CORNING MicroDEN Manuel d'instruction - Page 7

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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

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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.

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

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detach and suspend into solution.

Corner notch

on flask in

lower left

position

Figure 5. Set up for harvest (Day 6).

Inlet ports

are up

Outlet ports

are down

Remove tubing

from the tubing

holder