and large-scale (>1000 litre) production, suspension cultures are being used mostly. The
major concern in scale-up of suspension cultures is the supply of oxygen and removal of
carbon dioxide. Gas transfer may be enhanced by the direct sparging of gas into the
culture medium and by dispersing the gas bubbles using high agitation speeds. However,
animal cells are relatively fragile compared to microbial cells due to their larger size and
lack of a protective cell wall. As a consequence, the hydrodynamic forces generated
during sparging and bubble dispersion cause damage to the cells.
Oh et al. (1989, 1992) showed that in a 1.4 dm^3 bioreactor agitation up to 450 rpm
does not damage cells as long as no sparging is used. Likewise, Chisti (1993) found that
hybridoma cells can be cultured in a 0.3 m^3 stirred-tank reactor withstanding high (>1
m.sā^1 ) impeller tip speeds and fluid turbulence. Finally, Kunas and Papoutsakis (1990)
showed that as long as bubbles can not interact with a freely moving air-liquid interface,
agitation becomes detrimental only at agitation rates far higher than those generally used
in animal-cell bioreactors. Thus, it can be safely stated that cell death solely due to
agitation does not occur under normal conditions. In contrast, the hydrodynamic forces
associated with the presence of air bubbles have been shown to cause excessive damage
to animal cells in numerous studies. There have been many reports trying to elucidate the
mechanism of air-bubble-related cell death and model this process. For reviews the
reader is referred to Chalmers (1994, 1996), Hua et al. (1993), Papoutsakis (1991a,
1991b) and Wu (1995b). Although progress has been made, the information is still quite
scattered and incomplete. In this chapter the current knowledge is summarised and the
remaining questions with respect to bubble-related cell death are discussed.
For optimal design and operation of a reactor in terms of minimising shear-related cell
death one would like to be able to predict cell damage directly from design parameters
using a mechanistic model. Essentially three types of knowledge are required for such a
model (Zhang and Thomas, 1993a; Papoutsakis, 1991a). These concern knowledge about
(i) the hydrodynamics in the reactor; (ii) the response of the individual cells in a cell
population to hydrodynamic forces; and (iii) the mechanism by which the hydrodynamic
forces interact with the cells. Because information on all three aspects mentioned above is
not available, empirical models have been constructed just relating design parameters to
cell death. The parameters of these models are determined from experiments. The main
disadvantage of this approach is that extrapolation of the results to, for instance, different
cell lines, medium formulations, and reactor scales is not allowed. The response of
animal cells to hydrodynamic forces may range from increase in DNA-synthesis rate
(Lakhotia and Papoutsakis, 1992) and metabolic rates (Al-Rubeai et al., 1990) to cell
death through apoptosis, necrosis or direct lysis (Al Rubeai et al., 1995a, 1995b). Here
we will only consider cell death.
From the start of animal-cell culture substances have been added that protect the cells
against hydrodynamic forces (Papoutsakis, 1991b). Protective additives include Pluronic
polyols, derivatised cellulose, polyethylene glycol (PEG), polyvinyl alcohol (PVA),
dextran, and protein mixtures like serum. The protection mechanism is closely related to
the damage mechanism and may be attributed to (i) increasing the strength of the cells,
(ii) decreasing the hydrodynamic forces, and (iii) shielding the cells from the regions with
damaging hydrodynamic forces. Therefore, these compounds and their protection
mechanism will be treated throughout this chapter. In Table 15.1 some of the most
common additives are listed together with their physical properties.
Multiphase bioreactor design 454