successful thawing and good cell growth of the thawed cells needs to be demon-

strated for short and longer storage times. To avoid losses of cell banks, storage at

two locations should be considered.

5.4.3

PRECULTURE AND MAINTENANCE OF CELLS

To allow a continuous growth/maintenance, passaging of cells is necessary due to

depletion of nutrients, accumulation of toxic by-products, or limitation of the

growth surface (adherent cells). During cell passaging, old medium is withdrawn

and cells are seeded in fresh medium at a lower cell concentration. The passage

number describes the number of these subcultivations. Although the growth of

continuous cell lines is not limited per se, a regulatory limitation exists. For cells

that pose a potential tumorigenicity hazard, including Vero and MDCK cells, the

maximum allowed passage number for production is often limited to 20. In contrast,

new designer cell lines (e.g., PER.C6) grown in suspension are often very well

characterized and can be used for up to 100 passages [12]. The cell line, starting from

the working bank, undergoes several passages with increasing volume during seed

train expansion until inoculation at production scale. Furthermore, cells can be

maintained at a small volume, e.g., laboratory scale experiments or vaccine devel-

opment studies. Adherent cells are typically passaged once or twice a week, shortly

before they reach confluence. For passaging, the attached cells have to be detached by

proteases (e.g., trypsin). Use of trypsin (activity/concentration, incubation time, pH)

and inactivation of protease activity by addition of either serum or medium needs to

be optimized for the respective cell line, medium and the cultivation vessel (e.g., T-

flask, roller bottle, cell factory, microcarrier culture). A typical split ratio for adherent

cells is 1:4-1:10. For suspension cells, typical cell concentrations in batch mode reach

2E06-10E06 cells/mL with doubling times of 20−30 h. Suspension cells will be

subcultured every 3 and 4 or 2 and 3 days at the late end of the exponential growth

phase and are seeded usually at a cell concentration of 0.5-0.8E06 cells/mL, while a

small fraction of the cell suspension is filled up with fresh medium (split ratio: 1:4-

1:20). Preferably, no antibiotics are added to the cell culture medium to avoid

masking of contaminations. Some of these possible contaminations only become

evident when switching to active aeration and use of antibiotics can potentially create

resistances. Therefore, when using antibiotics, a clear strategy of changing antibiotics

in a regular mode needs to be followed.

Offline measurement of cell concentration is either done via trypan blue dyes to

discriminate between viable and non-viable cells (integrity of the membrane) or via

crystal violet by staining of cell nuclei. Cells are either counted manually using a

microscope or with dedicated cell counters. Cristal violet has the problem that some

cells have multiple nuclei, which can result in overestimation of cell concentrations.

The methods of cell counters need to be carefully set, so that cell aggregates or cell

debris are correctly determined. The typical standard deviation in measuring cell

concentration is 6−10% (but can easily increase up to 30% depending on the

method and the experience in case of manual cell counting). With increasing cell

concentrations, the needed dilution increases and with that the potential dilution

error. For bioreactors, various online options are available. Turbidity or optical

Upstream processing for viral vaccines

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