Wash To Lower Density Media; Defensive Washing Strategies - Thermo Scientific gibco CTS Rotea User Manual

Wash to lower density media

Transferring to a lower density media such as PBS can disrupt the fluidized bed due to the dramatically
increased buoyancy effects caused by the spinning CFC Chamber. When the lower density media
enters the tip of the CFC Chamber, cells can be entrained by the rapidly rising lighter media and
washed out of the chamber. This can often be clearly seen in the live video and can be quantified by
sampling the media exiting the chamber through line A or G. Sometimes simply increasing the G force
and/or reducing the flow rate will be enough to address this issue.
Note: Residual low-density wash media in the Pause loop and/or Bubble Trap can potentially disrupt
the fluidized bed as it enters the CFC Chamber. Allow time for the bed to re-settle when moving to a
Pause step.

Defensive washing strategies

The following defensive washing strategies can be applied when the properties of the new media are
different to the original media. They are also particularly helpful during process development when the
properties of the media and cells are not well defined.
Recover as concentrate to intermediate bag and dilute to new media
CTS ™ Rotea ™ Counterflow Centrifugation System Process Design User Guide
Image
Chapter 4 Protocol Building Blocks
Wash cells and buffer exchange
Details
After loading the cells into the CFC Chamber,
recover the cells as concentrate to an intermediate
bag connected to line E or F. Transfer additional new
media to the intermediate bag, re-priming the line
and diluting the concentrate such that its density is
similar to the new media.
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