Vesicle preparation

Vesicle production and preparation protocol

Day 1

Thaw batch of BHK21 cells and plate in 1xT75 in 12 ml media:

GMEM (https://www.thermofisher.com/order/catalog/product/11710035?SID=srch-srp-11710035)

2% TBP (https://www.thermofisher.com/order/catalog/product/18050039?SID=srch-srp-18050039)

2% HEPES (https://www.thermofisher.com/order/catalog/product/15630056?SID=srch-srp-15630056)

10% FBS

Day 2 (or 3)

Passage cells when they are ~80% confluent to 1xT175

• remove media
• wash with PBS
• 1 ml trypsin
• stop with 10 ml medium
• spin down 300 g 2 min
• properly resuspend 1 ml
• add 25 ml media (as before) in T175 flask
• add cells to the flask

Day 3

Cells should be ~70-80% confluent (critical not to use flasks that are fully confluent)

Transfection:

DNA – 1 mg/ml

DNA / transfectant ratio 1:2

Medium:

1. SF (serum free) GMEM - 2% TPB, 20 mM HEPES
2. FBS-GMEM - 2% FBS, 2% TPB, 20 mM HEPES

Transfection procedure

• 1.5 ml SF GMEM + 60 µg (60 µL) DNA – mix well in tube & vortex
• 1.5 ml SF GMEM  + 120 µL Lipofectamin 2000 (https://www.thermofisher.com/order/catalog/product/11668019?SID=srch-srp-11668019) – mix well in tube & vortex
• mix both together in 15 ml falcon & vortex
• incubate for 20 min
• wash cells with warm PBS
• exchange media in T-175 carefully for 25 ml FBS-GMEM
• vortex DNA/Lipofectamine mixture again and add carefully to cells
• shake gently to distribute transfection mix
• incubate for 2 h @ 37°C
• Wash with warm PBS twice
• Add 30 ml FBS-GMEM

Day 4

• Wash cells with warm PBS
• Exchange media for 30 ml warm SF GMEM

Day 5

Harvest & purification:

Buffer A: 20 mM HEPES pH 7.4; 130 mM NaCl (sterile filtered)

Buffer A+20% sucrose: dissolve 20 g sucrose (https://www.sigmaaldrich.com/DE/de/product/mm/107651) in 100 ml Buffer A > sterile filter solution

• Collect cell supernatant – use supernatant to wash cells gently and transfer the supernatant to 50 ml falcon

Avoid formation of foam or bubbles 

• centrifuge for 20 min @ 3000xg; 4°C
• Wash with EtOH and dry tubes (Polyallomer 35 x 89 mm 38.5 ml) for SW32 (https://www.beckman.de/centrifuges/rotors/swinging-bucket/369694) rotor
• Transfer supernatant to tube - 30 ml / tube
• Add 5 ml 20 % sucrose in buffer A at the bottom using a long, blunt end, metal needle with luer connection
• Load tubes carefully in buckets and balance
• Spin for 2 h @ 30 000 rpm (153 000xg max), 4°C
• Remove supernatant using aspirator (https://pr.vwr.com/store/product/11725661/vacusip-bench-top-aspiration-system-integra-biosciences)
• Remove residual liquid in the tube with lint-free tissue, but do not touch the bottom part of the tube
• Add 100 µL Buffer A
• Swirl gently and seal the tube with parafilm
• Put in fridge over night to swell

Day 6

• Carefully resuspend the vesicles by pipetting up and down without creating foam
• transfer vesicles to Eppendorf tube
• analyse by SDS PAGE followed by commassie stain or western blot

Remarks:

• For proteins that do form vesicles, still high expression yields are necessary
• Usually pellets are hard to see
• can be scaled down to T75 flasks using 25 µg DNA + 50 µL Lipofectamine > 10 ml SF media + spin in SW41 rotor @ 30 000 xg with 2 ml sucrose cushion
• Original method: Zeev-Ben-Mordehai T. et al. Extracellular Vesicles: A Platform for the structure determination of membrane proteins by cryo-EM. Structure 2014 Nov; 22(11):1687-1692