by Caitlin Smith
How do you separate different cell types in a complex suspension of cells? You may use different methods depending on whether your cells are a rare subset, or have a tendency to bind to anything. “The biggest challenge in cell separation is meeting the varying needs of the scientist,” says Vicki Stronge, product manager for immunology at StemCell Technologies. “It’s an exciting time in the field of cell separation. Researchers have come to expect robust, consistent and high-performing cell separation systems that enable them to perform cutting edge research quickly.” Franco Fracella, head of global e-marketing at Miltenyi Biotec, sees the widening range of separation targets exciting too. “The possibility to isolate even extremely rare immune cell subsets and progenitor cells with excellent purity, enabling us to perform a detailed analysis of their functional properties, tremendously increased our knowledge and understanding in different exciting research fields,” he says. This is possible because of the wealth of tools available for cell separation today.
Separation by negative selection
Negative selection allows researchers to deplete samples of unwanted cell types. “[This] is proving to be of great advantage to scientists who want untouched, functional cells that are ready for use directly in downstream assays,” says Stronge. “With its column-free EasySep® technology, StemCell’s cell separation kits are gentle on cells, perfect for sensitive, fragile or bulky cell types such as granulocytes or dendritic cells.” Their recent releases include kits for isolating antigen-presenting cells by negative selection, such as the EasySep® Human Plasmacytoid DC Enrichment Kit, and the EasySep® Mouse Monocyte Enrichment Kit. They have further optimized their EasySep® Human CD4+ T Cell Enrichment Kit for better recovery in fast, negative isolation of purified CD4+ T cells.
StemCell Technologies also offers a separation system for blood cells. Their RosetteSep® is an “immunodensity cell separation system that isolates specific cell types directly from human whole blood without the use of columns or magnets,” says Stronge. “RosetteSep® works by cross-linking unwanted cells to red blood cells, [and] purifying the desired cells during a standard FicollTM spin. Purities of up to 95% can be achieved without the use of magnetic particles, and with no need for special equipment.”
Separation by positive selection
Positive selection allows scientists to pull out specific known cell types, usually using an antigen on the cell surface. Many manufacturers of superparamagnetic beads make systems that allow the beads to be used for positive or negative selection. For example, BD Biosciences’ IMag Cell Separation System includes beads that are covalently labeled with monoclonal antibodies or streptavidin. Typically, the cell type of interest binds to the monoclonal antibody and is retained when a magnetic field is applied by the BD IMagnet, allowing other cells to be removed.
Miltenyi Biotec’s MACS cell separation system is similar in that it can be used for positive or negative selection, using labeled magnetic beads. Their MACS MicroBeads, about 50 nm in diameter, are labeled with the monoclonal antibody of interest. After binding to target cells, the beads are retained in a MACS column by the magnetic field imposed by the MACS separator. According to Miltenyi Biotec, the non-toxic MACS MicroBeads are biodegradable and do not compromise viability or cellular functions in subsequent flow cytometry or cell culture, for example.
Like many manufacturers of magnetic beads, Miltenyi Biotec’s system can be used to label almost any cell type for which you have a primary antibody. However, they also strive to supply labeled beads for nearly every interest. “We are continuously extending the range of cell separation products to new areas of scientific research,” says Fracella. Miltenyi Biotec recently released reagents for isolation of cells secreting IL17, which are important in inflammatory diseases. “The reagents are based on the unique cytokine secretion assay technology allowing the direct isolation of viable, cytokine-producing cells from single-cell suspensions of different tissues,” says Fracella. Miltenyi also released a kit to isolate basophils from peripheral blood for allergy research, and reagents to isolate neural mouse stem cells, glial progenitor, or microglial cells for neuroscience research. They also have two new kits for stem cell research: the human Anti-MSCA-1 MicroBead Kit (for mesenchymal stromal cell antigen-1, a marker for bone marrow MSCs with high proliferative potential); and mouse Anti-SSEA-1 MicroBeads.
(a division of Life Technologies) also makes magnetic beads, but they coat them with an inert polymer to preclude the possibility of metal leakage into solution. Their Dynabeads are further labeled with modified streptavidin. In the Dynabeads® FlowComp™ system, cells are incubated with modified-biotin-tagged antibodies to mark target cells. Subsequent addition of Dynabeads results in the targeted cells bound to the beads, which can be pulled down with magnetic force and unwanted cells removed in the supernatant. With the target cells isolated, the addition of FlowComp™ buffer releases the cells from the Dynabeads. Invitrogen’s system is especially suitable for those who might be concerned about beads remaining on cells. “The Dynabeads® FlowComp™ technology is a method for positive cell isolation directly from human whole blood or from mouse/human mononuclear cells (MNC), leaving your cells bead-free,” says Berit Reed, Dynal FlowCompTM product manager at Invitrogen. “The bead-free cells can then be directly analyzed by flow cytometry and used in any downstream application.”
Automated cell separation and sorting
Special equipment is usually required for automated separation, but some are seeing such an investment as an important milestone. “As cell separation becomes a routine function within many labs, researchers are looking to automate their cell separation systems, freeing up their time to prepare for subsequent experiments,” says Stronge. StemCell Technologies’ RoboSep® is a fully-automated cell separator, which as Stronge notes, is ideal for infectious disease labs who like to reduce handling times. “Using column-free EasySep® technology, RoboSep® performs all steps necessary to label and separate virtually any cell type by positive or negative selection on up to four samples simultaneously,” she says. “With customizable protocols, RoboSep® can be optimized for any cell separation, allowing automation to become a part of even the most specialized cell separation procedure.”
Miltenyi Biotec’s autoMACS™ Pro Separator is a high-speed automated cell sorter for positive or negative selection using MACS® technology. Up to six samples can be processed simultaneously. The autoMACS™ Pro Separator works with the over 200 MACS® cell separation reagents available, and can sort cells directly from whole blood or bone marrow. “Isolation of rare cell subsets can now also easily be achieved in an automated fashion using the autoMACS™ Pro Separator for highly reproducible results,” says Fracella, noting examples of sophisticated cell separation kits as the human CD4+ Central Memory T Cell Isolation Kit, the human CD4+ Effector Memory T Cell Isolation Kit, the human EPC Enrichment and Enumeration Kit, and the Diamond CD34 MicroBead Kit.
Fracella believes that a major obstacle standing between you and reliably separated cells is “the proper preparation of the starting material: single-cell suspensions from different tissues or peripheral blood,” he says. To facilitate this, Miltenyi Biotec developed the gentleMACS™ Dissociator, an instrument for automated tissue dissociation, whose optimized protocols give more reproducible results. Hopefully new tools such as these will help you separate your work from the rest.