Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra-assay (within-day, n = 6) and inter-assay (day-to-day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument-optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra-assay CVs were 1–12% for both EVs and artificial samples, measured on the same instrument. The overall day-to-day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument-optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument-optimised settings on the same instrument did not contribute to significant variation compared to the overall day-to-day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised execution of analysis, as well as monitoring longitudinal variation parameters on both biological and synthetic samples, should be highlighted.

Exosomes are here defined as extracellular vesicles (EVs) in the approximate size range of 30-100 nm in diameter, and are observed in most body fluids containing typical exosomal markers such as CD9, CD63, and CD81. Potential subpopulations of exosomes can be captured by targeting these markers using magnetic beads. Magnetic beads are versatile tools for exosome isolation and downstream analysis. Here, we describe the workflow of immuno magnetic isolation and analysis of exosomes by flow cytometry, Western immunoblotting, and electron microscopy.

Distribution of exosomes that contain CD19, CD20, CD24, CD37, and HLA-DR may intercept immunotherapy directed against these antigens, which is important to be aware of for optimal treatment. The use of an immunomagnetic separation platform enables easy isolation and characterization of exosome subpopulations for further studies of the exosome biology to understand the potential for therapeutic and diagnostic use.

This chapter describes the use of Dynabeads for cell isolation and expansion. Dynabeads are uniform polystyrene spherical beads that have been made magnetisable and superparamagnetic, meaning they are only magnetic in a magnetic field. Due to this property, the beads can easily be resuspended when the magnetic field is removed. The invention of Dynabeads made, by Professor John Ugelstad, has revolutionized the separation of many biological materials. For example, the attachment of target-specific antibodies to the surface of the beads allows capture and isolation of intact cells directly from a complex suspension such as blood. This is all accomplished under the influence of a simple magnetic field without the need for column separation techniques or centrifugation. In general, magnetic beads coated with specific antibodies can be used either for isolation or depletion of various cell types. Positive or negative cell isolation can be performed depending on the nature of the starting sample, the cell surface markers and the downstream application in question. Positive cell isolation is the method of choice for unprocessed samples, such as whole blood, and for downstream molecular applications. Positive cell isolation can also be used for any downstream application after detachment and removal of the beads. Negative cell isolation is the method of choice when it is critical that cells of interest remain untouched, i.e., no antibodies have been bound to any cell surface markers on the cells of interest. Some cell populations can only be defined by multiple cell surface markers. Such populations of cells can be isolated by the combination of negative and positive cell isolation. By coupling Dynabeads with antibodies directed against cell surface activation molecules, the beads can be used both for isolation and expansion of the cells. Dynabeads are currently used in two major clinical applications: 1) In the Isolex 300i Magnetic Cell Selection System for CD34 Stem Cell Isolation--2) For ex vivo T cell isolation and expansion using Dynabeads ClinExVivo CD3/CD28 for clinical trials in novel adoptive immunotherapy.

This chapter covers magnetic bead-based isolation and analysis of the smallest members of extracellular vesicles (EVs), the exosomes (30-150 nm), generally regarded to originate from the multivesicular bodies (MVBs). Also included, are descriptions of how to prepare samples prior to isolations. The magnetic bead-based isolation workflow is dramatically shortened both by omitting the pre-enrichment step and providing an option for a very short capture time. Three direct exosome isolation strategies are described: (1) "Specific and Direct," (2) "Semi Generic and Direct" and (3) "Generic and Direct" as well as exosome release from the magnetic beads. Detailed description of downstream exosome analysis is included covering flow cytometry, Western blot and electron microscopy. Finally, a description of exosome isolation from more complex starting material including urine and serum/plasma is discussed.

Adoptive immunotherapy with tumour‐reactive CD8+ cytotoxic T lymphocytes (CTLs) requires efficient in vitro approaches allowing the expansion of CTLs to large numbers prior infusion. Here, we investigated the antigen‐independent activation and the expansion of human T cells in peripheral blood mononuclear cells (PBMCs) and in tumour‐reactive CTLs using Dynabeads coated with monoclonal antibodies to CD3 and to the costimulatory molecules CD28 and CD137 (4‐1BB). T cells in PBMCs showed an increased expansion rate of 15‐ to 17‐fold during a 2‐week culture period using antibody‐conjugated beads with interleukin‐2 (IL‐2) added versus IL‐2 alone. No significant difference between CD3/CD28 beads and CD3/CD28/CD137 beads was observed (P = 0.4). In contrast, expansion of tumour‐reactive CD8+ CTLs over 2 weeks was more efficient using CD3/CD28/CD137 beads (14.4‐fold ±1.2) compared with CD3/CD28 beads (10.6‐fold ±0.7) (P = 0.03) and matched well to the control arm using weekly stimulation with tumour cells. Although all modes of in vitro stimulation decreased the expression of central memory markers CD62L and CCR7 on CTLs, bead‐activated cultures expressed consistently higher levels than tumour‐stimulated cultures. CTLs analysed after bead‐induced expansion versus weekly tumour stimulation showed equal IFN‐γ production in ELISPOT assay. Furthermore, cytotoxicity assays demonstrated an either unchanged or slightly reduced capability of tumour cell lysis for antigen‐independent stimulated CTLs versus those that maintained on weekly tumour stimulation, regardless of which type of beads was used. Our data suggest that the conjugation of anti‐CD137 antibodies to conventional CD3/CD28 beads results in a minor but significant increase in the expansion capacity for tumour‐reactive CD8+ CTLs.

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5118 Introduction Efficient methods for the reliable in vitro expansion of tumor-reactive T cells will surely broaden the applicability of adoptive T cell therapy in cancer. In this study we investigated the antigen-independent stimulation and expansion of human T cells in peripheral blood mononuclear cells (PBMC) and in long-term cultured tumor-reactive CD8+ T cell lines using superparamagnetic beads coated with antibodies to CD3 and the costimulatory molecules CD28 and CD137. Methods T cell numbers were measured in healthy donor PBMC after in vitro stimulation with Dynabeads® coated with CD3/CD28/CD137 versus Dynabeads® coated with CD3/CD28 (all beads +/- 100 U/mL IL-2) versus IL-2 alone at different bead/cell ratios (3:1, 1:1). Expansion was also analyzed in human renal cell carcinoma-reactive CD8+ T cell lines after restimulation with tumor cells (weekly), CD3/CD28 beads and CD3/CD28/CD137 beads, respectively (bead/cell ratio of 1:5, 100 U/mL IL-2 added). Expanded T cell lines were phenotyped for expression of activation, differentiation and homing molecules (i.e. CD27, CD28, CD45RA, CD45RO, CD57, CD62L, CD137, CCR7) and were also tested for function. Results T cells in PBMC showed an increased expansion rate of up to 17-fold during a 2-week culture period using beads with IL-2 added versus IL-2 alone (p<0.0001 for CD3/CD28/CD137; p<0.0001 for CD3/CD28). The difference between CD3/CD28/CD137 beads and CD3/CD28 beads was not significant (p=0.4). Bead/cell ratios of 1:1 and 3:1 expanded T cells in PBMC with similar efficiency. In addition, IL-2 was essential to obtain maximum T cell proliferation. Peripheral blood CD4+ and CD8+ T cells showed a strong increase of CD137 surface expression starting 12-24 hours upon stimulation, regardless which beads were used. In contrast to PBMC, tumor-reactive CD8+ T cell lines expanded more rapidly using CD3/CD28/CD137 beads versus CD3/CD28 beads (p=0.03). Stimulation with CD3/CD28/CD137 beads was comparably efficient versus the control arm using weekly addition of tumor cells and IL-2. Simultaneous addition of beads and tumor cells did not have a synergistic effect. CD8+ T cell lines analyzed 12 days after bead-induced in vitro expansion versus weekly tumor stimulation showed a comparable level of tumor reactivity in IFN-g ELISPOT assay. Phenotypically, expression of CD137 on CD8+ T cell lines showed maximum up-regulation 24 hours after beads stimulation and persisted for at least 72 hours. In contrast, cultures stimulated solely with tumor cells showed a much shorter and transient CD137 expression with an earlier peak level after 12 hours. Other phenotypic markers were similar on tumor-reactive T cell cultures, except for increased CD62L expression after bead-induced stimulation. Conclusion Antigen-independent in vitro expansion of T cells in PBMC was equally efficient using CD3/CD28 beads or CD3/CD28/CD137 beads, respectively. In contrast, we observed an increased growth rate for tumor-reactive CD8+ T cell lines when activated with CD3/CD28/CD137 beads compared to CD3/CD28 beads. Antitumor reactivity of T cell lines was maintained during the antigen-independent stimulation step. Bead activation was associated with increased expression of the lymph node homing receptor CD62L on antitumor CD8+ T cell lines, which indicates a central memory phenotype. Our data suggest that the conjugation of anti-CD137 antibodies to the traditionally used CD3/CD28 beads improves their expansion capacity for antitumor CD8+ T cell lines. Disclosures No relevant conflicts of interest to declare.