Thermo Scientific gibco CTS Rotea User Manual page 50

Chapter 4 Protocol Building Blocks
4
Priming
Prime Chamber and Line A
Start centrifuge rotation slow (10 G), continuing to pump the liquid through the CFC Chamber.
Centrifuge spinning will drive any air to the inside of the chamber, allowing it to be pushed out by
priming fluid entering the chamber. A volume trigger of 15 mL is usually enough.
Add Priming Volume
Calculate the amount of volume needed in bag A to complete priming and pump slightly more than
that volume from B to A. The same fluid path, pump speed, and centrifuge speed as the previous
step is maintained. To calculate this volume simply add together the priming volume required in the
subsequent priming steps.
Reverse flow and fill Bubble Trap
Reverse the pump to fill the Bubble Trap and prime the input line from bag B with priming fluid, typically
10–15 mL.
Prime remaining input lines
Any other lines through which product is drawn into the system must be primed, e.g. lines C, D, E and
F. Smaller volumes can usually be used now that the Bubble Trap and CFC Chamber have been filled.
Each line requires its own step. A to D is shown here as an example.
Note: It is particularly important to prime lines E and F when being used as inputs as they are not
protected by the Bubble Trap.
Pressure Prime
If lines E or F are not connected to a bag, they should be primed using a Pressure Prime step
type. This improves the performance and reproducibility of the Harvest step. In this example, Bag B is
selected as the origin bag and the Pressure Prime step type automatically primes lines not being used
within the Single-Use Kit. The centrifuge is ideally set at the same G as the prior step with all other
settings being default settings.
Prime Pause Loop
The recirculation loop is primed using a brief Pause step type running in the forward direction. Set a
small volume trigger or short timer trigger on a Pause step type with the centrifuge set to 10 G and
a low pump flow rate e.g. 25 mL/min. This will help to ensure that bubbles in the J–K line don't skip
across the bottom of the Bubble Trap and remain trapped in the recirculation loop.
50
CTS ™ Rotea ™ Counterflow Centrifugation System Process Design User Guide