Lentivirus Clarification; Separate Cells From Microcarriers - Thermo Scientific gibco CTS Rotea User Manual

• RBC's are typically smaller than PBMC's, but they are denser and non-spherical. In the Rotea
instrument RBC's behave in a similar way to lymphocytes and small monocytes when processed in
culture media or wash buffer. This means that using elutriation alone, the majority of RBC's will be
in the lymphocyte fraction.
Note: Lysing the RBC's enables them to be elutriated from the PBMC's. The density of the buffer
can also be manipulated, e.g. Ficoll to deplete RBC's from PBMC based on the density differential
• Granulocytes typically end up with the monocyte fraction due to their size and density

Lentivirus clarification

Lentivirus is very small relative to the HEK producer cells so can be elutriated leaving the concentrated
HEK cells in the CFC Chamber. In this instance, the product of interest is being elutriated from the input
material leaving the concentrated waste behind.

Separate cells from microcarriers

Microcarriers are very large e.g. 200 μm relative to the adherent cells they are producing. Once
separated from the microcarriers, the cells can be elutriated from the bed leaving the concentrated
microcarriers behind.
• The microcarriers will settle very rapidly in the CFC Chamber. To avoid pelletizing them in the tip, it
will be necessary to process at very low G and high flow rates.
• Despite the significant size difference between microcarriers and cells, it is difficult to guarantee
that there won't be any microcarriers in the output due to the potential catch points and dead
zones within the fluid path. Avoiding the Bubble Trap during processing can reduce the number of
trap points for microcarriers but requires careful attention to priming.
• The large diameter, quantity of microcarriers and batch volume typically being processed means
that the CFC Chamber is likely to require emptying on a frequent basis to complete a batch
• Since separation of adherent cells requires the addition of enzymes, the Rotea
also be used to wash away the enzyme
CTS ™ Rotea ™ Counterflow Centrifugation System Process Design User Guide
Chapter 4 Protocol Building Blocks
Elutriation
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