Axl Neurauter

Expression of B-Cell surface antigens in subpopulations of exosomes released from B-cell lymphoma cells

PURPOSE Exosomes are small (30- to 100-nm) vesicles secreted by all cell types in culture and found in most body fluids. A mean of 1 mL of blood serum, derived from healthy donors, contains approximately 10(12) exosomes. Depending on the disease, the number of exosomes can fluctuate. Concentration of exosomes in the bloodstream and all other body fluids is extremely high. Several B-cell surface antigens (CD19, CD20, CD22, CD23, CD24, CD37, CD40, and HLA-DR) and the common leukocyte antigen CD45 are interesting in terms of immunotherapy of hematologic malignant neoplasms. The established standard for exosome isolation is ultracentrifugation. However, this method cannot discriminate between exosome subpopulations and other nanovesicles. The main purpose of this study was to characterize CD81(+) and CD63(+) subpopulations of exosomes in terms of these surface markers after release from various types of B-cell lymphoma cell lines using an easy and reliable method of immunomagnetic separation. METHODS Western blotting, flow cytometry, and electron microscopy were used to compare the total preenriched extracellular vesicle (EV) pool to each fraction of vesicles after specific isolation, using magnetic beads conjugated with antibodies raised against the exosome markers CD63 and CD81. FINDINGS Magnetic bead-based isolation is a convenient method to study and compare subpopulations of exosomes released from B-cell lymphoma cells. The data indicated that the specifically isolated vesicles differed from the total preenriched EV pool. CD19, CD20, CD24, CD37, and HLA-DR, but not CD22, CD23, CD40, and CD45, are expressed on exosomes from B-cell lymphoma cell lines with large heterogeneity among the different B-cell lymphoma cell lines. Interestingly, these B-cell lymphoma-derived EVs are able to rescue lymphoma cells from rituximab-induced complement-dependent cytotoxicity. IMPLICATIONS 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.

Methods for the extraction and RNA profiling of exosomes

AIM To develop protocols for isolation of exosomes and characterization of their RNA content. METHODS Exosomes were extracted from HeLa cell culture media and human blood serum using the Total exosome isolation (from cell culture media) reagent, and Total exosome isolation (from serum) reagent respectively. Identity and purity of the exosomes was confirmed by Nanosight(®) analysis, electron microscopy, and Western blots for CD63 marker. Exosomal RNA cargo was recovered with the Total exosome RNA and protein isolation kit. Finally, RNA was profiled using Bioanalyzer and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) methodology. RESULTS Here we describe a novel approach for robust and scalable isolation of exosomes from cell culture media and serum, with subsequent isolation and analysis of RNA residing within these vesicles. The isolation procedure is completed in a fraction of the time, compared to the current standard protocols utilizing ultracentrifugation, and allows to recover fully intact exosomes in higher yields. Exosomes were found to contain a very diverse RNA cargo, primarily short sequences 20-200 nt (such as miRNA and fragments of mRNA), however longer RNA species were detected as well, including full-length 18S and 28S rRNA. CONCLUSION We have successfully developed a set of reagents and a workflow allowing fast and efficient extraction of exosomes, followed by isolation of RNA and its analysis by qRT-PCR and other techniques.

Magnetic bead-based isolation of exosomes.

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.

Direct Isolation of Exosomes from Cell Culture: Simplifying Methods for Exosome Enrichment and Analysis

Exosomes, (50-150 nm sized vesicles), are secreted by all cells and found in all body fluids investigated. Isolation and characterization of exosomes from cell culture systems and body fluids provide valuable information about the biological system. The level of exosomes in human serum will vary depending on many factors such as age, sex, time of sample collection (circadian rhythm/nutrition status) , and of particular interest during diseased conditions. Such information may possibly be used for early detection of disease, monitoring of disease and/or effect of treatment. The established standard for exosome isolation is differential ultracentrifugation a method which cannot discriminate between exosome subpopulations or other particles with similar size and density. Here we have established a direct method for specific isolation of exosomes from cell culture supernatant suitable for a range of different downstream applications. Magnetic beads targeting human CD9 or CD81, common exosomal markers, were used to first isolate and characterize pre-enriched exosomes addressing critical factors (volume, time and exosome concentrations) to establish optimal and comparable isolation conditions. Finally, exosomes were isolated and characterized directly from cell culture media. In conclusion, the data demonstrates the ability to capture exosomes directly providing the possibility to characterize and compare exosomes from different sources and increasing the compatibility in terms of applications.

Separation and expansion of human T cells

Publisher Summary This chapter describes techniques for separation and expansion of human T cells. There are two main strategies for isolating a specific cell type: a “positive selection” of cells of interest or a “negative selection” where unwanted cells are depleted. By positive selection, a specific cellular subset is isolated directly from a complex mixture of cells based on the expression of a distinct surface. By positive selection, a specific cellular subset is isolated directly from a complex mixture of cells based on the expression of a distinct surface antigen. The resulting immune complexes of beads and target cells are collected using a magnet. By negative selection, all unwanted cell types are removed from the sample by the magnetic beads. CD4+ T cells are isolated from buffy coat by positive selection using the Dynal CD4 positive isolation kit. The isolated CD4+ cells are then ready for further examination. The T-cell activation in vivo is initiated by the binding of T-cell receptors on its surface to appropriate peptide-HLA molecules on the surface of antigen-presenting cells (APC).