3.5

Media

Preparation/Hold

1. Basal culture media should be aseptically transferred into the

bioreactor. Shear protectant can be added to reduce stress to

the cells during the culture. When determining the amount of

basal media to be used, it is important to consider the maxi-

mum working volume of the bioreactor (usually around 2/3 of

the vessel volume) as well as the volume added by the inoculum

and by addition of feeds (up to ~40% of the final volume) and

acid/base (if applicable). Depending on the percentage of

volume removed for sampling during the process, it might be

required to adjust the feeding volume accordingly (see Note

24).

2. Before the inoculation, it is recommended to equilibrate media

inside the bioreactor. Temperature, as well as DO and pH

should reach their equilibrium or setpoint [23] (see Note 20).

3. DO probes should be calibrated with two points using air

saturated media as the upper point and “oxygen free media”

(either by removal of oxygen using nitrogen sparging or mim-

icking this by disconnection of the probe) as 0% point (see Note

21).

4. pH probes should be re-calibrated using an offline measure-

ment as autoclavation can have an impact on the calibration.

5. Feeds, as well as other supplements, need to be aseptically

connected to the bioreactor. This can also be delayed until

the actual addition of the feed (s) is required (see Note 3).

3.6

Inoculation

1. At the time of inoculation, pH, pCO2, and metabolite concen-

trations should be carefully checked and potentially regulated

to ensure a correct environment allowing cells to grow. Typical

physical parameters listed in Table 1 should be observed at this

particular step of a fed-batch process.

2. After counting cells from the expansion, the required amount

of inoculum to reach the target cell concentration is added to

the bioreactor. Adjusting the concentration of the cells to a

pre-determined value so that the same volume can be trans-

ferred for each inoculation simplifies this process and increases

reproducibility (see Notes 22 and 23).

3. Post-inoculation sampling is performed when the culture is

homogenized (after around 30 min). Homogeneity of the

bioreactor is important to ensure a representative sample is

obtained and to determine initial condition of the culture. If

the target seeding density is not reached, cells or media

(depending if the VCC is too high or too low) could be

added. In this case, the increase of the initial bioreactor volume

should be taken into account and the feed volume should be

adapted.

Benchtop Bioreactors in Mammalian Cell Culture: Overview and Guidelines

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