2. After 7 days postinoculation, reduce the percentage of FBS in

both chambers to 5% and use 50% DMEM/50% Advanced

DMEM with 1% CDM-HD or other appropriate media.

3. After 14 days post-inoculation, reduce the percentage of FBS in

both chambers to 1% and use 25% DMEM/75% Advanced

DMEM with 2% CDM-HD or other appropriate media.

4. After 21 days post-inoculation, use 100% Advanced DMEM

and 2% CDM-HD in both chambers, 1% FBS in the media

chamber, and exclude FBS from the cell chamber.

3.4

Continually

Harvesting EVs and

Monitoring Shed Cells

from the Bioreactor

(Fig. 2)

1. The CELLine AD 1000 bioreactor is opaque and must be

monitored using the conditioned media that is collected (vol-

ume and cell count).

2. Start collecting EVs after media adaptation has finished and the

cell chamber is serum-free.

3. Collection of conditioned media and replacement (cell cham-

ber) is done twice a week (can be done once a week or less

depending on cell type) and media replacement in the media

chamber is done once a week.

4. Loosen the cap of the cell chamber to prevent an air lock

(Fig. 2a). Transfer all media from the media chamber to a

waste container (Fig. 2b).

5. Aspirate 15 mL from the cell chamber using a 25 mL serologi-

cal pipette inserted into the black silicone cone (Fig. 2c–d).

Make a note of the volume (see Note 7).

6. Count the cell concentration and viability in the collected

conditioned media using Trypan Blue exclusion method and

a hemocytometer (see Note 8) or other suitable method

(Fig. 3a).

7. Immediately centrifuge the conditioned media at 2000
g to

remove cells and debris, then transfer supernatant to an ultra-

centrifuge tube. A 200
g spin prior to the 2000
g is

optional.

8. Gently wash the cell chamber 2–3
with 15 mL prewarmed

sterile PBS using a 25 mL serological pipette inserted into the

black silicone cone (Fig. 2e).

9. Add 15 mL of prewarmed media to the cell chamber using a

25 mL serological pipette inserted into the black silicone cone

(Fig. 2f). Add 500 mL of prewarmed media to the media

chamber (Fig. 2g) and return the bioreactor to a 5% CO2 and

37 ^C incubator (Fig. 2h).

10. Check the conditioned media for microbial contamination

microscopically if excessive cloudiness or discoloration is pres-

ent (see Note 9). Some fat producing cells may produce visible

amounts of lipids that cause cloudiness due to the high cell

density.

Production of Extracellular Vesicles Using a CELLine Adherent Bioreactor Flask

187