by Catherine Shaffer
Scientific advances in the area of stem cell therapies have created an increasing demand for laboratory tools for separating cells in body fluids and tissue cultures. Innovations such as tetrameric antibody and immunomagnetic labeling have revolutionized the field, streamlining experiments that once required many days and many sample preparation steps. Additionally, older techniques have been refined and improved for new applications.
Centrifugation
One of the oldest and most reliable methods for separating cells is centrifugation. The principle is based not only on separation by mass, but by shape as well. One method of centrifugation is sedimentation by cell density using an ordinary centrifuge tube. This can be done by benchtop or high performance centrifugation, depending on the degree of separation desired. Simple pelleting is often conducted on bench top centrifuges in centrifuge or microcentrifuge tubes. By using a number of spins on different instruments, you can refine the separation to zero in on cells of interest. “We accommodate a wide variety of tubes and labware as well as range of g-force to ensure accurate separation of mammalian and microbial cells alike,” says Barbara Keller, business manager for centrifugation at Beckman Coulter.
Where does flow cytometry fit in?
Cell elutriation, which uses a specially designed conical separation chamber, separates cells based on size rather than density. Both simple centrifugation and elutriation are nonspecific with regard to cell type. A number of technologies have been developed to improve the specificity of the separation. At the opposite end of the specificity spectrum (from centrifugation) is flow cytometry , which analyzes and separates cells one at a time. Often, it is desirable to combine methods in multiple steps to achieve optimal separation. Separating raw fluids or blood by flow cytometry would be a very tedious and time-consuming process. To enrich the samples in preparation for flow cytometry, or as a final separation in its own right, is the magnetic or immunological separation—basically mechanical methods that isolate cells of interest in a specific manner.
Magnetic Cell Separation
Magnetic cell separation is one of the easier and more popular methods to isolate cells and is available from a number of vendors, including Miltenyi Biotec, BD Biosciences, and StemCell Technologies. Antibodies are used to tag cells of interest with a small magnetic particle. In the Miltenyisystem, the sample is processed through a column that generates a magnetic field when placed within the separator instrument, retaining the labeled cells.
Other vendors, such as BD Biosciences and StemCell Technologies, offer simplified versions of the magnetic separator. Instead of a column and separator instrument, these systems use a simple magnet to directly retain the labeled cells within the tube, while the supernatant is drawn off. The BD IMag™ Cell Separation System can be used in a positive or negative selection manner. Negative or enrichment selection means that unwanted cells can be labeled (captured), leaving the cells of interest label-free. “With our magnetic cell separation system, it gives the researcher a very convenient method to pre-enrich their cell suspensions before they run any further downstream sorting through flow cytometry,” says Marie Hammonds, marketing product manager for BD Biosciences. The magnetic particles do not interfere with flow cytometry , nor do they interfere with cell growth, according to Hammonds, so cells that have been isolated using BD IMag™ system can be further cultured.
StemCell Technologies takes magnetic separation to the next level of convenience with RoboSep®, a fully automated version of the simple magnetic separation. The RoboSep® instrument adds all reagents required in the separation, eliminating time-consuming sample preparation steps.
The grandfather of magnetic cell separation technologies is the Dynal Dynabead® system, owned by Invitrogen. Dynal's innovation on magnetic cell separation is encasing the iron coating of the Dynabead® magnetic particle with a polymer coating to protect the cells from exposure to iron or dextran, which can have cytotoxic or immunogenic properties. Invitrogen's published statistics on recovery and purity are 83% (for CD4+ T cells) and 99%, respectively. Like the BD IMag™ and StemCell Technologies’ EasySep/RoboSep® systems, the Dynabead® system is column-free.
Magnetic separation has proven uniquely powerful and broadly applicable. Muniz and Leff of the University of Chicago developed a method for isolating eosinophils from human blood using a column-based negative magnetic separation that resulted in 70% recovery and 98% purity while retaining cell viability—all in less than four hours1. And Mavrou, et al., from the Athens University School of Medicine in Greece, developed a technique for potentially screening maternal blood for fetal aneuploidy using a magnetic cell separation system targeted to CD71 surface antigens of fetal nucleated red blood cells (NRBC). Their results showed that fetal aneuploidy resulted in a higher number of NRBCs in the blood, paving the way for the development of a screening test based on magnetic cell separation technology.2
StemCell Technologies also offers an efficient non-magnetic separation method, RosetteSep®. This proprietary technology was developed internally, based on work on tetrameric antibody complexes (TACs) done externally by Terry Thomas and Peter Lansdorpe at the Terry Fox Laboratories of the BC Cancer Agency. RosetteSep® works by linking unwanted cells in whole blood to the red blood cells using TACs. After labeling, the sample is layered over a buoyant density medium such as Ficoll. The labeled cells pellet with the RBCs when centrifuged, while the desired, unlabeled cells are recovered at the interface.
Applications for RosetteSep® include enrichment of lymphoid and myeloid cells for donor/recipient chimerism, T cell isolations for HIV/AIDS research, and many others. “It's very popular because it's fast,” says Steve Woodside, PhD, senior scientist at StemCell Technologies. “The cells you get back are not labeled with antibodies. You are getting rid of the cells you don't want. The cells you do want are untouched.”
Many of these techniques are most powerful in combination. Jing, et al., from the Cleveland Clinic, combined magnetic and TAC labeling to isolate hematopoietic progenitor cells by continuous magnetophoresis, resulting in purity up to 85% and yield as high as 84%3. This work represents an example of how these research techniques can be scaled up and adapted to clinical applications, where there is great need for diagnostic and therapeutic cell separation.
References:
1 NM Munoz, AR Leff, “Highly purified selective isolation of eosinophils from human peripheral blood by negative immunomagnetic selection,” Nature Protocols, 1(6):2613-20, 2006.
2A Mavrou et al, “Identification of nucleated red blood cells in maternal circulation: a second step in screening for fetal aneuploidies and pregnancy complications,” Prenatal Diagnosis, 27(2):150-3, Feb 2007.
3Y Jing et al, “Negative selection of hematopoietic progenitor cells by continuous magnetophoresis,” Experimental Hematology, 35(4):662-72, Apr 2007.
