Cell culture FAQ

Yes, the iCELLis™ nano bioreactor is a closed system and can be run outside a sterile environment.

There are three ways to estimate the cell density in the iCELLis™ 500 bioreactor (a Pall™ Life Sciences product).

Biomass probe: cell density may be correlated to the normalized capacitance measured by the biomass probe, provided that this correlation has been established experimentally during process development. Capacitance measurement is dependent not only on cell density but also on cell diameter, media composition, and other process parameters. Therefore, we recommend establishing the correlation specifically per process and per process step.

Metabolite concentration: the cell density may also be correlated to the concentration of certain metabolites (for example, glucose), provided that this correlation has been established experimentally during process development.

Oxygen consumption: for high cell densities, cell density value may also be correlated to the oxygen consumption, provided that this correlation has been established experimentally during process development. The oxygen consumption of a cell culture can be estimated by calculating the difference of dissolved oxygen (DO) measured by the probe in the media before it passes through the fixed-bed versus the probe in the media after it passes through the fixed-bed.

The most crucial factor to consider for an even distribution of cells is the quality of the biological material, that is, a good single-cell suspension inoculum. Clusters of cells present on the inoculum will create patches of cells in certain areas of the fixed bed, causing an uneven cell distribution. Secondly, the process parameters during inoculation will impact the distribution. Our applications team can work with users to provide recommended settings for each process. Contact us.

As noted in the operating manual USD 3277b, the compressed gas provided should be a clean, dry, analytical grade source of each compressed gas (O₂, CO₂, air, and optionally N₂) regulated between 0.2 MPa (2 bar, 29 psi) and 0.6 MPa (6 bar, 87 psi). Gases from local compressors or generators must be filtered and dried. This is required to prevent failure in the mass flow controllers and hardware.

Yes, the iCELLis™ nano has been used in GMP production by some of our customers. The software delivered with the iCELLis™ nano bioreactor system (a Pall™ Life Sciences product) is compatible with 21 CFR Part 11 requirements. Following the guidance of our Instructions for use, USD328 (mPathLink), the system can be implemented in a GMP environment.

As stated in the iCELLis™ 500+ GenQ bioreactors validation guide USTR 3393, in case of a one-point calibration in the range from 6.5 to 8.5, an accuracy of ± 0.1 is guaranteed for the pH sensor after gamma irradiation and with product calibration. For the DO sensor, an accuracy of ± 3.5% between 15% and 75% and of ± 7% between 75% and 150% after one-point calibration is provided after gamma irradiation and with product calibration.

Seeding densities as low as 500 cells/cm² may be used in iCELLis™ bioreactors (a Pall™ Life Sciences product). However, the use of extremely low seeding densities may result in a prolonged lag phase.

As a first approximation, it is accepted that:

• Seeding densities ranging from 15 000 to 30 000 cells/cm² can be used with no impact on lag phase.
• Seeding density can be reduced to 5000 cells/cm² without noticeable impact on the lag phase for most (but not all) processes.
• Reducing the seeding density below 3000 cells/cm² is likely to impact the lag phase.

We recommend that end users evaluate the impact of cell seeding density on cell growth kinetics and process productivity in the iCELLis™ nano bioreactor for their specific process.

The iCELLis™ technology allows for setting-up perfusion processes. The significance of the advantage is of course process dependent. Perfusion allows for better media-management during growth phase (that is, lower consumption or stable metabolite levels), as well as continuous harvest during production phase when the product is extracellular. This may increase titers when the product is unstable at cell culture temperatures or sensitive to impeller caused shear.

Cell densities are highly process dependent. Some cell lines (for example, HEK293 and Vero) may reach up to 800 000 cells/cm² in iCELLis™ bioreactors (a Pall™ Life Sciences product). Wang et al. reached 1 180 000 cells/cm² after 11 d (days) of growing 294Vec (1).

However, for most bioprocesses, extremely high cell densities negatively impact productivity. Furthermore, high cell densities are always achieved at the expense of a high media consumption, which may negatively impact process economics.

We recommend that users evaluate the actual impact of cell density on productivity as part of their individual process optimization. Contact us to request support.

The iCELLis™ nano bioreactor (a Pall™ Life Sciences product) features inline continuous monitoring of cell culture temperature, pH, dissolved oxygen (DO), and cell capacitance for estimating cell density. Aseptic sampling of supernatant through a needless swab-able valve is featured for offline monitoring of metabolites, pH verification, and titer follow-up. Sampling of carriers (within reason without limitation) from the top of the fixed bed is featured for offline counting of nuclei to follow-up on cell density.

The iCELLis™ technology was not designed for the production and recovery of viable cells, but rather of drug substances produced by the cells.

Depending on process circumstances (including factors such as cell type and media composition), it may be possible to recover a small portion of live cells, for example, for cell characterization. For more information on how to perform such trials contact us to request support.