9
8
5. Disconnect the seeding bottle and replace with the new storage bottle
containing differentiation medium.
◗
–
Disconnect the seeding bottle MPCs and connect the new storage bottle
MPCs to the inlet side tubing.
7. Priming
1. Ensure that the tubing is placed in both pumps and the pumps are clamped.
2. Turn the Corning® MicroDEN®
unit “ON,” and select “Prime” using the
function button.
3. The pumps will perfuse differentiation medium from the inlet bottle into the
flasks.
4. Stop perfusion by pressing the Corning® MicroDEN®
function button when
the flasks are filled with liquid.
Helpful Priming Tips
When beginning to perfuse liquid, or when the tubing is filled with air, angle the
left side inlet MPCs upwards so that air is completely removed from the MPCs
(Figure 4). If this is not performed, air will be trapped in the MPCs and can cause
an air bubble to perfuse into the flask.
8. Expansion
1. Turn the Corning MicroDEN
unit “OFF” and disconnect the power cord.
2. Transfer the MicroDEN
unit and flasks that were set-up from the biosafety
cabinet into the incubator.
3. Reconnect the power cord.
◗
–
The power cord can either be placed through the incubator port or passed
through the door gasket in the front. The gasket will seal around the cord to
minimize loss of incubator environmental conditions.
4. Turn the MicroDEN
unit “ON” and select “Culture” using the function button
on the unit.
◗
–
A timer will display elapsed time when culture begins, and the pumps will
perfuse differentiation medium at 8.0 µL/min. into each flask.
Medium Exchange (Day 3)
Each 3-day period requires 80 mL of differentiation medium to maintain
perfusion. Due to degradation of medium at incubator conditions, it is
recommended to refresh differentiation medium every 3 days.
NOTE:
The outlet bottle does not need to be emptied on Day 3.
1. Turn the Corning MicroDEN
unit “OFF”, disconnect the power cord, and
remove from the incubator.
2. Lightly spray the MicroDEN
system with 70% ethanol and place inside the
biosafety cabinet.
◗
–
Do not spray ethanol directly into the filters as wetting the filters will cause
them to restrict gas exchange, and the bottles can pressurize as medium is
perfused.
◗
◗
When beginning to perfuse liquid, or when the tubing is filled with air, angle
the left side inlet MPCs upwards so that air is completely removed from the
MPCs (Figure 4). If this is not performed, air will be trapped in the MPCs and
can cause an air bubble to perfuse into the flask.
Figure 4.
MPCs angled upward to remove air.
◗
◗
After seeding, a small air gap will remain at the top of the flask. The air gap is
intentional to ensure that cell solution does not perfuse into the outlet tubing.
◗
◗
If cell solution is perfused into the outlet tubing, reverse the tubing
orientation through the pump so that the colored tubing marker is on the
right side of the pump head. Select the “Seed” function to perfuse the cell
solution back into the flask. Be sure to switch the tubing orientation back to
the original position (colored tubing markers on the left of each pump) before
continuing. The pump rotates clockwise and liquid will flow from left to right
when set-up properly. Tubing from the inlet bottle should enter the left side of
the pump to perfuse into the flasks.
6. Differentiation Medium Loading
1. Place a new storage bottle in the biosafety cabinet.
2. Open the bottle.
◗
–
To unscrew the cap, turn the bottle while keeping the cap and tubing set
in place so the tubes don’t twist. (Figure 1). Take care not to touch the dip
tubes to any non-sterile surface.
3. Add 80.0 mL differentiation medium to the new bottle.
4. Insert tubing set and cap back onto the bottle. While pressing down on
the tubing set, rotate the orange cap to tighten the assembly to the bottle
(Figure 1). Place the bottle back into the thermoform tray.
