EVAnet (the network of Extracellular Vesicle research in Aarhus) is a cross-departmental initiative started at Aarhus University to bring together researchers and students working in the highly interdiscplinary field of extracellular vesicles (EVs). The aim is to boost our research at large by joining forces across different departments/centres through collaborations and student education.
There is absolutely no fee to be a network member! Just need to be associated with the EV research happening at Aarhus University. New students, from bachelors to PhDs, as well as experienced postdocs and senior researchers are all welcome to join us ;) Please simply send an e-mail to Yuya (yuya.hayashi@mbg.au.dk) indicating your AU ID and the research group you are affiliated with. All members are included in the mailing list.
We will cover basic aspects of EVs, which will include their nomenclature, biogenesis, their release and uptake as well as EV cargos. Different isolations methods will be presented such as ultracentrifugation, size exclusion chromatography and precipitation techniques and their pros and cons discussed. Different methods to identify, characterize and enumerate EVs will be presented and discussed as well as how to explore their content. As it is not a trivial task to work with EVs we will discuss critical things to consider during collection and isolation of EVs from various sample types and how to explore their function in vitro and in vivo. We will focus on human and model organism but also discuss non-model organisms, which are facing specific challenges. During the practical, you will learn to isolate EVs using size exclusion chromatography (qEV) and a precipitation technique. The size distribution and numbers of EVs in the samples isolated by the two methods will be examined using nanoparticle tracking analysis (NTA). Western blots will be used to identify classical EV makers.
You will be introduced to the field of EV rearch with the theorertical background and technical challenges in isolation and characterization of EVs. We will then discuss how to explore EV function in vitro and in vivo and, lastly the diagnostic and therapeutic potential of EVs (e.g. as drug delivery systems, vaccines). During the practical sessions, you will learn how to isolate EVs using size exclusion chromatography (qEV) and a polymer precipitation technique. The size distribution and numbers of EVs isolated will be determined using nanoparticle tracking analysis (NTA). Classical EV makers will be identified using Western Blot, and proteomics will be used to explore how different conditions affect the protein content of EVs. Though the focus of the course will be on mouse and human EVs, we will also cover and discuss EVs from non-model organisms, including outer membrane vesicles (OMVs) and some of their specific challenges and opportunities (e.g. drug delivery system). As many of the methods and considerations for working with EVs are the same irrespectively of their source, this course is relevant for most people interested or already active in the EV field.
This course provides hands-on introduction to flow cytometry in general which is necessary to understand how EVs can be analysed using regular flow cytometers. There is one lecture that specifically features analysis of EVs in flow cytometry, followed by an instrument demonstration of our state-of-the-art infrastructure, CytoFLEX nano, a flow cytometer specialized in EV analysis and other nano-sized particles.
In contrast to most basic flow cytometry courses and online resources, this intensive training course teaches key concepts by derivation from "first principles". The course thus covers the progression from the basic physics of light and fluorescence, through fluorochrome chemistry, spectral overlap and compensation, and antibody panel design, experiment design, flow cytometry controls, and data analysis. On the instrumentation side, the course provides a detailed understanding of the core components in modern flow cytometers, thus covering light detection principles, fluidics, optics and signal processing. Data analysis and compensation is taught by a "hands-on" approach via practical computer exercises with FlowJo software and generic, raw flow cytometry data files (participants are encouraged to bring their own PC and data, if relevant). Advanced data analysis approaches (clustering, dimensionality reduction, tSNE and more) are presented in the last part of the course. In addition, guidelines for publishing flow cytometry data will be covered.
Key words: Single/Bulk EV Analysis, Flow Cytometry, Imaging Flow Cytometry
EV characterization
Høegh-Guldbergs Gade 10
The FACS Core Facility provides access to well-maintained instrumentation and training as a fee for service.
Flow cytometer capable of resolving particles down to 160 nm on scatter. Johann Mar Gudbergsson has experience in analysing EVs with this.
Flow cytometer capable of resolving particles down to 100 nm only by scatter but smaller when using fluorescence. Link to the manufacturer's application note.
Imaging flow cytometer capable of resolving particles down to 100 nm only by scatter but smaller when using fluorescence. Yuya Hayashi has experience in analysing EVs with this. Link to a methodology paper on the analysis of EVs labelled by CD63-EGFP.
A Fluorescence-Activated Cell Sorter (FACS) which has small particle detection mode enabling it to resolve/sort particles down to 100-160 nm – EV-sorting on this instrument has not been tested yet.
A dedicated Small Particle Flow Cytometer capable of resolving particles down to 40 nm on scatter and to separate particle with a size difference down to 10 nm. Analysis of particles between 450-800 nm require authorization from the FACS Core Facility. Particles above 800 nm cannot be acquired on this instrument.
Key words: Exosomes, Intercellular communication, Schwann cells, Animal model
EV imaging, EV characterization
Høegh-Guldbergs Gade 10
Our genetic mouse model allows us to specifically visualize Schwann cell (SC)-derived EVs through fluorescent tag. Our aim is to investigate basic facts about SC-derived EVs, in particular their biogenesis, release and internalization in target cells. Future studies will determine SC-derived EVs involvement in axonal homeostasis and regeneration after injury.
Our current focus is the identification of an efficient and reproducible method for the isolation of SC-derived EVs from mouse peripheral nerves.
Tamoxifen-inducible transgenic mouse model expressing hCD63-copGFP tag on EVs membrane under the activity of Mpz promoter, specific for Schwann cells in peripheral nerves.
miRNA, Protein, EV characterization
Universitetsbyen 81
We are characterizing and studying foodborne extracellular vesicles mostly from milk (human, bovine, caprine). Main interest is intestinal uptake, bioactivity, and molecular nutrition, as well as impact of industrial processing on EV integrity. Techniques to gently isolate EV is a speciality. Testing on cells grown in culture constitute a major tool.
Macrophage-like cells (RAW264.7, THP-1), Intestinal cell lines (Caco-2, HT29, FH74 int, IEC-18, IPEC-J2), Entroendocrine cells and many others.
SEC, RP-HPLC, IEX, HIC, Affinity chromatography (attachment of self-selected ligands). Possibility for large-scale EV isolation.
By lactadherin-fluorophore conjugates (binds to phosphatidylserine in the phospholipid membrane).
miRNA, Synthetic EV, EV characterization, EV imaging
Gustav Wieds Vej 14
Extracellular vesicles, RNA therapeutics, synthetic biology, cellular interactions, microRNA, circRNA, aptamers, RNA biomarkers.
Nanoimager fluorescent and super-resolution microscopy with imaging modes for epifluorescence, total internal reflection (TIRF) and HiLo. The microscope has four laser lines 405 nm, 488 nm, 561 nm, and 640 nm. The microscope can image in real-time ideal for tracking of single molecules and is equipped with a temperature controller and a micro fluiding pumping system. Read more here (https://oni.bio/nanoimager/).
Contact person for ONI: Mette Malle <malle@inano.au.dk>
OBS: the microscope is not trivial to use and Mette is happy to collaborate and/or help with everything from guiding which fluorophores should be used, microscope surface preparation and imaging conditions.
For nanoparticle synthesis. Read more here (https://www.precisionnanosystems.com/platform-technologies/product-comparison/ignite).
For 2D single-cell spatial transcriptomics. Read more here (https://nanostring.com/products/cosmx-spatial-molecular-imager/).
For flow Induced Dispersion Analysis (FIDA) to study binding parameters in solution. Read more here (https://fidabio.com/products/)
Nanoparticle tracking analysis (NTA) for size and concentration determination (single-particle approach).
Dynamic light scattering (DLS) for determination of size distributions (ensemble approach, intensity biased).
Digital PCR for absolute quantification with multiplexing.
Key words: Stroke, Plasma EVs, miRNA, Cellular Models
miRNA, EV characterization
Aarhus University Hospital, Palle Juul-Jensens Boulevard 45
We study stroke diagnostics and treatment utilizing characterization of extracellular vesicles (EVs) in the blood as well as the vast number of secreted miRNAs and other small RNAs. We work closely together with clinicians to obtain acute blood samples and use a multitude of molecular biological techniques including EV isolation and characterization, cellular stroke model systems, RNA purification and quantification as well as bioinformatics.
Human brain microvascular endothelial cells (HBMECs).
For routine EV isolation.
Nanoparticle tracking analysis (NTA) for size and concentration determination (single-particle approach). Available at Blood & Biochemistry, AUH.
Key words: miRNA, Exosomes, Plants, Cultivated Meat, Bioactivity
miRNA, EV characterization
Agro Food Park 48
We do research within the context of food science. We are currently looking at EV and miRNAs in plants and their impact on health. Also, we are trying to understand the role of EVs and miRNA for myogenesis.
Primary cultures of satellite cells and hepatocytes. IPEC-J2, Caco2, HepG2, Hepa1-6, HepaRG and C2C12 lines are also available.
Gene-chip based mapping of miRNAs in tissue or fluids.
High-throughput automated microscopy combined with microplate reader, used for e.g. determination of EV uptake.
For ultracentrifugation of EVs.
Dynamic light scattering (DLS) for EV sizing.
ddPCR for determining the absolute quantity of miRNA content in EVs.
* All research infrastructure can be accessed through collaboration.
miRNA, Protein
Aarhus University Hospital, Palle Juul-Jensens Boulevard 45
RAW264.7, SIM-A9, THP-1, HEK293T, IPEC-J2, peripheral blood mononuclear cells (PBMCs), primary monocytes, adipose-derived stem cells (ASCs).
In vivo (pre-)labelling of EVs, engineered labelling of EVs, and engineered loading of therapeutic proteins into EVs.
Augmented COlorimetric NANoplasmonic (CONAN) method for determining purity and concentration of EVs using gold nanoparticles.
Key words: Brain-derived EVs, miRNA, Proteomics, Postmortem Brain, Substance Use Disorders, Mood Disorders
miRNA, tRNA, Protein, EV characterization
Aarhus University Hospital, Palle Juul-Jensens Boulevard 11 (FORUM)
Our project investigates miRNA-protein interactions in Brain-Derived Extracellular Vesicles (BDEs) from individuals with Substance Use Disorders (SUD), with and without Major Depression (MDD). We hypothesize these BDEs exhibit distinct profiles reflective of CNS pathophysiological changes due to substance use and neuro-inflammatory responses. Focusing on differential expression of miRNAs and proteins linked to neuro-inflammation, neurogenesis, and neuroplasticity, we aim to identify biomarkers for understanding underlying mechanisms in dual diagnosis and developing novel treatments. This includes validating plasma BDE findings with postmortem brain tissue EVs. This method offers the possibility for real-time tracking of disorder progression and treatment response, underscoring the importance of EVs in the field of Psychiatry.
Portable nanopore sequencing device for high-throughput, simultaneous DNA/RNA reads of fragments ≥20 base pairs.
Key words: Bioimaging, EVomics, EV-Protein Corona, EV Characterization, Tissue Regeneration, Macrophage & Endothelial Biology
miRNA, Protein, EV-protein corona, EV characterization, EV imaging
Universitetsbyen 81
We study EVs as the new frontier of cell-to-cell communication. For example, by which mechanisms can EVs find the target recipient cells via the bloodstream? What kind of messages are conveyed to regulate/support the recipient cells? With zebrafish as our little partners, we seek answers to these questions by nanoscience approaches, bioinformatics and 4D imaging of live transgenic embryos.
We have two approaches: fluorescent labelling of endogenous EVs and microinjection of (fluorescently labelled) EVs including, but not limited to, human-derived EVs. In both cases, EVs are imaged in live zebrafish embryos using optical microscopes to study the biodistribution, blood clearance kinetics, and interactions with cells of interest (e.g. macrophages and endothelial cells). Induction of TNF-alpha can also be visualized in real-time as an indicator of macrophage polarization in response to EVs. Collaborations on tissue injury models and EV injections are welcome.
ChromoTek’s GFP-trap Dynabeads for EV isolation from tissues (zebrafish embryos).
Through Embion. Cryo-electron microscopy (cryo-EM) and cryo-correlative light-electron microscopy (cryo-CLEM) for imaging EVs at ultrastructural resolution.
