single cell, the detector measures light scattering due to the particle. Depending on the fluorescent signal of the particle as received by the detectors, the instrument assigns a charge to the droplet (e.g. red particles might be assigned a negative charge, green particles might be assigned a positive charge, and unlabeled particles will remain uncharged). The charged droplets will then move through the deflectors, and the droplet will be deflected from the center flow stream and towards a plate of opposite charge, thus resulting in a sorted sample.

Magnetic-activated cell sorting (MACS) also requires labels, but instead of a fluorescent label, a magnetic label is used to tag cells. Once the sample to be sorted has been appropriately labeled with magnetic beads, the sample is then exposed to a magnetic field and the labeled cells migrate towards the magnetic field, allowing the labeled and unlabeled cells to be sorted. The configuration of this magnetic field may change. For example, the column-free approach applies the magnetic field to the vessel holding the mixed sample, causing the tagged cells to move toward the wall and allowing the user to remove the supernatant to collect the unlabeled cells. The column-based method, on the other hand, is a flow-through-based method that flows the mixed sample through a column. The magnetic field is applied to the column, causing the tagged cells to remain in the column and the unlabeled cells to flow through. When the magnetic field is released, the tagged cells are also released.

When should I choose FACS and MACS and when should I consider an alternative approach?

Though similar in function, FACS and MACS thrive in their respective applications. FACS, for example, is an excellent option for sorts that require high sensitivity and purity. FACS, compared to MACS, is also a safer option for fragile cell types. Sorts that do not require high purity and benefit significantly from greater throughput should yield to MACS as an option for cell sorting. Both FACS and MACS are integral technologies in biological research and the choice between the two is ultimately up to the needs of the user.

These technologies, though widely implemented, have several limitations. Perhaps most significantly, both techniques require labels. This means that a label must exist for the population you are interested in and be specific enough to distinguish the populations of interest. These limitations significantly hinder some fields of research, such as those involving rare cell types, stem cells, and precision medicine, where a marker does not exist or lacks sufficient specificity. Both methods are also greatly impacted by sample preparation, are highly susceptible to contamination, and may damage delicate cell types due to the forces and pressures associated with each method. There remains a need in the field of biomedical research for a gentle, label-free sorting technology and a means for improved sample preparation prior to FACS or MACS sorting.

For more on the limitations of using FACS or MACS as strictly sample preparation techniques for downstream culture and analysis, check out the blog post titled, “The Most Overlooked Challenge in Biomedical Research: Sample Preparation.”

FACS nor MACS address my cell sorting needs. What innovation does CytoRecovery bring to the table?

CytoRecovery’s technology opens the door to emerging fields and enhanced sample preparation with our innovative platform for label-free cell sorting. Even without a label, cells innately have unique electrical signatures that represent and change with their morphology. By applying an electric field gradient across the CytoChip^TM, cells in suspension are separated based on their individual electrical signatures. The phenomenon harnessed by the CytoR1^TM Platform causes cells of interest to migrate to specified areas of the chip. While cells of interest are maintained in designated regions in the CytoChip, unwanted cells are unresponsive to the applied electric field and continue flowing through the chip for waste collection. When the unwanted cells are cleared from the sample channel, the electric field can be turned off, and the previously trapped desired cells will be released to collect at the outlet of the device. CytoRecovery’s solutions birth innovation into the field of cell sorting by manipulating cells dependent on their inherent properties without any labels or modifications to the cell. CytoRecovery returns Your cells. As They Are. Sorted.

Using the CytoR1 Platform, users can sort cell suspensions without a label OR improve the quality of their sample prior to performing FACS or MACS. The CytoR1 platform has been used for high-purity label-free sorts such as stem cell isolation or characterizing metastatic potential of cancer stem cells, where well-defined labels were lacking.^1-3 The CytoR1 Platform is also a gentle option for even your most fragile cells with the capability to maintain >90% viability after sorting. Additionally, the CytoR1 Platform 

excels in sample preparation by removing non-viable cells and debris from a mixed sample, not only purifying the sample itself but also improving the sample for subsequent FACS and MACS facilitating easier multiplex sorting and heightened accuracy.

Key Takeaways:

• CytoRecovery’s CytoR1 Platform is a gentle, label-free cell sorting platform to be used independently or in conjunction with label-based sorters for improved accuracy in your cell sorting applications
• FACS and MACS require well-defined labels for sufficient cell sorts which, in turn, limits innovative research where these labels have yet to be discovered.
• FACS and MACS might not be the best option for delicate cells due to the force exerted on the cells during sorting. The CytoR1 Platform maintains and enriches for viable cells.

References:

1. Salmanzadeh, A., Sano, M. B., Gallo-Villanueva, R. C., Roberts, P. C., Schmelz, E. M., & Davalos, R. v. (2013). Investigating dielectric properties of different stages of syngeneic murine ovarian cancer cells. Biomicrofluidics, 7(1), 011809. https://doi.org/10.1063/1.4788921
2. Salmanzadeh, A., Kittur, H., Sano, M. B., Roberts, P. C., Schmelz, E. M., & Davalos, R. v. (2012). Dielectrophoretic differentiation of mouse ovarian surface epithelial cells, macrophages, and fibroblasts using contactless dielectrophoresis. Citation: Biomicrofluidics, 6, 22811. https://doi.org/10.1063/1.3699973
3. Alinezhadbalalami, N., Douglas, T. A., Balani, N., Verbridge, S. S., & Davalos, R. v. (2019). The feasibility of using dielectrophoresis for isolation of glioblastoma subpopulations with increased stemness. ELECTROPHORESIS, 40(18–19), 2592–2600. https://doi.org/10.1002/elps.201900026