SEC-based Isolation of Microglia-derived Extracellular Vesicles: A Technique to Isolate Extracellular Vesicles from Microglia Conditioned Culture Media

Abstract

Source: Lemaire, Q. et al. Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment. J. Vis. Exp. (2020)

This video describes a method based on size-exclusion chromatography (SEC) to isolate extracellular vesicles or EVs derived from the brain immune cells―the microglia. The EVs act as molecular cargo to deliver mediators, which regulate an immune response. Therefore, the isolated EVs can be used as an in vitro model to study the microglia-neuron crosstalk during a neuronal immune response.

Protocol

1. Primary Culture of Microglia/Macrophages

1. Primary culture of microglia
   1. Culture commercial rat primary microglia (2 x 10⁶ cells) (see the Table of Materials) in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% exosome-free serum, 100 U/mL of penicillin, 100 μg/mL streptomycin, and 9.0 g/L glucose at 37 °C and 5% CO₂.
   2. Collect the conditioned medium after a 48 h culture and proceed to the isolation of EVs.
2. Primary culture of macrophages
   1. Culture commercial rat primary macrophages (1 x 10⁶ cells) in the medium provided by the manufacturer (see the Table of Materials) at 37 °C and 5% CO₂, with exosome-free serum.
   2. Collect the conditioned medium after a 24 h culture and proceed to the isolation of EVs.

2. Isolation of EVs

1. Pre-isolation of EVs from conditioned medium
   1. Transfer the conditioned culture medium from microglia or macrophage cultures (steps 1.1.2 or 1.2.2) into a conical tube.
   2. Centrifuge at 1,200 x g for 10 min at room temperature (RT) to pellet the cells.
   3. Transfer the supernatant into a new conical tube. Centrifuge at 1,200 x g for 20 min at RT to eliminate apoptotic bodies.
   4. Transfer the supernatant into a 10.4 mL polycarbonate tube and transfer the tube into a 70.1 Ti rotor. Ultracentrifuge at 100,000 x g for 90 min at 4 °C to pellet the EVs.
   5. Discard the supernatant and resuspend the pellet containing EVs in 200 μL of 0.20 μm filtered phosphate buffer saline (PBS).
2. Isolation of EVs
   1. Preparation of the home-made size exclusion chromatography column (SEC)
      1. Empty a glass chromatography column (length: 26 cm; diameter: 0.6 cm) (see the Table of Materials), wash and sterilize it.
      2. Place a 60 μm filter at the bottom of the column.
      3. Stack the column with a cross-linked agarose gel filtration base matrix to create a stationary phase of a 0.6 cm diameter and a 20 cm height.
      4. Rinse the phase with 50 mL of 0.20 μm filtered PBS. Store at 4 °C to be used later if necessary.
   2. Place the resuspended EV pellet on top of the stationary phase of the SEC column.
   3. Collect 20 sequential fractions of 250 μL while continuing to add 0.20 μm filtered PBS on the top of the stationary phase to prevent drying of the column. Store the fractions at -20 °C if necessary.
      NOTE: A longer storage than one week can be performed at -80 °C to maintain the EV integrity for molecular analysis.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rat Macrophage & Microglia Culture Medium	Cell Applications	R620K-100	Cell origin: Normal healthy Rat bone marrow
Rat primary microglia	Lonza	RG535
Sepharose CL-2B	GE Healthcare	17014001
Ultracentrifuge	Beckman Coulter	A95765
Ultracentrifuge Rotor 70.1 Ti	Beckman Coulter	342184
Glucose	Sigma-Aldrich	G8769
Phosphate Buffer Saline	Invitrogen	14190094	No calcium, No magnesium
Polycarbonate centrifuge tubes	Beckman Coulter	355651
Penicillin-Streptomycin	Gibco	15140-122
Exo-FBS	Ozyme	EXO-FBS-50A-1	Exosome depleted FBS
DMEM Gibco 41966029
Centrifuge	Eppendorf	5804000010
CO2 Incubator	ThermoFisher
pluriStrainer M/ 60 μm	pluriSelect	43-50060
MonoP FPLC column	GE Healthcare		No longer available