3. Collect the 15 mL preculture cell suspension with a 25 mL

serological pipette, open the cell chamber (lower compart-

ment) and inoculate it by inserting the pipette into the black

silicone cone (Fig. 2f). Close the cell chamber by completely

tightening the cap.

4. Add 450 mL of additional media into the media chamber and

then completely tighten the cap (Fig. 2g).

5. If bubbles are present in the cell chamber, tilt the reactor so

that the bubbles move toward the black silicone cone and

remove the bubbles using a 25 mL serological pipette.

6. If droplets of media are spilled near the black silicone interface,

use an ethanol-soaked wipe to clean the area.

7. Gently place the bioreactor into a standard 5% CO2 and 37 ^C

incubator (Fig. 2h).

3.3

Adapting Cells to

Long-Term Bioreactor

Media (See Note 6)

1. Every 3–4 days, collect 15 mL conditioned media (Fig. 2c)

using a 25 mL serological pipette inserted into the black sili-

cone cone, gently wash cell chamber 2–3
with 15 mL pre-

warmed sterile PBS (Fig. 2e), and refill with 15 mL prewarmed

media based on adaptation schedule (Fig. 2f). Every 7 days

discard the 500 mL media in the media chamber (Fig. 2b) and

refill with prewarmed media (Fig. 2g). Continual use of 1% PS

in both chambers is optional but encouraged.

Fig. 2 Step-by-step process of continually harvesting EVs from the CELLine AD 1000 bioreactor. (a) Loosening

the cell and media chamber caps, (b) removing media from the media chamber, (c) aspirating conditioned

media from the cell chamber, (d) transferring conditioned media to a falcon tube for centrifugation, (e)

washing the cell chamber with PBS, (f) adding fresh media to the cell chamber, (g) adding fresh media to the

media chamber, and (h) returning bioreactor to incubator

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