The isolation of specific cells is the prerequisite step for many research endeavors, including those studying cell function or signal and gene expression. Cell sorting techniques that enable the rapid and accurate enrichment of target cell populations in a complex heterogeneous solution are therefore an area of substantial interest.
Clinical and experimental studies often require highly purified cell populations. This means tha purified cells produced by a cell sorting technique should be of high purity, and also should be easily recoverable and produced in a significant quantity. Overall, a main goal of any cell sorting technique is to develop and optimize a highly efficacious and throughput process. Based on the different principles used, cell sorting techniques can be categorized into two general categories:
- Cell sorting techniques based on physical properties: Cell sorting may utilize physical factors of the target cell including density, size, electric charges, and deformability. Two of the most common techniques include density gradient centrifugation and membrane filtration. The advantage of physical cell sorting techniques is that target cells do not require labeling, meaning that the structure does not need to be chemically ;modified for the procedure.
- Cell sorting techniques based on biological characteristics: Cell sorting techniques may also be achieved through affinity-based methods, which rely on the tendency of a compound or molecule to chemically attract and react with the target cells. Two common methods include Fluorescence-Activated Cell Sorting (FACS) and Magnetic-Activated Cell Sorting (MACS). These techniques are highly specific, and have versatile use in line with other methods.
Density gradient centrifugation, membrane filtration and MACS are categorized as Bulk sorting in which all the target cells are collected together. FACS, a specific type of flow cytometry, is a single cell sorting technique in which each cell is analyzed individually. The “cell enrichment” process is the act of purifying cells for downstream applications such as qRT-PCR, or for further flow-based experiments. Compared to bulk sorting, single cell sorting results in more homogeneous and highly enriched cell populations. All cell sorting techniques, however, work by disrupting and dissociating the cells of interest from their surrounding microenvironment.
Traditional Cell Sorting Techniques
Density gradient centrifugation is commonly used for the initial separation of whole blood into plasma, white blood cells (WBCs), and red blood cells (RBCs). This type of cell separation technique relies on a centrifugal force to push larger, more dense cells to the bottom of the containment vessel while the least dense cells settle towards the top. This technique uses a density gradient media, like Ficoll or Polymorphprep, to aid in the separation and cell recovery process. From least to most dense cells layer in the vessel as plasma, WBCs, density media, then RBCs, respectively.
Alternatively, membrane filtration is a process that uses a selective barrier to separate cells or other particulates based on their size. This cell sorting technique uses a membrane with a known, uniform, pore size and the separation process can either occur through force or gravity. In essence, particulates and cell debris that are smaller than the membrane pores pass through the membrane, while the target cells that are larger than the pores remain trapped on the other side. Membranes can be made out of a number of materials, including those that are synthetic or natural, and come in a variety of pore sizes. Membrane filtration and density gradient centrifugation are both useful cell separation techniques for the rough separation of cells, however, both methods are laborious and error-prone. Therefore, more specialized cell separation techniques using affinity methods are often needed.
Fluorescence Activated Cell Sorting (FACS)
Cell sorting by flow cytometry is a powerful technique that can provide a large amount of information in a short amount of time. FACS is a specialized type of flow cytometry that adds a degree of functionality to the assay, and is an ideal quantification method for multiplex immunoassays. In FACS, resuspended cells are incubated with fluorophore-labeled antibodies, also termed dyes or fluorochromes, before undergoing cell sorting. These antibodies are specific to surface antigens on the target cells, and because each antibody has a different emission wavelength, are uniquely identifiable by their fluorescence in subsequent analysis.
In FACS, first the cell solution is incubated with the labeling fluorophores (e.g., with protein A), and then sent through the flow cytometer where each cell is analyzed one at a time. Each cell moves through a laser excitation area, individually, where the laser excites the fluorophores bound to the cell surface. The resulting fluorescent emission is recorded, and the cell is directed either into a collection tube or a discard tube according to user-defined parameters. Multiple cell types can be enriched in a single run, and quantitative information about cell count and percent of total population are simultaneously recorded.
FACS provides a wealth of information, and although it is an incredibly precise cell sorting technique is also relatively expensive. The machine itself is often prohibitively pricey and requires extensive training for use and data analysis. Many institutions have a flow cytometry research core facility that charges by the hour for use of their machines, and these rates can also be quite high. For this reason, many laboratories look towards magnetic-based cell sorting techniques.
Magnetic Activated Cell Sorting (MACS)
MACS is a bulk enrichment cell sorting technique that can ensure isolation of the desired cell populations with extremely high purity. MACS equipped with superparamagnetic nanoparticles introduces an even higher degree of specificity to the cell sorting technique. Superparamagnetic nanoparticles (also, magnetic beads) are made of a core of iron oxide, typically magnetite (Fe3O4). Magnetite is not innately magnetic, but under the influence of an external magnetic field will become magnetized which polarizes affixed cells. These magnetic beads are coated with silica or a polymer surface to prevent clumping. A well-chosen coating provides a rich affinitive surface for the covalent attachment of functional groups on the magnetic beads such as antibodies, enzymes, lectins, or streptavidin. Like FACS, MACS uses antibody attachment, increasing specificity to target cells. In the process antibodies are attached to magnetic beads, the beads are incubated with the target cell solution, and the cells with surface antigens complementary to the antibodies will bind to form a cell-bead conjugate. The conjugates are then enriched by MACS.
Magnetic cell sorting techniques are a good choice when specificity is desired. Other bulk sorting methods such as filtration, sedimentation, and centrifugation-based experiments will not provide you with this level of precision. MACS is also rapid and efficient, and the use of advanced biomagnetic separation systems can increase sorting accuracy by providing empirical standard curves and optical real time monitoring of the cell sorting process. Additionally, advanced biomagnetic separation systems are engineered to provide gentle and consistent magnetic forces throughout the working volume. This overcomes the issue of cell loss and cell death caused by physical, shear, or centripetal stress by torah conventional cell sorting techniques.
How Much Do Cell Sorting Techniques Cost?
Flow cytometers can cost from just below $100,000 to over $250,000. The cheapest flow cytometers are only optimized for one type of cell marker and contain a single laser or cell detection system. Flow cytometers in the mid-range between $100,000 and $160,000 generally have two lasers and can detect a broader range of fluorescence and therefore colors. For example, Bio-rad has a machine that detects 4 colors, and the Scindo XT detects up to 8 colors. More expensive machines have more lasers and features that can support additional sample formats. For practicality and affordability, it is no surprise that more and more laboratories are switching to magnetic separation-based cell sorting techniques. Magnetic separators, comparatively, will only cost you between $1000 to $10,000. Magnetic separation is a specific, simple, cost-effective, rapid, and efficient process when care is taken to develop and finely tune a cell sorting strategy. Download our basic guide to magnetic bead cell separation to learn more about magnetic cell sorting techniques.
Published on august 10, 2023 and updated on august 08, 2024.
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