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Large-scale cell isolation is commonly performed in labs and core facilities for cell banking and drug discovery, and as a critical step in cell therapy manufacturing. Current methods can be a significant bottleneck in a lab’s workflow, often requiring a full day for sample processing and cell isolation. To address this need, we have developed two methods: manual column-free isolation using the Easy 250 EasySep™ magnet, and automated cell isolation in a closed system using RoboSep™-C. The Easy 250 magnet allows users to isolate cells from a full leukopak of up to 20 billion cells in under 30 minutes by simply pipetting out their target cells. Negative selection protocols have been optimized to achieve 95.3% T cell, 97.2% CD4+ T cell, 91.9% CD8+ T cell, 99.4% B cell, 96.5% NK cell, and 91.5% monocyte purities. Positive selection protocols obtain 95.4% CD3+ cell, 94.4% CD4+ T cell, 93.9% CD8+ T cell, and 96.2% CD14+ cell purities. RoboSep™-C automates this cell isolation procedure, along with the cell washing steps for sample preparation, in as little as 50 minutes. The instrument features a scale tower, pump and clamp modules to direct fluid through defined paths, and a magnet for separation of labelled cells. The system uses a sterile single-use tubing set that incorporates a cartridge for cell washing and concentration, and a magnet chamber for cell separation. Starting with fresh leukopaks, we obtained 95.9% T cell, 95.8% CD4+ T cell, and 89.6% CD8+ T cell purities following negative selection, and 92.0% CD4+ T cell and 89.5% CD8+ T cell purities following positive selection. These approaches offer efficient and user-friendly cell isolation that allow researchers to scale up their operations, and can be easily integrated upstream of existing workflows.

Isolating cells from large volume-samples such as leukapheresis or whole blood bags is a common procedure that precedes many immunological studies. Working with large volumes can be challenging as the sample often needs to be split and processed in parallel, which can be tedious and may delay downstream studies. The EasySep™ T75 Magnet simplifies cell separation procedures when processing up to 225 mL of leukapheresis sample or 125 mL of whole blood, and is based on column-free immunomagnetic EasySep™ cell isolation technology. Negative selection protocols have been optimized for isolation of human T cells (purity: 95.3 ± 2.5%, n=5), CD4+ T cells (purity: 97.2 ± 1.4%, n=4), CD8+ T cells (purity: 91.9 ± 2.2%, n=5), and monocytes (purity: 91.5 ± 1.0%, n=3). Positive selection protocols have been optimized for isolation of CD3+ cells (purity: 95.4 ± 2.9%, n=9), CD4+ cells (purity: 88.8 ± 3.2%, n=3), CD8+ cells (purity: 95.1 ± 2.4%, n=6), and CD14+ cells (purity: 96.2 ± 0.7%, n=3). Protocols for depletion of red blood cells (RBCs) (residual RBCs: 2.5 ± 1.7%, n=5) and enrichment of peripheral blood mononuclear cells from whole blood (purity: 99.2 ± 0.5%, n=5) have also been developed. Notably, these protocols can be completed in 20 – 26 minutes, making the EasySep™ T75 Magnet the fastest and simplest method for isolating highly purified immune cells from large-volume whole blood and leukapheresis samples.

Peripheral blood circulating T cells can be subdivided into either antigen experienced effector and memory T cells or naïve T cells that have not yet encountered their cognate antigen. Naïve T cells are valuable tools when studying mechanisms of T cell activation and are required for foundational research into infectious diseases, cancer and transplantation. We describe here a simple immunomagnetic cell isolation protocol for the isolation of untouched human naïve T cells from fresh or previously frozen PBMC or leukapheresis samples. The column-free, negative selection procedure involves labelling and removing unwanted cells using bispecific antibody complexes that crosslink cell surface antigens to magnetic particles. The labelling cocktail is composed of antibodies that target non-T cells, memory T cells, and an optional cocktail for depleting gamma delta T cells. Briefly, the procedure involves a five minute incubation with the antibody cocktail followed by the addition of magnetic particles and two three minute magnetic separations in a hand-held EasySep™ magnet. Following the second magnetic separation, the untouched naïve T cells are simply poured off into a new tube and are ready for use. We define naïve T cells phenotypically as CD3+CD45RA+CD45RO-CD197+. Starting from fresh PBMC, we were able to obtain purities of 96.1 ± 2.3% (mean ± SD, n=14) and recoveries of 62.6% ± 24.5% (mean ± SD, n=14). Additionally, isolated naïve T cells are functional and respond by upregulating the expression of CD25 and CD69 upon stimulation. Our EasySep™ Human Naïve Pan T Cell Isolation Kit offers a fast and easy isolation method for naïve T cells that is suitable for downstream applications such as flow cytometry, cell culture or DNA/RNA extraction.

Tissue specific research often requires mechanical and/or enzymatic digestion to isolate and study certain cell types. The digestion can be a harsh process resulting in a significant number of dead cells in the final cell suspension. Subsequent analysis by flow cytometry is difficult to interpret due to non-specific binding of antibodies to dead cells and dead cell auto fluorescence. Factors released by dead cells can also interfere with downstream assays, complicating the study of primary tissues. During apoptosis, the cell membrane loses its phospholipid asymmetry resulting in exposure of negatively charged phospholipids on the cell surface. Relocation of phosphatidylserine (PS) to the outer leaflet of the cell membrane is a well-established marker of apoptosis. By targeting exposed PS with Annexin V we have developed a rapid method (EasySep™) to immunomagnetically remove dead cells from primary tissue samples. Performance of this kit was examined on various mouse and human tissue types. Using this method, we were able to improve viability of a single cell suspension of mouse lungs digested with collagenase/hyaluronidase from an initial viability of 39.6 ± 12.3% AnxV−/PI− to 70.9 ± 11.8% AnxV−/PI. From 1×10^8 total start cells, 1.43 ± 0.68 ×10^7 live cells were recovered (n=10). From human polymorphonuclear leukocytes cultured overnight, viability was improved from 23.7 ± 9.8% AnxV−/PI− to 67.7 ± 12.1% AnxV−/PI− with recovery of 1.27 ± 0.52 ×10^7 live cells from 1×10^8 total start cells (n=7). This equates to removal of 88.9 ± 8.3% and 91.8 ± 5.3% dead cells respectively. Since live cells are untouched, subsequent isolation of desired cell types can be performed, resulting in a more viable population of cells for downstream applications.

Regulatory T cells (Tregs) are a specialized subset of T cells that play a key role in immune regulation. Harnessing the suppressive function of Tregs is a major area of interest as they hold great potential for the treatment of autoimmune disorders. The first step towards quality Treg research is the isolation of highly purified, functional Tregs. With unique cell isolation platforms, STEMCELL has developed a full range of products for the rapid and efficient isolation of highly functional Tregs from virtually any peripheral blood sample. RosetteSep is an antibody mediated buoyant density centrifugation method used to isolate unlabelled cells specifically from whole blood or buffy coat samples. EasySep is an immunomagnetic cell separation method used to isolate cells from fresh or previously frozen PBMCs. Treg pre-enrichment is achieved by antibody mediated crosslinking of unwanted cells to either red blood cells (RosetteSep) or magnetic particles (EasySep) allowing their removal by Ficoll centrifugation or magnetic separation, respectively. RosetteSep or EasySep pre-enriched Treg populations consist of CD4+, CD4+CD127low or CD4+CD127lowCD49d- T cells. Pre-enriched Tregs can be further purified using EasySep positive selection to isolate Tregs expressing high levels of cell surface CD25. Purities of 85% +/- 10% CD4+CD25highFOXP3+ human Tregs can be achieved depending on the Treg population. From start to finish, Treg isolations can be completed in less than 3 hours.