Extracellular Vesicle Core

About Us

The CHLA Extracellular Vesicle Core provides the research community with expertise, optimized tools and emerging technologies to support research in the developing fields of Extracellular Vesicle and nanoparticles research. The core provides isolation, characterization, and analysis of extracellular vesicles and other nano-sized particles (e.g. viruses, liposomes, nanobubbles). Established in July 2018, this is the first core in the country dedicated to the rapidly evolving field of Extracellular Vesicle research and applications.

Services

The CHLA Extracellular Vesicle (EV) Core provides the research community with expertise, optimized tools and emerging technologies to support research in the developing fields of Extracellular Vesicle and nanoparticles research.

Provided services:

  • EV isolation with a choice of standard and up-to-date methodologies.
  • EV analysis by Nanoparticle Tracking Analysis (NTA) with Nanosight NS300 instrument
  • EV characterization by
    • ExoView R100 platform: based on Single Particle Interferometric Reflectance Imaging Sensor (SP- IRIS) technology
    • Classical SDS-PAGE and immunoblotting
  • Exosomal RNA analysis by TaqMan-based quantitative real-time PCR
  • Generation of EV-depleted FBS.
  • User accessible instruments:
    • Beckman Ultracentrifuge
    • KrosFlo Tangential Flow Filtration syste

This Core participates in the CHLA Core Pilot Program. To learn more click here.

CHLA welcomes external users to utilize our Core facilities. Please contact Paolo Neviani, PhD at pneviani@chla.usc.edu or call 323-361-8564.

  • Fee Structure
  • Equipment and Software
  • References and Useful Publications
  • Core Acknowledgment in Publications

For Principal Investigators based at The Saban Research Institute.

Service Instrument / Notes Rates for CHLA
Nanosight Tracking Analysis (NTA) - Full Service NanoSight NS300 $125/hour
Nanoparticle Tracking Analysis (NTA) - Trained User NanoSight NS300 $50/hour
ExoView - Full Service ExoView R100 $125/sample
ExoView - User-assisted (user provides chips) ExoView R100 $60/sample
Instrument Use Only – Trained user ExoView R100 $125/hour
Extracellular Vesicle Isolation - Full service (includes NTA) Choice of isolation steps:
1. Size Exclusion Chromatography
2. Density gradient ultracentrifugation
3. Differential ultracentrifugation
4. Affinity purification
1-step: $115/sample
2-step: $150/sample
Extracellular Vesicle Isolation only (NTA excluded) Choice of isolation steps:
1. Size Exclusion Chromatography
2. Density gradient ultracentrifugation
3. Differential ultracentrifugation
4. Affinity purification
1-step: $55/sample
2-step: $90/sample
RNA Isolation QC by Nanodrop $15/sample
qPCR full service (user provides TaqMan® primer/probes assays) BIoRad CFX96 Touch $2/replicate
Instrument use only BIoRad CFX96 Touch $15/hour
Immunoblotting for EV characterization According to ISEV guidelines
up to 17 samples per gel
up to 4 primary antibodies
$280/gel
EV-depleted FBS 50ml aliquots $75/aliquot
Tangential Flow Filtration System (user provides filters and other supplies) KrosFlo TFF by SpectrumLabs Complimentary
Beckman Ultracentrifuge - requires IBC and Biohazard use approval Beckman Coulter Optima XE-90K Complimentary
Instrument training fee Required for:
NanoSight NS300
ExoView R100
$125/hour

Access for Non-Hospital Researchers

Access to ECV Core facilities for researchers at USC and other institutions can be arranged. Interested researchers should contact the Technical Director to inquire about availability and rates.

NanoSight NS300

Image result for nanosight ns300

The Malvern Panalytical NanoSight NS300 uses the technology of Nanoparticle Tracking Analysis (NTA). This unique technology utilizes the properties of both light scattering and Brownian motion in order to obtain the size distribution and concentration measurement of particles in liquid suspension. A laser beam is passed through the sample chamber, and the particles in suspension in the path of this beam scatter light in such a manner that they can easily be visualized via 20x magnification microscope onto which is mounted a camera. The camera operates at 30 frames per second (fps), capturing a video file of the particles moving under Brownian motion. The software tracks many particles individually and using the Stokes-Einstein equation calculates their hydrodynamic diameters.

  • Automated analysis of the size distribution and concentration of all types of nanoparticles and extracellular vesicles from 20nm to 1000nm in diameter.
  • Equipped with a 532 nm green laser and a 565LP fluorescence filter for the detection of fluorescently labeled vesicles (e.g. CellMask Orange, PKH26, Dil dyes).

Bio-Rad fraction collectors (Model 2110) and chromatography columns

Chromatography columns packed with Sephacryl S-300 high resolution size exclusion chromatography resin are used to isolate exosomes (50-150 nm in size) from body fluids or from the supernatant of cell cultures. After the elution of several fractions by gravity flow, the presence of EVs is be confirmed by protein determination or Nanoparticle Tracking analysis (see above).

ExoView R100

ExoView R100 Photo 1.pngExoView R100 Photo 2.png

The ExoView instrument is a step forward in characterization capabilities within the extracellular vesicle field. The fully automated platform, based on Single Particle Interferometric Reflectance Imaging Sensor (SP- IRIS) technology, provides multi-level and comprehensive measurements for exosome particle size analysis, exosome count, exosome phenotype, and biomarker colocalization. The ExoView platform provides previously unattainable information in a single and bias free sample workflow.

ExoView works without the need for sample purification and brings the researcher one step closer to analyzing a sample in its natural state. ExoView is an affinity-based technology that allows specific populations of exosomes and other extracellular vesicles to bind in a multiplexed manner to a functional ExoView® chip. The fully automated instrument can measure 9 samples automatically, direct from sample, saving cost, time, and reducing purification biases.

Advantages:

  1. Biomarker colocalization:
    • Quantify relative protein expression of up to 4 proteins per exosome
  2. Count extracellular vesicles:
    • Count the number of antigen-positive exosomes directly from sample, no purification required
  3. Extracellular vesicle size:
    • Acquire high-resolution measurements of the size of individual antigen-positive exosomes
  4. Extracellular vesicle cargo:
    • Probe for exosomal luminal proteins and cargo
  5. Fluorescence:
    • Detect lowest abundance proteins with single binding event sensitivity across three color channels
  6. Streamlined workflow:
    • Automated analysis of up to 9 samples with single button sample analysis
  7. Purification not required:
    • Detect the changes in your sample, not the biases from your purification technique
  8. Multiplexed sample analysis:
    • Characterize multiple populations of exosomes from a single sample using up to 6 surface markers

For more information: www.nanoviewbio.com

Beckman Coulter Optima XE-90K

Beckman Coulter Optima XE-90K Photo.png

The Beckman Optima™ XE-90K Preparative Ultracentrifuge is suitable for separation of small particles such as small extracellular vesicles, viruses, viral particles, proteins and/or protein complexes, lipoproteins, RNA, and plasmid DNA.

Several rotors (swing-out and fixed angle) for different applications are available. Please inquire with core for user training.

Characterization of Exosomes by Immunoblotting with Bio-Rad Protean and Trans-Blot System

Exosome-containing samples are characterized by immunoblotting with previously validated antibodies.

Four markers are analyzed according to the International Society of Extracellular Vesicles https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4275645/:

  1. Transmembrane or lipid-bound extracellular proteins (e.g. CD9, CD63 or CD81); Argues presence of a membrane in the isolate; present or enriched in EVs/exosomes.
  2. Cytosolic proteins (e.g. TSG101, Alix); With membrane- or receptor-binding capacity; present or enriched in EVs/exosomes.
  3. Intracellular proteins (e.g Calnexin); associated with compartments other than plasma membrane or endosomes; absent or under-represented in EVs/exosomes.
  4. Extracellular proteins (e.g. Fibronectin-1); binding specifically or non-specifically to membranes, co-isolating with EVs; variable association with EVs.
  5. Apolipoproteins (e.g Apo-B100); for complex biofluids such as plasma or serum the presence of low-density lipoproteins (VLDLs, LDLs) is an indication of purity of the EV preparation.

Tangential Flow Filtration – KrosFlo TFF by SpectrumLabs

Tangential Flow Filtration Photo.png

Tangential flow filtration (TFF) is a rapid and efficient method for separation and purification of biomolecules. See how it works here.

It can be applied to a wide range of biological fields such as immunology, protein chemistry, molecular biology, biochemistry, and microbiology. TFF can be used to concentrate and desalt sample solutions ranging in volume from a few milliliters to several liters. It can be used to fractionate large from small biomolecules, harvest cell suspensions, and clarify fermentation broths and cell lysates.

For example, TFF can be used to concentrate large volumes of EV-containing samples (e.g. conditioned medium) to a more manageable volume prior to EV isolation. Please inquire with the core for user training.

  1. Considerations on how to pick the best isolation methods.
Method Advantages Disadvantages
Differential ultracentrifugation Standard method; most commonly used until recently. Isolation from large volumes. Lengthy (4-5h).
Low recovery (5-25%).
Co-precipitation of protein aggregates, nucleosomal fragments.
EVs may be damaged by shearing force (100,000g).
Density Gradient on Sucrose or Iodixanol cushion Highest degree of purity based on density. Very lengthy (16+ hours), very sensitive to centrifugation time. Complex. Not easily scalable. Sucrose toxicity.
Size Exclusion Chromatography Highest degree of purification based on size; vesicles not affected by shearing force, gentle. Large scale. Superior reproducibility. Highest degree of purification based on size; vesicles not affected by shearing force, gentle. Large scale. Superior reproducibility.
Immuno-affinity sorting High purity based on surface markers Not suitable for large samples; low yield.
  1. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Thery et al, JEV. 2018 Nov 23;7(1):1535750. doi: 10.1080/20013078.2018.1535750 (https://www.tandfonline.com/doi/full/10.1080/20013078.2018.1535750)

How to cite the Core in publications:

This work was performed with the support of the ExtraCellular Vesicle Core at The Saban Research Institute of Children's Hospital Los Angeles. 

Publications made possible by Core support:

  • Xin Tang, Cheng Chang, Jiacong Guo, Vadim Lincoln, Mei Chen, David T. Woodley, and Wei Li. Department of Dermatology and the Norris Comprehensive Cancer Centre, University of Southern California Keck Medical Centre. Tumour-Secreted Hsp90a on the External Surface of Exosomes Mediates Tumour-Stromal Cell Communication via Autocrine and Paracrine Mechanisms. Sci Rep. 2019 Oct 22;9(1):15108
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805946/
     
  • Paolo Neviani, Petra M Wise, Mariam Murtadha, Cathy W Liu, Chun-hua Wu, Ambrose Y Jong, Robert CSeeger and Muller Fabbri Natural Killer-derived exosomal miR-186 inhibits neuroblastoma growth and immune escape mechanisms. Cancer Res December 12 2018 DOI: 10.1158/0008-5472.CAN-18-0779
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428417/