Fundamental research often involves the study of isolated cell populations. It is these enriched populations that enable researchers to make new discoveries about cell function, signaling, gene expression, fate decisions, and much more. Techniques for the rapid and accurate enrichment of target cell populations are an area of great interest. Cell sorting techniques fall into two general categories: bulk sorting and single cell sorting. In bulk cell sorting all of the target cells are collected in one sweep, whereas in single cell sorting every cell is individually analyzed. There are multiple methods of bulk cell sorting: filtration, centrifugation, and magnetic cell sorting. The main single cell sorting method is flow cytometry or fluorescence activated cell sorting. While cell sorting can be very accurate, it is hard to say that a sorted cell population is “pure”. Instead, the collected population is referred to as “enriched”. In general, single cell sorting results in highly enriched cell populations that are more homogeneous than those obtained via bulk sorting methods.
Centrifugation and Filtration
These bulk cell sorting techniques rely on cell characteristics like size and density. Filtration requires a membrane with a consistent pore size smaller than the target cell, but larger than the unwanted debris. Membranes with a variety of pore sizes are available to select from. Centrifugation is commonly used for initial separation of blood into plasma, white blood cells, and red blood cells. Density gradient centrifugation improves the separation process by adding a material with a density between the white blood cell fraction and the red blood cell fraction; this allows for a clean separation between the two. The heavier, or more dense cells fall to the bottom during centrifugation, while the less dense plasma remains at the top. These techniques are useful for rough separation of cells; more specialized techniques are needed for enriching specific cell types based on cell surface markers.
Magnetic Cell sorting
Magnetic cell sorting is a bulk enrichment technique that, can be highly specific. Magnetic sorting using superparamagnetic nanoparticles introduces a high degree of specificity to a cell enrichment protocol. Superparamagnetic nanoparticles are made of a core of iron oxide, typically magnetite (Fe3O4), which is not innately magnetic, but becomes magnetized by an applied magnetic field. These particles or beads are coated with silica or a polymer surface to prevent clumping, and a well-chosen coating also provides a rich surface for the covalent attachment of functional groups and antibodies. The attachment of antibodies provides the superparamagnetic particles with specificity. The functionalized particles 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 enriched by magnetic cell separation. Magnetic cell sorting is a good choice when specificity is desired. Other bulk sorting methods such as filtration, sedimentation, and centrifugation cannot achieve such specificity. Magnetic sorting is rapid and efficient. Advanced biomagnetic separation systems can increase sort accuracy by providing standard curves and optical monitoring of the sorting process. Additionally, advanced separation systems are engineered to provide gentle and consistent magnetic forces throughout the working volume to increase cell viability.
Flow Cytometry fluorescence-based cell sorting
Cell sorting by flow cytometry analyzes each cell individually. This is an extremely powerful technique that can provide a large amount of information at once. Flow cytometry is an ideal quantification method for multiplex immunoassays. The cells are incubated with fluorophore-labeled antibodies before the sort. The antibodies are specific to surface antigens on target cells. Each antibody has a different emission wavelength and is uniquely identifiable. One method of precisely labeling antibodies with fluorophores is on-bead labeling with protein A.
After incubation with the labeled antibodies, the cell solution is sent through the flow cytometer. This machine guides the solution through a micron-sized nozzle one cell at a time. Each cell moves through a laser excitation area, where the laser excites the flurophores bound to the cell surface. The fluorescent emission is recorded, and the cell is directed either into a collection container or a discard container according to user-defined parameters. Multiple cell types can be enriched in a single run, and quantitative information about cell numbers and percent of total population are simultaneously recorded.
Flow cytometry is a highly informative method, and is the most precise cell sorting technique, but it is also very expensive. The machine itself is often prohibitively expensive, and requires a trained operator. 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 are turning back toward magnetic sorting techniques. Magnetic separation is specific, rapid, and efficient when care is taken to develop and finely tune a sorting strategy.