11
10
3. Place a new storage bottle in the biosafety cabinet and unbag.
4. Disconnect the filled waste bottle on the right side of the unit and replace
with the new storage bottle for cell collection (harvest).
5. Connect the power cord, turn the MicroDEN
unit “ON”, and select “Harvest”
using the function button.
6. Differentiation medium remaining in the left side inlet bottle will perfuse
into the flasks.
7. As medium is perfusing, carefully remove the flasks from the thermoform tray
and rotate so that the outlet port is down and the inlet port is up (Figure 5).
NOTE:
It may be necessary to remove the tubing from the right side tubing
holder.
8. Allow perfusion to continue when the inlet bottle is empty of differentiation
medium. Air will perfuse into the flasks and medium will drain into the
harvest bottle.
9. Remove the flasks from the thermoform tray and slide the flasks side-to-side
as medium drains from the flasks into the harvest bottle (Figure 6).
◗
–
The sliding motion causes turbulence that allows the cultured cells to
detach and suspend into solution.
Corner notch
on flask in
lower left
position
3. Place a new storage bottle in the biosafety cabinet and unbag.
4. 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.
5. Add 80.0 mL differentiation medium to the new bottle.
6. Insert tubing set and place the 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.
7. Disconnect the empty bottle and replace with the new storage bottle
containing differentiation medium.
◗
–
Disconnect the empty bottle MPCs and connect the new storage bottle
MPCs to the inlet side tubing.
8. Transfer the Corning® MicroDEN®
system from the biosafety cabinet into the
incubator.
9. Connect the power cord, turn the unit “ON”, and select “Culture” using the
function button on the unit.
◗
–
A timer will display elapsed time when culture begins; the pumps will then
perfuse differentiation medium at 8.0 µL/min. into each flask.
Imaging
The flasks can be imaged using most standard microscopes. The flasks do not
need to be removed from the consumable assembly for imaging. If the entire
consumable assembly does not fit in the microscope for imaging, the flasks can
be removed from the thermoform tray. Take care not to accidentally disconnect
the tubing outside a sterile environment. Alternatively, the flask can be dis-
connected from the tubing for imaging. To maintain sterility, sterile filters or
Luer Lock caps should be placed on the inlet and outlet ports of the flasks.
The tubing can also be capped using sterile filters or Luer Lock caps. Be sure to
employ proper aseptic techniques.
9. Harvest (Day 6)
The Corning MicroDEN
unit has a “Harvest” function that perfuses cold buffer
from the inlet bottle to remove cells from the flask. A new storage bottle can
be used for both buffer (inlet) and cell collection (outlet) by disconnecting the
MPCs and replacing with a new bottle.
1. Turn the 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 the
buffer is perfused.
Outlet ports
are down
Inlet ports
are up
Figure 5.
Set up for harvest (Day 6).
Remove tubing
from the tubing
holder
