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Chapter 4 Protocol Building Blocks
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Multi-bite processing
Multi-bite processing
Overview
The CFC Chamber in the Single-Use Kit has a working volume of 10 mL which is often sufficient to
collect and concentrate all of the cells in a batch.
The size of the CFC Chamber enables Rotea
• Operate at high G's (this results in a relatively high throughput rate and the ability to concentrate
small cells)
• Process as few as 50 × 10
Where the number of cells in a batch exceeds the capacity of the CFC Chamber, the Rotea
can be programmed to automatically load and empty the CFC Chamber multiple times ("bites") using
Loops.
Determine the number of cells per bite
Cell loading
While 5 billion T-cells have been successfully loaded and recovered in a single CFC Chamber load, this
can only be achieved by loading very slowly and at high G which isn't always practical. Using more
"typical" loading conditions for 10 to 20 μm cells, the CFC Chamber can hold 2 to 3 billion cells before
overwhelming the chamber.
This is a good starting point for process development but ultimately it is dependent on what you are
processing and the settings that you choose to run at.
Note: All cells being loaded into the CFC Chamber need to be included in the cell number estimate,
for example, if you are processing PBMC or leukopak, then you need to include the number of RBC's.
In the case of buffy coat, the number of RBC's typically far exceeds the number of PBMC's making
processing of buffy coat difficult.
Elutriation
Elutriating cells from the fluidized bed requires relatively high pump speeds and low G force that expand
the bed. If there are too many cells in the bed prior to the elutriation step, the bed can expand beyond
the 10 mL working volume and cells you want to retain will be washed out. Process development runs
are recommended to find the right balance between elutriation speed and the size of bed that can be
sustained at that speed.
Note: One common strategy is to slowly increase the elutriation flow rate as the volume of the fluidized
bed reduces through elutriation of cells.
The other side to elutriation is when there are too few cells in the bed for the elutriation settings.
Elutriation settings are bed size dependent. If, when using a multi-bite process, the final bite is a small
number of cells, then the subsequent elutriation step may not perform as expected.
Best practice for multi-bite processing is to set a bite volume so the last bite before the bubble sensor
trigger will be 70% to 90% of the normal cells per bite. This can be manipulated by adjusting the
volume per bite or the volume of a dilution step.
92
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instrument to:
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cells which is ideal for autologous samples and process development
CTS
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Rotea
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Counterflow Centrifugation System Process Design User Guide
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instrument
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