15. Appropriate recording of usage of both DO and pH probes is

highly recommended as each sterilization could impact their

performances.

16. During the first check of the pH, the probe signal value could

be off, this is because the probe is in air and not in media.

17. In case of magnetically driven systems, be careful with regard to

the speed and the strength of the magnet as decoupling can

occur.

18. The topic of gas transfer in bioreactors has been extensively

studied and is highly complex, in particular when it comes to

adjustment between scales. The values provided in Table 1 can

only serve as a starting point and will certainly need adaption

for a particular cell line and equipment.

19. DO setpoints can be reached by letting cells consume oxygen

present in the media or by sparging nitrogen in the media. A

constant air flow through the sparger usually helps the control

of DO percentage at the beginning of the process.

20. If extended storage of media before addition is required, pay

attention to the significant light sensitivity of many cell culture

media and limit exposure to temperatures above 2–8
C as

much as possible. Depending on the volume of media and

mixing time of the system, equilibration time could last from

1 h to multiple hours. An equilibration period of more than

1 day could also help to detect a potential contamination

before the inoculation.

21. When using CO2 (typically 5–8%) during the equilibration

step, this needs to be taken into account for the calibration of

the DO probe, resulting in an initial calibration value of

92–95%.

22. Careful manipulation of the inoculum is critical as cells are

quite sensitive to shear stress. Also, during the transfer cells

are not well oxygenated and the temperature is difficult to

maintain. This step should be performed as quickly as possible.

If the transfer is performed using a peristaltic pump, the liquid

flow must be low in order not to damage cells.

23. The initial seeding cell density as well as split ratio have a

substantial impact on the performance of the culture and

have received increased attention especially in the context of

process intensification [26]. This topic is furthermore compli-

cated by the fact that equipment availability will have a strong

impact on potential initial cell densities and state of the inocu-

lum (amount of fresh media, DO), especially at larger scales. As

with many setpoints, performing a round of experiments in a

less resource intensive format like shake flask to assess general

trends is highly advisable.

Benchtop Bioreactors in Mammalian Cell Culture: Overview and Guidelines

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