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EVAnet

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.

Info

EVAnet Meetings

EVAnet Meetings consist of two forums:

  • [Network meeting] All members are invited to discuss network-related issues and research infrastructure
  • [Student meeting] Particularly for students and postdocs, to network and discuss science in a casual atmosphere

Topics for Network meetings:

  • Facilitating of collaborations between network members
  • Sharing of equipment and technical support with co-authorship
  • Joint application for research & infrastructure grants
  • Announcement of events, recruitments and open positions

Topics for Student meetings:

  • Sharing good papers (Journal club) or own technical experience
  • Trouble shooting on technical issues
  • Networking among students & postdocs who are actually in the lab

Frequency and Format:

  • Bi-monthly
  • Places to be rotated among MBG, iNANO, Biomed, Clin and Omiics
  • Network meeting (1 h): Project presentation (30 min) + Discussion forum
    * Can be adjusted if there are urgent topics to discuss.
  • Student meeting (1 h): Presentation of a scientific article with a focus on the methods, or presentation of own methods if trouble-shooting is needed (45 min) + Networking time (15 min)

Meeting Schedule

Location, Date & Time – Presenters/Hosts

  • 5th EVAnet Meeting at Biomed on 25 Sep 2023, 13.00-15.00
    – Johann Gudbergsson (Biomed) & Ziyu Huang (Food)
  • 6th EVAnet Meeting at MBG on 21 Nov 2023, 12.00-14.30
    – Jan Trige Rasmussen (MBG) & Elisabeth (iNANO)
  • 7th EVAnet Meeting on TBA (Jan 2024)

Become a Network Member

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.

Contact Organizers

Overview of Our Research Infrastructure

EV Isolation and Purification

Ultracentrifugation

  • Beckman Coulter Optima L-100K, Type 60 Ti rotor (iNANO, MBG)
  • Beckman Coulter XP900, Type 70 Ti rotor (Food) – through collaboration with Martin Krøyer Rasmussen

Specialized size-exclusion chromatography (SEC)

  • Izon qEV SEC (AUH) – through collaboration with Kim Ryun Drasbek
  • Large-scale SEC (MBG) – through collaboration with Jan Trige Rasmussen

EV Characterization

Nanoparticle tracking analysis (NTA)

  • Malvern NanoSight LM10-HS (iNANO)
  • Malvern NanoSight NS300 (AUH)

Dynamic light scattering (DLS)

  • Malvern ZetaSizer Nano (iNANO)
  • Malvern ZetaSizer Pro (Food) – through collaboration with Martin Krøyer Rasmussen

Flow cytometry (FCM) and cell sorter (FACS)

  • Sony ID7000 Spectral Cell Analyzer (FACS Core Facility, Biomed) – paid access or service
  • NovoCyte Quanteon (FACS Core Facility, Biomed) – paid access or service
  • Amnis ImageStreamX MkII Imaging Flow Cytometer (FACS Core Facility, Biomed) – paid access or service
  • Invitrogen Bigfoot Spectral Cell Sorter (FACS Core Facility, Biomed) – paid access or service

EV Imaging

Super-resolution microscopy

  • Oxford NanoImaging (ONI) microscope (iNANO) – through collaboration with Jørgen Kjems

Cryo-electron microscopy (Cryo-EM) and Cryo-correlative light-electron microscopy (Cryo-CLEM)

  • Titan Krios G3i (Embion) – paid access or service
  • Titan Krios (Embion) – paid access or service
  • Leica THUNDER Imager Cryo-CLEM (Embion) – paid access or service

High-throughput automated imaging

  • BioTek Cytation 5 Cell Imaging Multimode Reader (Food) – through collaboration with Martin Krøyer Rasmussen

EV Cargo Analysis

High-throughput analysis of miRNA

  • RNAseq and bioinformatics (Omiics ApS) – paid service
  • GeneChip miRNA Arrays (Food) – through collaboration with Martin Krøyer Rasmussen
  • Oxford Nanopore Technologies MinION (AUH) – through collaboration with Rodrigo Grassi-Oliveira

Absolute quantitation of miRNA

  • Bio-Rad Droplet Digital PCR (Food) – through collaboration with Martin Krøyer Rasmussen

Model Organisms

Cell lines and primary cells

  • RAW264.7, SIM-A9, THP-1, HEK293T, IPEC-J2, Caco2, HT29, FH74 int, IEC-18, HepG2, Hepa1-6, HepaRG, C2C12, adipose-derived stem cells (ASCs), peripheral blood mononuclear cells (PBMCs), primary monocytes, human brain microvascular endothelial cells (HBMECs), primary satellite cells, primary hepatocytes – see individual research profiles below.

Non-mammalian

  • Zebrafish (MBG) – through collaboration with Yuya Hayashi

Education

Extracellular Vesicles – An Introduction

Course level

  • PhD (2.6 ECTS)

Semester

  • Spring (3 days)

Course organiser

  • Peter Lindberg Nejsum (Faculty of Health)

Brief description

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.

Extracellular Vesicles in Health and Disease

Course level

  • Master (5 ECTS)

Semester

  • Summer course (3 weeks)

Course organiser

  • Peter Lindberg Nejsum (Faculty of Health)

Brief description

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.

Associated Research Groups

FACS Core Facility – Fluorescence/Scatter-Based Analysis of Extracellular Vesicles

Key words: Single/Bulk EV Analysis, Flow Cytometry, Imaging Flow Cytometry

  • Department of Biomedicine


Anja Bille Bohn

Imaging Flow Cytometry - Flow cytometry specialist

EV Expertise

EV characterization

Laboratoy Location

Høegh-Guldbergs Gade 10

Research Area

The FACS Core Facility provides access to well-maintained instrumentation and training as a fee for service.


Sony ID7000 Spectral Cell Analyzer

Flow cytometer capable of resolving particles down to 160 nm on scatter. Johann Mar Gudbergsson has experience in analysing EVs with this.

NovoCyte Quanteon

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.

Amnis ImageStreamX MkII Imaging Flow Cytometer

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.

Invitrogen Bigfoot Spectral Cell Sorter

A Fluorescence-Activated Cell Sorter (FACS) which has small particle detection mode enabling it to resolve/sort particles down to 100-160 nm – but the instrument is very new and we did not have time to test it yet. In theory, it should be possible to sort EVs on this.

Schwann Cell-Derived Extracellular Vesicles in Axonal Homeostasis and Regenerative Processes

Key words: Exosomes, Intercellular communication, Schwann cells, Animal model

  • Department of Biomedicine

  • DANDRITE


EV Expertise

EV imaging, EV characterization

Laboratoy Location

Høegh-Guldbergs Gade 10

Research Area

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.


Mouse model

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.

Molecular Nutrition Aspects of Extracellular Vesicles

Key words: 

  • Department of Molecular Biology and Genetics


EV Expertise

miRNA, Protein, EV characterization

Laboratoy Location

Universitetsbyen 81

Research Area

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.


Cell lines

Macrophage-like cells (RAW264.7, THP-1), Intestinal cell lines (Caco-2, HT29, FH74 int, IEC-18, IPEC-J2), Entroendocrine cells and many others.

Chromatography

SEC, RP-HPLC, IEX, HIC, Affinity chromatography (attachment of self-selected ligands). Possibility for large-scale EV isolation.

Specific EV labelling

By lactadherin-fluorophore conjugates (binds to phosphatidylserine in the phospholipid membrane).

Key words: EV, miRNA, Biomarker, Visualization, Tracking, RNA Therapeutic, Delivery Vesicle

  • Interdisciplinary Nanoscience Center (iNANO)

  • Department of Molecular Biology and Genetics


EV Expertise

miRNA, Synthetic EV, EV characterization, EV imaging

Laboratoy Location

Gustav Wieds Vej 14

Research Area

Extracellular vesicles, RNA therapeutics, synthetic biology, cellular interactions, microRNA, circRNA, aptamers, RNA biomarkers.


Oxford Nanoimager (ONI)

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.

Precision NanoSystems NanoAssemblr Ignite

For nanoparticle synthesis. Read more here (https://www.precisionnanosystems.com/platform-technologies/product-comparison/ignite).

NanoString CosMx Spatial Molecular Imager

For 2D single-cell spatial transcriptomics. Read more here (https://nanostring.com/products/cosmx-spatial-molecular-imager/).

Fida Biosystems Fidabio

For flow Induced Dispersion Analysis (FIDA) to study binding parameters in solution. Read more here (https://fidabio.com/products/)

Malvern NanoSight LM10-HS

Nanoparticle tracking analysis (NTA) for size and concentration determination (single-particle approach).

Malvern ZetaSizer Nano

Dynamic light scattering (DLS) for determination of size distributions (ensemble approach, intensity biased).

Extracellular Vesicles and Micro RNAs in Stroke Diagnosis and Treatment

Key words: Stroke, Plasma EVs, miRNA, Cellular Models

  • Department of Clinical Medicine


EV Expertise

miRNA, EV characterization

Laboratoy Location

Aarhus University Hospital, Palle Juul-Jensens Boulevard 45

Research Area

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.


Primary cells

Human brain microvascular endothelial cells (HBMECs).

Izon qEV size-exclusion chromatography

For routine EV isolation.

Malvern NanoSight NS300

Nanoparticle tracking analysis (NTA) for size and concentration determination (single-particle approach). Available at Blood & Biochemistry, AUH.

Micro RNA and Extracellular Vesicles in Food Science

Key words: miRNA, Exosomes, Plants, Cultivated Meat, Bioactivity

  • Department of Food Science


EV Expertise

miRNA, EV characterization

Laboratoy Location

Agro Food Park 48

Research Area

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.


Cell lines and primary cells

Primary cultures of satellite cells and hepatocytes. IPEC-J2, Caco2, HepG2, Hepa1-6, HepaRG and C2C12 lines are also available.

GeneChip miRNA Arrays

Gene-chip based mapping of miRNAs in tissue or fluids.

BioTek Cytation 5 Cell Imaging Multimode Reader

High-throughput automated microscopy combined with microplate reader, used for e.g. determination of EV uptake.

Beckman Coulter XP900, Type 70 Ti rotor

For ultracentrifugation of EVs.

Malvern ZetaSizer Pro

Dynamic light scattering (DLS) for EV sizing.

Bio-Rad Droplet Digital PCR

ddPCR for determining the absolute quantity of miRNA content in EVs.

* All research infrastructure can be accessed through collaboration.

Key words: 

  • Department of Clinical Medicine


EV Expertise

miRNA, Protein

Laboratoy Location

Aarhus University Hospital, Palle Juul-Jensens Boulevard 45

Research Area


Cell lines and primary cells

RAW264.7, SIM-A9, THP-1, HEK293T, IPEC-J2, peripheral blood mononuclear cells (PBMCs), primary monocytes, adipose-derived stem cells (ASCs).

EV labelling

In vivo (pre-)labelling of EVs, engineered labelling of EVs, and engineered loading of therapeutic proteins into EVs.

The CONAN assay

Augmented COlorimetric NANoplasmonic (CONAN) method for determining purity and concentration of EVs using gold nanoparticles.

Brain-Derived Extracellular Vesicles in Psychiatry

Key words: Brain-derived EVs, miRNA, Proteomics, Postmortem Brain, Substance Use Disorders, Mood Disorders

  • Department of Clinical Medicine
    (Translational Neuropsychiatry Unit)


EV Expertise

miRNA, tRNA, Protein, EV characterization

Laboratoy Location

Aarhus University Hospital, Palle Juul-Jensens Boulevard 11 (FORUM)

Research Area

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.


Oxford Nanopore Technologies MinION

Portable nanopore sequencing device for high-throughput, simultaneous DNA/RNA reads of fragments ≥20 base pairs.

Profiling EV RNA Content using RNA Sequencing

Key words: EVs, RNA, miRNA, tRNA, circular RNA, mRNAs, Biomarker

  • Omiics ApS


Yan Yan

Co-founder & CSO, Omiics ApS

e-mail

yan.yan@omiics.com

address

INCUBA - Katrinebjerg
Åbogade 15
8200 Aarhus N

EV Expertise

miRNA, tRNA

Laboratoy Location

Gustav Wieds Vej 14

Research Area

We have experience in purifying EVs from different types of material using different methods. We are specialized in using RNA sequencing to profile the RNA content in EVs, which can be used for the investigation of biomarkers, EV biogenesis and new drug development. There are different types of RNAs can be investigated in the sequencing data, including miRNAs, tRNA derived small RNAs, tRNAs, circular RNAs, mRNAs and long non-coding RNAs. Beside RNA sequencing, we also can help set up other RNA quantification methods, such as digital PCR, qPCR and nanostring.


RNAseq

Omiics can offer RNA sequencing service to EV samples and also offer suggestions on EV samples preparation for RNA sequencing. We have established bioinformatics pipeline for analyzing the small RNAs (miRNAs, tRNA derived small RNAs and other small RNAs) and long-RNAs (mRNAs, circular RNAs and long non-coding RNAs) in EV samples. The analysis include: quantification of different types of RNAs, differential expression analysis, pathway analysis and so on.

A Zebrafish Approach to Extracellular Vesicle Biology

Key words: Bioimaging, EVomics, EV-Protein Corona, EV Characterization, Tissue Regeneration, Macrophage & Endothelial Biology

  • Department of Molecular Biology and Genetics

  • Interdisciplinary Nanoscience Center (iNANO)


EV Expertise

miRNA, Protein, EV-protein corona, EV characterization, EV imaging

Laboratoy Location

Universitetsbyen 81

Research Area

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.


Zebrafish models

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.

Immunoprecipitation of EVs

ChromoTek’s GFP-trap Dynabeads for EV isolation from tissues (zebrafish embryos).

Electron microscopy of EVs

Through Embion. Cryo-electron microscopy (cryo-EM) and cryo-correlative light-electron microscopy (cryo-CLEM) for imaging EVs at ultrastructural resolution.