Rotea Bed Establishment

One of the challenges encountered with small cells at low concentrations is persuading them to form an initial fluidised bed.

Rather than accumulating within the chamber, the cells seem to remain suspended and wash directly through the chamber.

Normal practice is to set up a recirculation path so any cells not captured are returned to the input bag and not lost. Nevertheless, the bed can struggle to form, and sometimes, magically, a bed suddenly appears.

Once a stable bed is established, capture efficiency often changes dramatically. This suggests that bed formation itself is a critical process objective.

Difficulties in bed establishment

During evaluation of a dilute stream of small cells suspended in relatively dense media, cell losses to the chamber outlet were monitored directly. In the absence of a stable bed, cells were continuously lost. When a bed formed spontaneously, losses ceased immediately.

The challenge was to establish that bed.

A number of strategies were tested, including disrupting the flow through the chamber to allow the cells to settle. This was encouraging but not robust; there were not always sufficient cells in the chamber.

Part of the testing included injecting some previously concentrated cells into the flow stream. A bed formed reliably when seeded this way.

The strong motivation to continue was the excellent functionality once the bed was formed.

The hidden bed

On another occasion, Rotea was challenged with a very small number of precious T-cells. There was an alarming lack of visible bed. Despite the apparent absence of a visible bed, the process achieved an 85% recovery. This phenomenon is referred to here as the “hidden bed”—a dispersed population that is not visually obvious but is nevertheless retained successfully.

The cell simulation in the Rotea Protocol Review tool gives some insight into this behaviour, highlighting that light cells may be retained in the upper regions of the chamber. If there are not many of these cells, no bed will be visible yet they are present.

Coriolis flow

Of concern in these circumstances is the swirl that is evident in the chamber when no bed is visible. Cells can be seen to swirl up into the chamber and back around towards the tip. Under these conditions, cells caught in that circulating flow can be lost if they are in proximity to the chamber outlet. This secondary circulation is consistent with the influence of Coriolis forces acting on the upward bulk flow.

As the bed becomes established, dramatically fewer cells are visibly entrained within the circulating stream. This may indicate that the circulating region becomes smaller, or that the circulation itself is reduced.

Whether the growing bed suppresses this circulation directly, or simply confines it to the upper chamber, remains an interesting question.

Video

Chamber flow before bed

The key observation here is the change in the swirling flow within the chamber as the bed begins to accumulate. The growing bed appears to establish a different flow regime for particles entering the chamber, coinciding with a marked improvement in cell retention.

Simulation of the chamber process

The Rotea Protocol Review tool provides some insights into these processes.

A single cell type in a cell population is defined as a user-defined histogram of cell sizes. In this case, a normal distribution across the diameter range. Other cell types may overlap this definition, and some heavier or lighter elements of a cell type might be separated by the process.

The simulator predicts both where different cell sizes are retained within the chamber and the local retention capacity responsible for that behaviour.

In this example the simulated cells are intentionally defined as both small and light to represent a particularly challenging population. Under these process conditions the cells cannot settle until they are well up into the chamber. Larger cells accumulate lower in the chamber while the smallest cells are retained progressively higher, producing a widely dispersed, very low concentration bed.

Although almost invisible to the operator, this dispersed population can still be retained successfully, providing an explanation for the “hidden bed” observed experimentally.

The ‘Anqi Li’ method

Anqi Li (Ref. 1) encountered similar challenges while processing placenta-derived human amnion epithelial cells (hAECs). She developed a robust loading strategy using a recirculation flow path:

• Run the pump quickly to ensure some 40 million cells are in the chamber.
  • [recirc 2000G x 40ml/min x 100ml volume trigger]
• Reduce the pump speed to allow the cells to settle and a bed to form.
  • [loading 2000G x 30ml/min to bubble sensor trigger]

The objective is not simply to change the flow rate, but to ensure that sufficient cells are present within the chamber for a stable bed to become established.

Once this initial bed has formed, cells are no longer readily washed from the chamber. Even cell populations with marginal bed-forming behaviour can often be retained successfully because the established bed provides a more stable operating condition for subsequent cell capture.

The common objective is therefore bed establishment—creating a sufficiently large initial bed from which efficient concentration can continue.

Preserving an established bed

When processing large culture volumes, the chamber may need to be recovered several times before the complete suspension has been processed. For cell populations with marginal bed-forming behaviour, completely emptying the chamber can mean repeating the difficult bed establishment process after every recovery.

A useful strategy is to perform a partial recovery before returning the process to the loading state, or to a short recirculation stabilisation step, allowing the disturbed bed to re-establish before processing continues.

Although each recovery briefly disrupts the bed, preserving a significant proportion of the established bed allows the process to continue with minimal interruption while maintaining the favourable bed conditions that have already been created.

Bed Builder

If establishing the initial bed is the challenge, an obvious question follows: can the bed simply be created deliberately?

For example, density- and size-controlled beads with non-adherent surface coatings can be used. They need to be pre-sterilised in a bag for kit attachment. Begin the process after priming by loading the beads to form a bed. The target cell stream can then be loaded and accumulate reliably in the presence of the bed. The challenge is how to separate the bed-forming beads from the target population.

Elutriation

The target cells can be elutriated from the beads. If the wash prior to elutriation is into a low-density medium, the cells are more inclined to form a bed without the bead bed. Elutriation commonly requires around 100–150 ml of fluid volume to ensure completion. Selecting heavier beads and higher flow rates may reduce that elutriation volume. The beads are then recovered to a bag before the target cells are re-loaded for concentration. This may be a good approach for capturing low-concentration flow streams such as perfusion culture or flow-based cell selection procedures.

Post-culture separation

In some cases, all cells and beads can be captured together and transferred to expansion culture, where the increased cell count (and perhaps cell size) facilitates a robust selection operation at the conclusion of culture.

Conclusion

Creating and maintaining a stable bed is the key to processing low-concentration cell populations with marginal bed-forming behaviour. Some successful strategies are described here, all very dependent on the cell type, process goals, and surrounding constraints.

References

1. LI, ANQI (2022). Developing Automated Solutions for Cell Manufacturing Using Counterflow Centrifugation Technology. Monash University. Thesis.