2

Materials

2.1

Cell Culture

1. Obtain cells, media, flasks, and CELLine AD 1000 bioreactor

flask (see Notes 1 and 2).

2. Culture materials.

(a)

Sterile PBS.

(b)

DMEM, RPMI or other media.

(c)

Advanced media (optional).

(d)

Fetal bovine serum (FBS) or other appropriate serum.

(e)

Penicillin–streptomycin (PS).

(f)

CDM-HD serum replacement (see Note 3).

2.2

EV Isolation

Materials

1. 35 nm qEV Original size exclusion chromatography (SEC)

column or equivalent column

2. Eppendorf 1.5 mL LoBind tubes.

3. Ultracentrifuge tubes, ultracentrifuge, and fixed-angle rotor

(Beckman Avanti J-30I or 100,000
g capable equivalent).

2.3

Characterization

Materials

1. Bicinchoninic acid (BCA) kit or comparable protein assay.

2. NanoSight NS300 or comparable nanoparticle counting and

sizing system.

3. Transmission electron microscopy (TEM) reagents (copper

grids, 2% uranyl acetate).

4. Western

blot

reagents

(blocking

solution,

antibodies,

membranes).

5. 4% glutaraldehyde.

3

Methods

3.1

Preparation

of CELLine AD 1000

Bioreactor Flask

(Figs. 1 and 2)

1. Add 50 mL of prewarmed inoculation culture media in the

media chamber and let the semipermeable membrane equili-

brate for 5 min.

3.2

Inoculation

of Cells (See Note 4)

1. Obtain at least 2.5
10⁷ viable cells from a preculture in a

growth phase and resuspend the cells in 15 mL media contain-

ing 10% FBS and 1% PS (this equates to approximately 5–8

T175 flasks of cells depending on the cell type and confluence).

2. Always loosen the media chamber cap when exchanging fluid in

the cell chamber to prevent an air lock (Fig. 2a) which can

damage the delicate semipermeable membrane (see Note 5).

Production of Extracellular Vesicles Using a CELLine Adherent Bioreactor Flask

185