by Caitlin Smith
The once onerous lab task of cell separation has finally turned a corner, according to Vicki Stronge, product manager for immunology at StemCell Technologies. “It has gone from being a time-consuming, cumbersome and laborious task that could take anywhere from a few hours to a full day,” she says, “to a rapid, simple, and efficient technique that can be accomplished in as little as twenty minutes, usually in a single step.”
Equally exciting are the applications that the new and faster cell separation technologies can support. “The biggest challenge is aligning cell separation techniques with new technologies, and making sure the process itself does not introduce issues, for example, microbial contamination, endotoxins, or RNAse with downstream applications,” says Mark Frei, technical marketing specialist for hematology and histology at Sigma-Aldrich. “The cell separation technology used for [cell-based therapies] is one of the most exciting developments we see today. Stem cell therapy has proven effective in the treatment of damaged or diseased tissues. Bone marrow harvesting and transplantation has been used successfully for many years in the treatment of leukemia and other cancers. More recent applications involve harvesting bone marrow from donor patients, isolating and amplifying the stem cells, and intravenously injecting them into patients with once-incurable diseases. As parenteral products can cause severe harm or life-threatening health risk to the patient, stringent technique is important when isolating cells for culture.”
This article looks at some of the latest technologies and instrumentation available for cell separation in terms of centrifugation, visual separation, and immunomagnetic separation.
Centrifugal separation
Density gradient centrifugation is one of the most accepted means of separating cells, and while not technically new, it does support new research areas in genomics and proteomics by supplying those fields with purified cells. “Density gradient centrifugation is commonly used to separate lymphocytes and other mononuclear cells from whole blood, and the most common separation mediums used are aqueous solutions of high molecular weight polysaccharides and iodinated nonionic compounds,” says Mark Frei, technical marketing specialist for hematology and histology at Sigma-Aldrich. However, he notes, “the technique is cumbersome and slow, and will result in impure cell segregation and poor cell recoveries if not precisely carried out.”
To help you successfully carry it out, Sigma-Aldrich offers the Accuspin System-Histopaque-1077, a chamber comprised of two tubes separated by a porous barrier containing the density gradient solution Histopaque 1077. “Whole blood can be easily poured into the upper chamber without risk of mixing with the density gradient solution,” says Frei. “Upon centrifugation, the whole blood descends through the barrier to contact the Histopaque 1077 and the elements of greater density displace the Histopaque 1077, giving a clear separation of the blood components. The dense band of lymphocytes and other mononuclear cells may be collected by simply pouring off the contents of the upper chamber. Accuspin System-Histopaque-1077 maximizes cell recoveries and greatly simplifies whole blood processing, reducing technique complexity and operator variability. The system allows processing large numbers of samples at a time and is ideal for clinical trials and research studies.”
Visual separation
Visual isolation of cells you are interested in is facilitated by staining agents that mark your cells—and leave your cells intact after separation. “The Streptamer® technology is the only technology that offers reversible reagents for staining and isolation of cells; i.e. the reagents can be completely removed from the cells,” says Gisela Mäck, product manager for cell TAGnologies at IBA. “This leaves the cells uncompromised so that unmanipulated and naturally occurring cells can be isolated and used for research or cell therapy.” After separation, the staining reagent rapidly dissociates in the presence of added d-biotin, which competes for binding with the Streptamer® labeling tools. The Streptamer® system allows you to isolate T-cells, for example, that are phenotypically and functionally the same as cells not treated with Streptamer®. Mäck thinks that this is “an exciting new development since it allows the researcher to study cells in their native state and use them for cell therapy,” by adoptive T-cell transfer for viral infections or cancer.
Immunomagnetic separation
Many companies have tools for immunomagnetic separation, such as magnetic beads coated with monoclonal antibodies that identify epitopes on a cell type’s surface. For example, BD Biosciences offers reagents for magnetic isolation of many cell types, including CD4+ T cells, NK cells, dendritic cells, and hematapoetic progenitor cells. BD IMag beads, ranging in size from 0.1 to 0.45 um in diameter, are optimized for positive or negative selection, depending on the cell type you are trying to isolate. Separation occurs using either the BD IMagnet™ direct magnet, or a magnetic separation column. Cells captured by the antibody-bead complexes can be run through a flow cytometer with the beads still attached.
“Magnetic separation is a fairly mature technology,” says Cyndy Lane, marketing product manager for research immunology at BD Biosciences. “However, incremental changes are improving the quality. There is a greater selection in kits designed for the separation of specific cell types. In addition, competition is improving the pricing for researchers. The biggest challenge is obtaining highly purified populations of cells. Most researchers are addressing this by using separation as a step prior to fluorescence-activated cell sorting (FACS) or using multi-step cell separation.”
StemCell Technologies also sells kits for the separation of a wide range of cell types, and indeed is still seeing improvement in quality and speed. “This year, we are releasing improved versions of three of our most popular kits, EasySep® Isolation Kits for human B cells, T cells and CD8+ T cells,” says Stronge. “These isolation kits have been optimized so that the entire separation can take place in just 20 minutes in a single step from PBMCs, with purities of up to 98% and excellent recoveries.”
StemCell Technologies is also making it easier to separate cells within a complex mixture such as whole blood. Their RosetteSep® removes unwanted cells by crosslinking them to red blood cells using tetrameric antibody complexes. “Any RBC-containing sample is simply incubated with RosetteSep® cocktail for 20 minutes prior to a density-gradient centrifugation, and the purified cells are collected at the plasma:density medium interface,” says Stronge. “Customers who use RosetteSep® love the fact that there is no additional magnetic cell separation step needed to get highly purified cells.”
According to Stronge, a challenge in cell separation is the isolation of rare cell types and those with heterogeneous phenotypes, usually requiring both negative and positive selection. “For example, the isolation of regulatory T cells (Tregs) has typically been a long and cumbersome process that would require performing at least three cell isolation steps, and would take up most of the day,” she says. “StemCell has addressed this challenge by combining its two rapid and simple cell separation platforms, RosetteSep® and EasySep®. With StemCell’s Complete Kits for the isolation of Tregs, the procedure is now a simple two step process: pre-enrichment of cells by negative selection with RosetteSep®, followed by positive selection of CD25high cells with EasySep®. The entire procedure from whole blood or buffy coat to purified Tregs takes only three hours, unlike other existing methods that take up to five hours to complete.” StemCell has just released two new Complete Kits for the isolation of Tregs directly from whole blood or buffy coat: the Complete Kit for Human CD4+CD127lowCD25+ T Cells, and for Human CD4+CD127lowCD49d-CD25+ T Cells.
Stronge believes that cell separation still comes down to helping the researcher work: “When researchers don’t have to think about how they perform cell separation, that’s when we know we have helped them the most.”