packed-bed or fluidized-bed reactors or hollow fiber bioreactors (HFBR) allow

cultivation of adherent cells with low shear stress at very high cell concentrations

including options for medium replacement or feeding. Nevertheless, there is a shift

towards suspension cells as scale-up and passaging is significantly easier, cell

growth is rapid, and very high cell concentrations by perfusion cultivations can be

achieved. Furthermore, suspension cell based processes have many advantages

regarding online monitoring and control. Typical cell lines used for viral vaccine

and vector production, including MDCK [16] and HEK293 cells [17], but also

designer cell lines (AGE1.CR.pIX [18], EB66 [19], CAP [20]) were recently

adapted to suspension growth under serum-free conditions for high titer production

of viral vaccines.

Adaptation to suspension growth can be very tedious and lengthy. Moreover, it is

not clear which of the various approaches proposed in literature will eventually

achieve the desired result. Currently, it seems reasonable to start parallel adaptations

with cell lines from different sources (i.e., cell culture collections), with different

passage histories or in different media to increase the rate of success. One adaptation

approach that is often used follows a two-step adaptation. Starting with adherent cells

that are cultivated in serum-containing media, the serum content in the medium is first

stepwise reduced (serum wheaning) by diluting with serum-free or chemically defined

medium. Next, confluent cells are maintained in T-flasks by continuous refreshment

of the medium over several weeks. By reaching a super-confluency state, cells start to

form aggregates above the confluent layer and in the supernatant. These cell spheroids

are then cultivated under agitation for several passages in spinner flasks aiming for

single-cell growth in suspension culture (separation of small and large aggregates)

[21,22]. Moreover, adaptation by a direct transfer of cells to a new medium might be

successful [23]. Alternatively, suspension growth can be triggered by targeted

transfection as shown for HEK293, AGE1.CR, and PER.C6 cells (Ad5 genes E1A

and E1B, [24]) or MDCK cells (siat7e gene [25]). Finally, whatever approach was

chosen, the stability of single-cell growth over several runs should be tested and the

doubling time should remain between 20−30 hours.

For commercial application, suspension cells have been mainly used for the

production of recombinant proteins or veterinary vaccines (BHK21, against foot

and mouth disease [26] and rabies [27]). A major concern regarding the use of

suspension cell lines for vaccine production for human use is traceability, risk of

adventitious agents and tumorigenicity/cancerogenicity. Due to enormous progress

made in methods to allow for rigorous cell line characterization, suspension cells

have been established for human influenza virus production, e.g., Optaflu^® (MDCK

cells, Novartis) licensed in 2007 [28] or Flucelvax Tetra (MDCK cells, Seqirus),

both currently available in Europe [29] and the United States [30]. Nevertheless,

drawbacks of cultivation with suspension cells are the risk of cell aggregation and

the requirement for cell retention devices for perfusion cultivation or the medium

exchange prior to infection (minor challenge for adherent cells). However, scaling

up of adherent cells is significantly more complicated and labor intensive. For

adherent cells, the maximum cell concentration is restricted by the provided surface

area, which must be increased during scale-up. Therefore, microcarrier systems

were established to increase the surface/volume ratio. Here, cells grow on

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