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A Novel System for High-Throughput Cell Isolation Directly from Blood in 25 Minutes

  • Document # 28726
  • Version 1.1.0
  • Sep 2016

This technical bulletin presents the new RosetteSep™ and SepMate™ system for fast, efficient and high-throughput cell isolation from whole blood samples. It features data from Dr. Ajay Jain’s lab at the University of Maryland School of Medicine, which used this system to reduce NK cell isolation time from four hours to a single hour for a 450 mL unit of blood.

The Need For High-Throughput Cell Isolation

Immune cell isolation plays an important role in areas such as drug discovery and development, vaccine research, and translational immunology. For example, isolating immune cells is routinely necessary in order to evaluate the efficacy of immunostimulatory compounds, assess the immunogenicity of vaccine candidates and investigate interactions between the immune system and infectious agents.1-4

The movement towards more physiologically relevant assays based on primary human cells has created the need for a fast and efficient method of isolating immune cells from large numbers of whole blood samples.2,4 To facilitate this type of high-throughput cell processing, STEMCELL Technologies Inc. has developed a new system for rapid and efficient cell isolation directly from whole blood in as little as 25 minutes.

The RosetteSep™ and SepMate™ System for Cell Isolation Directly from Whole Blood

The RosetteSep™ and SepMate™ system combines a unique immunodensity cell isolation reagent (RosetteSep™) with a specialized cell processing tube (SepMate™) to reduce the number of steps needed for cell isolation. This new system saves time, minimizes variability between users and allows efficient, high-throughput sample processing. It also minimizes the risk of activating or damaging cells by isolating untouched and highly purified cells without columns or immunomagnetic beads.

Cells isolated with the RosetteSep™ and SepMate™ system are immediately available for use in a variety of downstream assays such as cytotoxicity testing, compound screening, and other applications where it is important to obtain viable, functional cells with minimal manipulation.

Case Study: A High-Throughput Method for Isolation of Natural Killer Cells and Lymphocytes for Assessment of In Vitro Cytotoxicity

Traditional methods for isolating lymphocytes from blood are laborious and time-consuming, requiring precision and technical expertise. These methods typically involve isolating peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation (DGC) before enriching specific cell subpopulations using immunomagnetic column-based systems. Using these conventional methods, isolating highly purified natural killer (NK) cells from a 450 mL unit of blood is a lengthy process (taking up to 4 hours), and it is difficult to process multiple samples quickly and efficiently (Jain et al., in prep).5

The new RosetteSep™ and SepMate™ system allows faster and more efficient blood processing without compromising cell function or performance in downstream assays. At the University of Maryland School of Medicine, Dr. Ajay Jain and colleagues routinely isolate NK cells from large numbers of human samples. In order to streamline their workflow and achieve higher-throughput sample processing, Jain’s lab has adopted the new RosetteSep™ and SepMate™ cell isolation system in place of their previous, column-based method.

Evaluation of the RosetteSep™ and SepMate™ System

Dr. Jain’s group found that isolation of NK cells was significantly faster with the RosetteSep™ and SepMate™ system: a 450 mL unit of blood could be split into multiple samples and processed in a single hour, compared to four hours using DGC/immunomagnetic selection (Jain et al., in prep).

Furthermore, NK cells isolated by RosetteSep™ and SepMate™ were highly purified (86.7% as assessed by flow cytometry) with intact cytolytic function. Using in vitro assays, Jain et al. showed that isolated cells retain the capacity to kill HT9 colon cancer cells or K563 leukemia cells (Figure 1).

Flow cytometric evaluation of NK cell activation markers demonstrated that NK cells isolated using RosetteSep™ and SepMate™ have similar expression profiles similar to cells isolated using DGC/immunomagnetic selection (Figure 2).

 In vitro cytotoxicity against HT29 colon cancer cells and K562 target cells with NK cells isolated by DGC/immunomagnetic selection (left panel) versus RosetteSep™ and SepMate™ (right panel).

Figure 1. In vitro cytotoxicity against HT29 colon cancer cells and K562 target cells with NK cells isolated by DGC/immunomagnetic selection (left panel) versus RosetteSep™ and SepMate™ (right panel).

In vitro conditions include culture with media alone (open circles), 10 μg/mL human IgG1 isotype control (open triangles), or 10 μg/mL cetuximab (open diamonds). Figure courtesy of Dr. Ajay Jain.

Surface expression of NK cell activation markers as evaluated by flow cytometry.

Figure 2. Surface expression of NK cell activation markers as evaluated by flow cytometry.

Expression is shown as mean fluorescence intensity (MFI). Figure courtesy of Dr. Ajay Jain.

The SepMate™ tubes… provide a mechanical barrier between the purified effector cells and the density gradient medium. The desired cell populations remain above the barrier and can be poured out of the tube with no technical expertise. This method allows for the concurrent rapid purification of NK cells and CD8+ and CD4+ T cells from multiple human donors, which makes downstream applications (i.e. flow cytometry, ELISA, assessment of in vitro cytotoxicity) more practical to perform in a high-throughput manner.

– So et al., 2013.

Conclusions

The RosetteSep™ and SepMate™ system allows efficient negative selection of lymphocytes from whole blood in a quarter of the time needed for an alternative method, requires no technical expertise, and yields fully functional cells. This new system makes it possible to rapidly isolate NK cells or other immune cells from many samples at once, facilitating high-throughput cytotoxicity testing and other downstream assays. Scroll down for more information on the RosetteSep™ and SepMate™ procedure.

How the RosetteSep™ and SepMate™ System Works

RosetteSep™ isolates highly purified cells directly from blood by crosslinking unwanted cells to red blood cells, forming immunorosettes (Figure 3). These immunorosettes pellet during density gradient centrifugation, leaving untouched and highly purified target cells at the interface between the plasma and the density gradient medium.

Using the new SepMate™ tube makes the RosetteSep™ procedure even faster and easier. The SepMate™ insert creates a physical barrier between the sample and density gradient medium, allowing the sample to be rapidly pipetted or poured into the tube. Centrifugation time is reduced to just 10 minutes, and purified target cells can simply be poured into a new tube. SepMate™ can also be used on its own for hassle-free PBMC isolation from whole blood in just 15 minutes.

Free samples and further information: www.rosettesep.com

Figure 3. Image of a blood sample after addition of the RosetteSep™ cocktail, and prior to density gradient centrifugation.

Advantages of the RosetteSep™ and SepMate™ System

  • FAST AND EASY. Isolate cells from whole blood in as little as 25 minutes with RosetteSep™ and SepMate™, or use RosetteSep™ on its own with standard density gradient centrifugation.
  • NO SPECIAL TRAINING OR EQUIPMENT. RosetteSep™ and SepMate™ can be used by anyone with minimal training. No columns, magnets or other special equipment are required.
  • HIGHLY VIABLE AND FUNCTIONAL CELLS. Isolated cells are functional, flow cytometry-compatible, and unlabeled with antibodies or magnetic beads.

The Novel RosetteSep™ and SepMate™ Procedure

The Novel RosetteSep™ and SepMate™ Procedure

Select RosetteSep™ Products

RosetteSep™ Product Listing

1. For labeling 40 mL of blood.
2. For labeling 200 mL of blood.
3. For CD2+ or CD19+
4. This product carries the CE marking.
5. Can be added to a standard RosetteSep™ cocktail, if not already present, or used alone.

Fresh Human Peripheral Blood Products1

RosetteSep™ Product Listing

1. Fresh products currently available in the United States and Canada (excluding Quebec). Please contact Product & Scientific Support (techsupport@stemcell.com) for more information.
2. ACDA - Acid-Citrate-Dextrose Solution A; CP2D - Citrate-Phosphate-Double-Dextrose.

SepMate™ Products

SepMate™ Product Listing

1. SepMate™ (IVD) is only available in select regions where it is registered as an In Vitro Diagnostic (IVD) device for the isolation of mononuclear cells from human whole blood or cord blood by density gradient centrifugation.
2. SepMate™ for Research Use Only (RUO) is available in regions where SepMate™ is not registered as an IVD device.

Density Gradient and Freezing Media Products

SepMate™ Product Listing

References

  1. WHO Guidelines on Nonclinical Evaluation of Vaccines. WHO Technical Report Series, No. 927, 2005
  2. Slater, K. Curr Opin Biotechnol 12: 70-74, 2001
  3. McMillin DW, et al. Blood 119(15): e131-e138, 2012
  4. Saez-Cirion A, et al. Nat Protoc 5: 1033-1041, 2010

Further Reading: Selected Recent RosetteSep™ Cytotoxicity Publications

  1. Chu CC, et al. J Exp Med 209(5): 935-945, 2012
  2. Newel EW, et al. Immunity 36(1): 142-152, 2012
  3. Pietra G, et al. Cancer Res 72: 1407-1415, 2012
  4. Rafi q S, et al. Leukemia 26(7): 1720-1722, 2012
  5. Alvarez-Breckenridge CA, et al. Nature Medicine 18(12):1827-34, 2012
  6. Sieni E, et al. PLoS ONE 7(9): e44649. doi:10.1371/journal.pone.0044649, 2012
  7. Lanzi G, et al. J Exp Med 209(1): 29-34, 2012
  8. Denman CJ, et al. PLoS ONE 7(1): e30264. doi:10.1371/journal.pone.0030264, 2012
  9. Trotta R, et al. Blood 117: 2378-2384, 2011
  10. Molon B, et al. J Exp Med 208(10): 1949-1962, 2011
  11. Fregni G, et al. Clinic Cancer Res 17: 2628-2637, 2011
  12. Herman SE, et al. Blood 116: 2078-2088, 2010
  13. Wu RP, et al. PNAS 107(6): 7479-7484, 2010
  14. Natesampillai S, et al. PLoS Pathogens 6(11): e1001213.doi:10.1371/journal.ppat.1001213, 2010
  15. Shi Y, et al. Leukemia 24: 1588-1598, 2010
  16. Loser K, et al. PLoS ONE 5(2): e8958. doi:10.1371/journal.pone.0008958, 2010
  17. Lucas DM, et al. PLoS ONE 5(6): e10941. doi:10.1371/journal.pone.0010941, 2010
  18. Ikeda H, et al. Clinic Cancer Res 15: 4028-4037, 2009
  19. Balsamo M, et al. PNAS 106(49): 20847-20852, 2009
  20. Wang X, et al. Haematologica 94: 470-478, 2009
  21. Lucas DM, et al. Blood 113: 4656-4666, 2009
  22. Trifari S, et al. Nature Immunology 10: 864-871, 2009
  23. Haniffa M, et al. J Exp Med 206(2): 371-385, 2009
  24. Critchley RJ, et al. PNAS 106(22): 9010-9015, 2009
  25. Chen Q, et al. PNAS 106(51): 21783-21788, 2009