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Cell isolation with Dynabeads

By 05.06.2022

How to Get the Cells You Need with Dynabeads?

Conventional cell isolation procedures can be time-consuming and delicate. Nevertheless, with Dynabeads cell isolation, you can observe reproducibility and quality for positive selections, negative selections, and more. See how to level up your cell separations.

The separation, isolation, and analysis of specialized cell types is an essential part of basic biological research, therapeutics discovery and development, and clinical diagnostics. Glean biological insights from a pure population by isolating specific cells. Thus, you don’t have to deal with functional signals and DNA, RNA, or protein biomarkers from other cell types. Those signals can complicate downstream data analysis.

Multicellular organisms typically have tens to hundreds of different cell types. Consequently, this presents a significant challenge for the isolation of a single subpopulation. Many methods for isolating cell types use a broad range of samples. But the volume of citations using Dynabead™ products speaks for itself.

Dynabeads unlock consistent results with magnetic cell separation

Dynabeads are micron-sized, monodispersed, superparamagnetic beads, making them particularly well-suited for biological separation and isolation.1 They can:

  1. Enable magnetic separation from other cell types while maintaining viability and integrity of target cell populations
  2. Deliver consistent and reproducible binding and yield
  3. Have defined non-porous, surface chemistry, helping minimize aggregation, non-specific binding, and exposure of cells to metals
  4. Customize your cell type of interest and downstream applications

In comparison, target cells can endocytose competing nanometer-sized beads. Once inside of cells, the beads are difficult to remove following isolation and cause negative downstream effects on cell activity. Dynabeads are now used widely across R&D applications, with over 70,000 research publications reporting their use. See our product guide titled Healthy cells in = good data out.

Three Common Cell Isolation Strategies

Cell isolation with Dynabeads relies on selective and specific antibodies that target cell surface markers on specific cell types.1 Cells in the supernatant can be isolated using antibodies. This is done in a simple tube-based workflow, using a magnetic stand or rack to pull Dynabeads to the side of a tube, isolating them from the other cell types within the sample.

Positive Cell Isolation

In positive cell isolation, antibody coated Dynabeads bind directly to the target cell type in a sample. Other cell types are removed in the supernatant. As a result, for downstream applications your desired cell type can then be gently released from the magnetic beads using DETACHaBEAD Reagent. If you need high-purity, viable cell populations, a positive selection strategy is ideal.

Dynabeads products for cell separation

Use of magnetic separation helps ensure that cell isolation is not affected by passage through a dense column and that the cells’ native characteristics are preserved.

One drawback to positive isolation is that it requires direct antibody binding to cell surface markers, which can activate certain cell types. Thus, in these cases, a negative selection strategy is more appropriate (see below). However, for some biological applications, activation may be a convenient and desirable step in your workflow. Take, for instance, the Dynabeads® Human T-Activator CD3/CD28, coated covalently with both ɑ-CD3 and ɑ-CD28 antibodies. In effect, the Dynabeads CD3/CD28 are used to mimic antigen-presenting cells. The entire cell is isolated using human mononuclear or peripheral blood mononuclear cells (PBMCs) from whole blood as input. Therefore, for activation of T-cells2 each antibody provides a primary and co-stimulatory signal.2 Furthermore, expansion can be stimulated by the addition of IL-2 and activated T-cells can then be used to study T-cell receptor (TCR) signaling, differentiate into helper T-cells, and more.

Negative Cell Isolation

In contrast, negative cell isolation uses Dynabeads linked to an antibody cocktail targeting all unwanted cells in a sample, leaving target cells present in the supernatant. Target cells are untouched, optimal for fragile cell types or those that can be activated by antibody binding. Therefore, removal of supernatant can give you the high-purity (antibody- and bead-free) cell populations you need for downstream applications.

Whole populations of T-cells can be isolated from PBMCs through negative selection, using an antibody mix that targets CD14, CD16, CD19, CD36, CD56, CD123, and CD235a. Thus, removing B-cells, NK cells, monocytes, platelets, dendritic cells, granulocytes, and erythrocytes.1 Consequently, the resulting T-cells can be directly used in functional or flow cytometry assays.

One variation on negative selection is the depletion of a single cell type, rather than a complex mixture of various cell types. For instance, if you want to deplete a blood sample of CD4+ or CD8+ T-cells, you can use a negative selection strategy with Dynabeads™ CD4 or Dynabeads™ CD8, respectively.

Combining Negative and Positive Cell Isolation 

Either positive or negative isolation strategies can be used for a broad range of downstream applications. This includes flow cytometry and DNA, RNA, or protein isolation. But what if you need to isolate a subpopulation of cells, such as regulatory T-Cells (Tregs), that have no single defining cell surface marker?

For subpopulations such as these, you can use a combination of positive and negative selection strategies. First, negative selection using a Dynabeads™ Untouched™ Human CD4 T Cells Kit can be done – which leaves CD4+ T-Cells-  followed by positive selection using Dynabeads™ CD25. Using this approach, Tregs which express both CD4 and CD25 can be enriched from 1-3% of the PBMC cell population to 95-98%.3

A broad range of Dynabead products exist. They are fit for a wide range of research applications requiring mouse or human cell isolations. Most importantly, they are useful if you are dealing with viscous sample types, like whole blood. In addition, if you want to use your own antibody, there is a flexible portfolio of streptavidin- and species-specific secondary antibody-coated or surface-activated Dynabead products.

Clinical Research Applications of Dynabeads

Engineered T-cells expressing a chimeric antigen receptor (CAR) have emerged as a promising adoptive cell treatment for multiple types of cancer. To date, the FDA has approved five CAR T-cell therapies with promising clinical results and additional interest from the broader biopharmaceutical community.

Reproducible production of CAR T-cells, under current good manufacturing practices (cGMP), is a critical consideration. Quality issues can lead to the exposure of patients to unnecessary safety risks. Early in the manufacturing process, following the collection of PBMCs from a patient, T-cells need to be selected and activated. Clinical-grade magnetic beads, CTS™ Dynabeads™ CD3/CD28 have been used in a number of clinical studies for ex vivo T-cell activation, prior to retroviral transduction with CAR cDNA.4 Their use has been indispensable for the safe, high-quality manufacture of these revolutionary therapeutics.

Start Your Cell Isolations Right

Dynabeads have become the cornerstone of many mammalian cell isolation workflows. They consistently deliver high purity and yield and are fast, easy to use, and flexible for many different protocols. Thermo Fisher Scientific offers a robust portfolio of Dynabeads for positive and negative selection across wide-ranging biological applications. Dynabeads are also automation-ready and can be combined with the KingFisher family of instruments to deliver reproducible results, eliminate cross-contamination, and cut the hands-on time down to as low as 40 minutes total.

For more articles on Dynabeads check out:

This article is for research use only. Not for use in diagnostic procedures.

References:

  1. Neurauter AA, Bonyhadi M, Lien E, et al. Cell isolation and expansion using Dynabeads. Adv Biochem Eng Biotechnol. 2007;106:41-73. doi:10.1007/10_2007_072
  2. Li Y, Kurlander RJ. Comparison of anti-CD3 and anti-CD28-coated beads with soluble anti-CD3 for expanding human T cells: differing impact on CD8 T cell phenotype and responsiveness to restimulation. J Transl Med. 2010;8:104. Published 2010 Oct 26. doi:10.1186/1479-5876-8-104
  3. Arruvito L, Sanz M, Banham AH, Fainboim L. Expansion of CD4+CD25+and FOXP3+ regulatory T cells during the follicular phase of the menstrual cycle: implications for human reproduction. J Immunol. 2007;178(4):2572-2578. doi:10.4049/jimmunol.178.4.2572
  4. Wang X, Rivière I. Clinical manufacturing of CAR T cells: foundation of a promising therapy. Mol Ther Oncolytics. 2016;3:16015. Published 2016 Jun 15. doi:10.1038/mto.2016.15

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