3.9 Media
Replenishment
1. Turn off the peristaltic pump and close the stopcock valve
above the CR.
2. Move the media tank to the BSC. Wipe the cap assembly, top of
the tank, and the bottle of fresh medium with 70% alcohol.
Allow to air dry.
3. Open the tank and pour in fresh medium. Replace the cap, wipe
the tank and tubing with a disinfectant wipe, and then move
the tank back to the shelf.
4. Open the stopcock valve and restart the peristaltic pump.
3.10 Culture
Monitoring
1. Every 24–48 h move the cartridge to the BSC as described in
ECS sampling (Subheading 3.8) and mix the ECS culture back
and forth between fresh syringes attached to the microclaved
ECS ports (see Note 17).
3.11 Waste Disposal
1. Waste medium that has passed through the system is collected
in a sterile 4 L plastic bottle. When the bottle needs to be
replaced ( 2/3 full), turn off the peristaltic pump and use a
tubing clamp on the waste line to isolate the waste bottle.
2. Move the waste bottle to the BSC and wipe the cap and top
with 70% alcohol. Replace the bottle with a fresh, sterile waste
bottle. Wipe both bottles and associated tubing with a disinfectant wipe, return the new waste container to the shelf/floor,
and release the tubing clamp. Restart the peristaltic pump.
3. Add bleach to the spent medium in the original waste bottle to
a final concentration of at least 10%. Wait for a minimum of
60 min. Discard.
3.12 Termination
of Experiment
and Disassembly
of HFS
1. Remove medium from the input tank but continue flow
through the system to empty the flow path of medium.
2. Close both terminal ports on the HFC and move the HFC
cassette to the BSC.
3. Inside the BSC, load a 10 mL syringe with bleach solution (use
enough bleach to give a final concentration of at least 10%) and
attach it to the microclave on one of the ECS ports. Attach an
empty 10 mL syringe to the other ECS port. Open the ECS
ports on the cartridge and push the bleach solution gently
through the cartridge into the empty syringe. Mix the contents
back and forth several times (see Subheading 3.7, step 8). Leave
the bleach in contact with the ECS and fibers for at least
an hour.
4. Remove the cassette from the system. Detach the tubing
connected to the duet pump from the top of the CR cap
assembly. Place the cartridge cassette in a biohazard bag and
autoclave.
726
A. Brett Mason and Ve ´ ronique Dartois
Replenishment
1. Turn off the peristaltic pump and close the stopcock valve
above the CR.
2. Move the media tank to the BSC. Wipe the cap assembly, top of
the tank, and the bottle of fresh medium with 70% alcohol.
Allow to air dry.
3. Open the tank and pour in fresh medium. Replace the cap, wipe
the tank and tubing with a disinfectant wipe, and then move
the tank back to the shelf.
4. Open the stopcock valve and restart the peristaltic pump.
3.10 Culture
Monitoring
1. Every 24–48 h move the cartridge to the BSC as described in
ECS sampling (Subheading 3.8) and mix the ECS culture back
and forth between fresh syringes attached to the microclaved
ECS ports (see Note 17).
3.11 Waste Disposal
1. Waste medium that has passed through the system is collected
in a sterile 4 L plastic bottle. When the bottle needs to be
replaced ( 2/3 full), turn off the peristaltic pump and use a
tubing clamp on the waste line to isolate the waste bottle.
2. Move the waste bottle to the BSC and wipe the cap and top
with 70% alcohol. Replace the bottle with a fresh, sterile waste
bottle. Wipe both bottles and associated tubing with a disinfectant wipe, return the new waste container to the shelf/floor,
and release the tubing clamp. Restart the peristaltic pump.
3. Add bleach to the spent medium in the original waste bottle to
a final concentration of at least 10%. Wait for a minimum of
60 min. Discard.
3.12 Termination
of Experiment
and Disassembly
of HFS
1. Remove medium from the input tank but continue flow
through the system to empty the flow path of medium.
2. Close both terminal ports on the HFC and move the HFC
cassette to the BSC.
3. Inside the BSC, load a 10 mL syringe with bleach solution (use
enough bleach to give a final concentration of at least 10%) and
attach it to the microclave on one of the ECS ports. Attach an
empty 10 mL syringe to the other ECS port. Open the ECS
ports on the cartridge and push the bleach solution gently
through the cartridge into the empty syringe. Mix the contents
back and forth several times (see Subheading 3.7, step 8). Leave
the bleach in contact with the ECS and fibers for at least
an hour.
4. Remove the cassette from the system. Detach the tubing
connected to the duet pump from the top of the CR cap
assembly. Place the cartridge cassette in a biohazard bag and
autoclave.
726
A. Brett Mason and Ve ´ ronique Dartois