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Esther Nolte-’t Hoen, Utrecht University, The Netherlands
Basic science
cancer
(cardio)vascular
hypoxia
reproduction/pregnancy
tissue repair and remodelling
body fluids / biomarkers
therapeutics...
ISEV 2013
cancer
(cardio)vascular
hypoxia
reproduction/pregnancy
tissue repair and remodelling
body fluids / biomarkers
th...
Flow cytometry
Single vesicle analysis
novel characterization
methods
standardization in
measurement
EV isolation and char...
EV isolation
Buzz words 2014:
• Size exclusion chromatography
• Comparative analysis
- Purpose: Separate EV from contamina...
Buzz words 2014:
• Size exclusion chromatography
• Comparative analysis
Sucrose / iodixanol
Gradient separation
Ultracentr...
Standardization
Topics 2014:
-Inter- and intra-individual variability
-Pre-analytical variables:
- Freeze/thaw (blood, mil...
EV characterization
Buzz words 2014:
• At the single vesicle level
• Comparative analysis
- (fluorescent mode) NTA
- t-Res...
EV characterization
Buzz words 2014:
• At the single vesicle level
• Comparative analysis
- (fluorescent mode) NTA
- t-Res...
EV cargo selection and release
Release:
- Live cell fluorescence imaging (e.g. pH sensitive optical reporter)
- Substances...
EV function
STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY
Key player ISEV 2014:
Mesenchymal Stem Cell
Physiological role Tissue...
Remarkable new insights
Release of organelles via EV:
Proteasomes (T cell lines)
Mitochondria (from MSC to macrophages)
1
Remarkable new insights
Facts:
- Pub-crawl driven research
- Consumption of high numbers of EV
- No pro-inflamm effects
Re...
Thank you all!
ISEV 2014 - Basic Science Wrapup (E. Holte t'-Noen)
ISEV 2014 - Basic Science Wrapup (E. Holte t'-Noen)
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ISEV 2014 - Basic Science Wrapup (E. Holte t'-Noen)

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Wrapup of basic science presentations at ISEV2014. For more information go to www.isev.org

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ISEV 2014 - Basic Science Wrapup (E. Holte t'-Noen)

  1. 1. Esther Nolte-’t Hoen, Utrecht University, The Netherlands Basic science
  2. 2. cancer (cardio)vascular hypoxia reproduction/pregnancy tissue repair and remodelling body fluids / biomarkers therapeutics pathogen-derived EV immune system nervous system isolation characterization RNA biogenesis Late breaking Clinical ISEV 2014: Basic science topics
  3. 3. ISEV 2013 cancer (cardio)vascular hypoxia reproduction/pregnancy tissue repair and remodelling body fluids / biomarkers therapeutics pathogen-derived EV immune system nervous system isolation characterization RNA biogenesis Late breaking ISEV 2014: Basic science topics Clinical
  4. 4. Flow cytometry Single vesicle analysis novel characterization methods standardization in measurement EV isolation and characterization
  5. 5. EV isolation Buzz words 2014: • Size exclusion chromatography • Comparative analysis - Purpose: Separate EV from contaminants - Fluids: - Prostate fluid - Blood plasma - Milk - Cell culture supernatant - Big advantage: low labor intensity - Future: - Different packing material to remove different contaminants? - Loss of EV? How to assess this?
  6. 6. Buzz words 2014: • Size exclusion chromatography • Comparative analysis Sucrose / iodixanol Gradient separation Ultracentrifugation ? hrs Matrix-based isolation SE chromatography Differences in: - Particle yield - Enrichment of certain EV populations - Contamination with non-EV - EV functionality Future: - Adjust method of choice to research question? - Serial usage for optimal purification and subpopulation isolation? EV isolation
  7. 7. Standardization Topics 2014: -Inter- and intra-individual variability -Pre-analytical variables: - Freeze/thaw (blood, milk) - Shear stress in needles (blood) - Handling time and temperature before/during EV isolation (blood, milk) - Timely removal of interfering protein complexes (e.g. THP in urine and casein/whey in milk) -Quality Control (e.g. protein/lipid ratios) - Standardization of measurements (?)
  8. 8. EV characterization Buzz words 2014: • At the single vesicle level • Comparative analysis - (fluorescent mode) NTA - t-Resistive Pulse Sensing (qNano) - Flow Cytometry - (cryo) EM - Asymmetric flow-field-flow fractionation - Small Angle X-ray Scatter - Dynamic Light Scattering - Atomic Force Microscopy - On-chip size/conc measurement - …. Compare: EV characteristics (e.g. size) EV concentration
  9. 9. EV characterization Buzz words 2014: • At the single vesicle level • Comparative analysis - (fluorescent mode) NTA - t-Resistive Pulse Sensing (qNano) - Flow Cytometry - (cryo) EM - Asymmetric flow-field-flow fractionation - Small Angle X-ray Scatter - DLS - New high-end cytometers & labeling strategies to use conventional cytometers - Comparison of different flow cytometers - Crucial settings for correct measurements - EV sorting High-throughput analysis of EV contentsEV numbers
  10. 10. EV cargo selection and release Release: - Live cell fluorescence imaging (e.g. pH sensitive optical reporter) - Substances inducing EV release Biogenesis: - C-Elegans and Drosophila as model organisms (e.g. RNAi screens) - EV from plasma membrane versus MVB & different types of MVB EV contents: -Subpopulations analysis: Lipidomics / proteomics / transcriptiomics -Overlap RNA species in EV from mammalian cells, parasites, fungi, bacteria! -Mechanisms for RNA sorting
  11. 11. EV function STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY Key player ISEV 2014: Mesenchymal Stem Cell Physiological role Tissue repair Other therapeutic effects Osteogenic differentiation of MSC Ischemia cancer angiogenesis Irradiation damage GvHD Skin burns nephrotoxicity Muscle injury
  12. 12. Remarkable new insights Release of organelles via EV: Proteasomes (T cell lines) Mitochondria (from MSC to macrophages) 1
  13. 13. Remarkable new insights Facts: - Pub-crawl driven research - Consumption of high numbers of EV - No pro-inflamm effects Release of organelles via EV1 Yeast-derived EV in beer 2
  14. 14. Thank you all!

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