Isolation and Characterization of Exosomes from Cultures of Tissue Explants and Cell Lines

Lucia Mincheva‐Nilsson1, Vladimir Baranov1, Olga Nagaeva1, Eva Dehlin1

1 Department of Clinical Microbiology, Division of Clinical Immunology, Umeå University, Umeå
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.42
DOI:  10.1002/cpim.17
Online Posting Date:  November, 2016
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Abstract

Exosomes are specialized, nanometer‐sized extracellular vesicles of endosomal origin actively secreted into the extracellular space by a variety of cells under normal and pathological conditions. Exosomes have recently emerged as important intercellular communicators and modulators of diverse mechanisms and cellular responses. Characterization of their composition and function will open possibilities for new diagnostic methods and promising therapeutic approaches based on nanobiology. This unit provides a standard isolation procedure for purification of exosomes based on density gradient ultracentrifugation with sucrose. The process of isolating exosomes relies on obtaining proper source fluids/supernatants as well as qualitative and quantitative assessment of the isolated vesicles. The methodological procedures here can be divided in three parts: (1) pre‐isolation procedures aiming to obtain fluids containing exosomes, with a focus on protocols for organ explants and cell cultures; (2) a procedure for exosome isolation with several gradient alternatives; and (3) post‐isolation procedures for estimating the purity and yield of the exosomal fraction. © 2016 by John Wiley & Sons, Inc.

Keywords: organ explant culture; extracellular vesicles; exosomes; sucrose gradient; density ultracentrifugation; flow cytometry; negative contrast staining; TEM

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparation of Exosome‐Containing Conditioned Medium from Tissue Explants
  • Basic Protocol 2: Preparation of Exosome‐Containing Conditioned Medium from Cell Cultures
  • Basic Protocol 3: Exosome Isolation Based on Ultracentrifugation and Sucrose Density Gradient
  • Basic Protocol 4: Flow Cytometry Analysis of Exosome Surface Proteins Using Antibody‐Coated Latex Beads
  • Basic Protocol 5: Negative Contrast Staining of Whole‐Mount Exosomes for Tem Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Exosome‐Containing Conditioned Medium from Tissue Explants

  Materials
  • Freshly dissected animal or human tissue
  • Explant culture medium (see recipe)
  • HBSS (see recipe)
  • Tissue culture hood
  • 90‐mm (or larger) sterile Petri dish
  • Sterile forceps and scissors or scalpel
  • 12‐well tissue culture plate
  • 37°C, 5% CO 2 incubator
  • Sterile Pasteur pipets
  • Protease inhibitor (e.g., AEBSF, Pefablock, Roche)

Basic Protocol 2: Preparation of Exosome‐Containing Conditioned Medium from Cell Cultures

  Materials
  • Adherent or non‐adherent cell lines of interest
  • Growth medium
  • PBS, pH 7.2‐7.5 (see recipe)
  • Exosome‐free medium: commercial serum‐free culture medium or regular culture medium with up to 10% (v/v) exosome‐free serum (see recipe)
  • Protease inhibitor (e.g., AEBSF, Pefablock, Roche)
  • Ionomycin, calcium salt (Sigma‐Aldrich)
  • Tissue culture flasks
  • 37°C, 5% CO 2 incubator
  • Tissue culture hood

Basic Protocol 3: Exosome Isolation Based on Ultracentrifugation and Sucrose Density Gradient

  Materials
  • Conditioned medium (see protocol 1 or 2)
  • PBS, pH 7.2‐7.5 (see recipe)
  • Vybrant DiI cell‐labeling solution (Molecular Probes, optional)
  • Gradient solutions (choose one; see step 7):
    • 20% and 40% (w/v) sucrose in PBS (see recipe for 40% solution)
    • 0.25 and 2 M sucrose solutions
    • OptiPrep Density Gradient Medium (60% [w/v] iodixanol, Sigma‐Aldrich)
    • Sucrose cushion: 30% (w/v) sucrose in PBS made with D 2O
  • Complete Protease Inhibitors (Roche)
  • 50‐ml sterile polypropylene centrifuge tubes
  • General purpose centrifuge
  • 0.2‐μm filter, bottle‐top (PES, Nalgene) or syringe‐type with 20‐ to 50‐ml syringe
  • 38.5‐ml Beckman Ultra‐Clear centrifuge tubes (25 × 89 mm)
  • 12.5‐ml Beckman Ultra‐Clear centrifuge tubes (14 × 89 mm)
  • Ultracentrifuge with SW 32 and SW 40 swinging‐bucket rotors (Beckman)
  • 1.5‐ml microcentrifuge tubes (optional)
  • 15‐ml gradient mixer with peristaltic pump and 10‐cm‐long glass capillary tube (optional)
  • NanoSight or ZetaView instrument for nanoparticle tracking analysis (NTA)
  • BCA kit for protein concentration

Basic Protocol 4: Flow Cytometry Analysis of Exosome Surface Proteins Using Antibody‐Coated Latex Beads

  Materials
  • MES buffer (see recipe)
  • Latex beads (Invitrogen Molecular Probes)
  • 1% (w/v) bovine serum albumin (BSA, fraction V) in PBS, filtered (0.2‐μm pore size)
  • Antibodies for coating beads
  • Ig isotype control antibody
  • PBS, pH 7.2‐7.5 (see recipe)
  • 100 mM glycine in PBS, filtered (0.2‐μm pore size)
  • Isolated exosomes (see protocol 3)
  • Fluorescent dye–conjugated revealing antibodies
  • 0.5‐ and 1.5‐ml polypropylene microcentrifuge tubes
  • Sonicating water bath
  • Tube rotator
  • Flow cytometer/fluorescence activated cell sorter (FACS)
NOTE: Perform all washing steps with cold buffer and use filtered solutions throughout the procedure to remove any particles that could interfere with FACS analysis.

Basic Protocol 5: Negative Contrast Staining of Whole‐Mount Exosomes for Tem Analysis

  Materials
  • Isolated exosomes (see protocol 3)
  • 4% (w/v) PFA (see recipe)
  • PBS (see recipe)
  • 1% (v/v) glutaraldehyde (see recipe)
  • Uranyl oxalate solution, pH 7 (see recipe)
  • 4% (w/v) uranyl acetate solution (see recipe)
  • 2% (w/v) methyl cellulose solution (see recipe)
  • Sterile forceps
  • Formvar/carbon‐coated EM grids
  • Sterile Petri dishes with lids
  • Parafilm
  • Sterile glass dish with lid
  • Filter paper (Whatman)
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Figures

Videos

Literature Cited

Literature Cited
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