Exosome Isolation: A Density-based Ultracentrifugation Technique Using Sucrose Cushion to Separate Exosomes from Conditioned Media of Cultured Cells

Overview

This video describes a density-based ultracentrifugation technique for isolating exosomes from conditioned media harvested from human embryonic kidney cell cultures using a 25% (w/w) sucrose cushion prepared in deuterium oxide. The isolated exosomes can be used for downstream applications.

Protocol

1. Exosome Isolation onto a Sucrose Cushion

1. Pre-clear the harvested conditioned media (CM) by differential centrifugation and filtration as follows.
   1. Centrifuge at 500 x g for 5 min at 4 °C. Transfer the supernatant into a new tube and discard the pellet. Repeat this centrifugation step once more, recovering the supernatant and discarding the pellet.
   2. Centrifuge the supernatant from step 1.1. at 2,000 x g for 15 min and 4 °C and then discard pellet. Filter the recovered supernatant once through 0.22 µm filters.
2. During pre-clearing, prepare 25% (w/w) sucrose solution in deuterium oxide by accurately weighing out 1.9 g (± 0.001 g) of sucrose in a universal tube, and then topping up with deuterium oxide until the weight reaches 7.6 g (± 0.001 g).
3. Fill up an ultracentrifuge tube (see Table of Materials) with 22.5 mL of pre-cleared CM. Make up the CM to 22.5 mL with 0.22 µm-filtered PBS if the current volume is less than that.
4. Place a glass pipette (see Table of Materials) in the tube and, through it, add 3 mL of sucrose solution so that the solution forms a separate layer beneath the CM.
5. Carefully place the tube containing layered CM/sucrose solution into the bucket of a swing-out rotor (see Table of Materials) and secure the bucket into the rotor.
6. Place the rotor into the ultracentrifuge (see Table of Materials) and spin at 100,000 x g for 1.5 h at 4 °C.
7. Collect 2 mL of the sucrose layer and add this to an ultracentrifuge bottle (see Table of Materials) containing 20 mL of filtered PBS for a washing step.
8. Place the tubes into a fixed-angle rotor (see Table of Materials) and spin at 100,000 x g for 1.5 h at 4°C.
9. Carefully remove the supernatant with a 10 mL serological pipette and resuspend the pellet with 400 µL filtered PBS. Keep this exosome stock at 4 °C or -80 °C for short-term and long-term storage respectively.

Materials

Name	Company	Catalog Number	Comments
Sucrose	Fisher Scientific	S/8600/60	1 kg
Deuterium oxide (D2O)	Sigma-Aldrich	151882	250 g
1X Phosphate Buffered Saline	Thermo Fisher Scientific	10010015	500 ml
Millex-GP Syringe Filter Unit 0.22 µm	Millipore	SLGP033RS
Ultracentrifuge	Beckman Coulter	Optima XPN-80
Swing-out rotor	Beckman Coulter	SW45 Ti
Fixed-angle rotor	Beckman Coulter	Type 70 Ti
Ultracentrifuge tubes	Beckman Coulter	355631	Polycarbonate. Max. fill 32 ml
Ultracentrifuge bottles	Beckman Coulter	355618	Polycarbonate. Min. fill 16 ml, max. fill 25 ml
Centrifuge	Eppendorf	5810R
Glass pipettes	Fisher Scientific	1156-6963
25% w/w sucrose cushion			Add 1.9 g (± 0.001 g) of sucrose in a universal tube, and top up with D2O until the weight reaches 7.6 g (± 0.001 g). This makes ~6ml of the 25% w/w sucrose cushion.