ends of the input tube and the two tubes that circulate medium
to the duet pump must always be set lower than the waste tube.
8. Connect tubing with a wider internal diameter (e.g. 0.125 in
ID) than the input L/S-14 tubing (0.063 in ID), to the 3-stop
pump tubing that will be placed in the peristaltic pump channel
controlling waste output. The waste line must be long enough
to reach from the CR in the incubator to the peristaltic pump
and to allow the cassette to be moved to the BSC for sampling/
maintenance.
9. Attach waste line tubing to the outflow end of the 3-stop pump
tubing to reach the cap of the 4 L waste bottle. Place in a spill
tray on a lower shelf or on the floor.
10. Assemble the autoclave-proof components of the primary flow
path to make a complete HF system (see Note 5). Cover
connection ports for the HF cartridge cassette and any open
ports on the 3-way stopcock (e.g., those to be used for microclave attachment) with aluminum foil.
11. Sterilize the system using a dry autoclave cycle.
12. Inside a BSC, spray the sterilized, cooled components liberally
with 70% ethanol then air dry for a few minutes before exposing the junctions to make connections. Use careful aseptic
handling technique.
13. Fill the CR with sufficient medium to establish both the desired
working volume inside the CR and charge the HF cartridge
and tubing.
14. Link the HF cartridge cassette to the CR via the two Luer ports
on the CR headpiece and fill the cartridge by manually squeezing the duet pump tubing until no more bubbles are expelled
into the CR.
15. Fill the ECS in the HF cartridge with medium using two Luerlock syringes attached to the external ports on the cartridge
housing (see Note 6).
16. Place the HF cartridge(s) and duet pumps in a large upright
incubator with glass door or window (Fig. 2). Position the CR
on top of a stir plate next to the duet pump. Stabilize the CR by
placing a lead collar around the container.
17. Place peristaltic pumps and media/waste reservoirs on secure
shelving positioned between the incubator and the BSC
(Fig. 2) (see Note 7).
3.2 Priming
the System
1. Turn on the duet pump and insert the HF cartridge cassette so
that the short section of pump tubing on the cassette slots into
the piston channel.
720
A. Brett Mason and Ve ´ ronique Dartois
to the duet pump must always be set lower than the waste tube.
8. Connect tubing with a wider internal diameter (e.g. 0.125 in
ID) than the input L/S-14 tubing (0.063 in ID), to the 3-stop
pump tubing that will be placed in the peristaltic pump channel
controlling waste output. The waste line must be long enough
to reach from the CR in the incubator to the peristaltic pump
and to allow the cassette to be moved to the BSC for sampling/
maintenance.
9. Attach waste line tubing to the outflow end of the 3-stop pump
tubing to reach the cap of the 4 L waste bottle. Place in a spill
tray on a lower shelf or on the floor.
10. Assemble the autoclave-proof components of the primary flow
path to make a complete HF system (see Note 5). Cover
connection ports for the HF cartridge cassette and any open
ports on the 3-way stopcock (e.g., those to be used for microclave attachment) with aluminum foil.
11. Sterilize the system using a dry autoclave cycle.
12. Inside a BSC, spray the sterilized, cooled components liberally
with 70% ethanol then air dry for a few minutes before exposing the junctions to make connections. Use careful aseptic
handling technique.
13. Fill the CR with sufficient medium to establish both the desired
working volume inside the CR and charge the HF cartridge
and tubing.
14. Link the HF cartridge cassette to the CR via the two Luer ports
on the CR headpiece and fill the cartridge by manually squeezing the duet pump tubing until no more bubbles are expelled
into the CR.
15. Fill the ECS in the HF cartridge with medium using two Luerlock syringes attached to the external ports on the cartridge
housing (see Note 6).
16. Place the HF cartridge(s) and duet pumps in a large upright
incubator with glass door or window (Fig. 2). Position the CR
on top of a stir plate next to the duet pump. Stabilize the CR by
placing a lead collar around the container.
17. Place peristaltic pumps and media/waste reservoirs on secure
shelving positioned between the incubator and the BSC
(Fig. 2) (see Note 7).
3.2 Priming
the System
1. Turn on the duet pump and insert the HF cartridge cassette so
that the short section of pump tubing on the cassette slots into
the piston channel.
720
A. Brett Mason and Ve ´ ronique Dartois