Microcarrier culture

Concerning adherent cells there are few studies in the literature. Some of them deal with the influence of stirrer speed on microcarrier cultures. Most studies using defined forces are from medical research. These studies, as well as those with production cells, use different types of exposure systems based on the parallel plate theory. They investigate the influence of stress on cell morphology and viability which is most important for arteriosclerosis research. 

Two different concepts are described in the literature for investigating the influence of shear stress on adherent cells Microcarrier cultures in stirred reactors and defined stress levels in flow chambers. 

ORDINARY DIFFERENTIAL EQUATION MODELS 17.3.1 Contact Inhibition in Microcarrier Cultures of MRC-5 Cells... 

Growth inhibition on microcarriers cultures can be best quantified by cellular automata (Hawboldt et al., 1994 Zygourakis et al., 1991) but simpler models have also been proposed. For example, Frame and Hu (1988) proposed the following model... 

Forestell, S.P., N. Kalogerakis, L.A. Behie and D.F. Gerson, "Development of the Optimal Inoculation Conditions for Microcarrier Cultures", Biotechnol. Bioeng., 39.305-313(1992). 

Kalogerakis, N. and L.A. Behie, "Oxygenation Capabilities of New Basket-Type Bioreactors for Microcarrier Cultures of Anchorage Dependent Cells", Bioprocess Eng., 17, 151-156(1997). 

Kalogerakis, N., and Behie, L. A., Oxygenation Capabilities of New Generation Three Phase - Two Region Bioreactors for Microcarrier Cultures of Animal Cells, Fluidization VIII, (C. Laguerie, and J. F. Large, eds.), p. 441, Engineering Foundation, Tours, France (1995)... 

Venkat, R. V., Stock, L. R., and Chalmers, J. J., Study of Hydrodynamics in Microcarrier Culture Spinner Vessels A Particle Tracking Velocimetry Approach, Biotechnol. Bioeng., 49 456 (1996)... 

Croughan, M. S., J.-F. Hamel, and D. I. C. Wang, "Hydrodynamic Effects on Animal Cells Grown in Microcarrier Cultures," Biotech. Bioeng. 29 (1987) 130-141. 

Butler M, Spier RE (1984), The effects of glutamine utilization and ammonia production on the growth of BHK cells in microcarrier cultures, J. Biotechnol. 1 187-196. 

Butler M, Thilly WG (1982), MDCK microcarrier cultures seeding density effects and amino acid utilization, In Vitro 18 213-219. 

Butler M, Imamura T, Thomas WG, Thilly M (1983), High yields from microcarrier cultures by medium perfusion, J. Cell. Sci. 61 351-363. 

Croughan MS, Hamel JF, Wang DI (1987), Hydrodynamic effects on animal cells grown in microcarrier cultures, Biotechnol. Bioeng. 29 130-141. 

Wang Y, Ouyang F (1999), Bead-to-bead transfer of Vero cells in microcarrier culture, Cytotechnology 31 221-224. 

Table 18.1 compares the relationship between cell culture surface area and bioreactor volume in many different culture systems usually used with adherent cells. For microcarriers, this coefficient might reach 60 cm2/ml of medium for culture area prepared with 10 mg of microcarriers per milliliter. For Roux bottles, this coefficient is around 3 cm2/ml. In cultures initiated with 2 mg of microcarriers per milliliter of medium, high cell densities of even 3 X 106 cells/ml are often reached, compared with smaller cell densities from 2 to 3 X 105 cells/ml usually observed in Roux bottle systems. Another great advantage of the use of microcarrier culture systems is the possibility of preparing cell cultures with hundreds or even thousands of liters (Montagnon et al., 1984). 

Butler M (1987), Growth limitations in microcarrier cultures, Adv. Biochem. Eng. Biotechnol. 34 57-84. 

Montagnon B, Vincent-Falquet JC, Fanget B (1984), Thousand litre scale microcarrier culture of VERO cells, Dev. Biol. Stand. 55 37-42. 

Reuveny S, Coret R, Freeman A, Kotler M (1985), Newly developed microcarrier culturing systems an overview, Dev. Biol. Stand. 60 243-253. 

The advantage of microcarrier culture methods can be obtained for suspension cells by their entrapment within a variety of different beads. 

Gregoriades, N., J. Clay, N. Ma, K. Koelling, and J.J. Chalmers. 2000. Cell damage of microcarrier cultures as a function of local energy dissipation created by a rapid extensional flow. Biotechnol Bioeng 69 171-182. 

Westergaard N, Sonnewald U, Peterson SB Schousboe A (1991) Characterization of microcarrier cultures of neurons and astrocytes from cerebral cortex and cerebellum. Neurochemistry Research 16 919-923. 

For batch microcarrier culture. Equations 4.2.23. 2.28 apply with Z) = 0. However, the contribution from free cells in Equations 4.2.25 and 4.2.26 will be greater because free cells are not removed from the system. High values may lead to aggregate formation, which further complicates analysis (see below). 

If is obtained from experiments in stirred suspension or microcarrier cultures, as described above, then, it may be possible to estimate from Q- lqu- However, care must be exercised because can be altered by changes in the culture environment. For example, qy for cells immobilized in agarose beads or hollow fibres may be different from qy for the same cells grown in the same medium in a stirred suspension reactor (Shirai et al, 1988 Wohlpart et al, 1991). In addition, qy may change over time due to changes in cell, nutrient and byproduct concentrations. Analysis of cell density and immobilization effects is complicated by the presence of nutrient concentration gradients. However, stirred vessels with cells immobilized... 

Viable fraction of free cells in microcarrier culture (dimensionless)... 

True specific growth rate of attached cells in microcarrier culture (h" ) Apparent specific growth rate of attached cells in microcarrier culture (h ) Apparent specific growth rate based on effective total cell concentration (hr ) True specific growth rate of free cells in microcarrier culture (h )... 

There is no substantial literature on direct sparging of non-porous microcarrier cultures. As is discussed in section 4.6, the difficulty is that the presence of bubbles induces bead flotation, i.e. attachment of beads to bubbles, and the formation of large bead-bubble aggregates that tend to rise and accumulate at the surface of the culture vessel, which is a highly undesirable characteristic. Nevertheless, it is possible slowly to sparge microcarrier cultures without undue cellular injury if suitable surfactants/antifoams (e.g. Pluronic F-68 or Medical Emulsion AF see section 4.6) are used. 

In microcarrier cultures, cell injury is due to the interactions (collisions) between microcarrier beads on which cells are attached, interactions between microcarriers and small turbulent eddies and interactions between microcarriers and bioreactor internals, such as the impeller and various probes (Papoutsakis, 1991a). To protect cells against injury in microcarrier bioreactors, an increased medium viscosity has been the only documented and studied medium alteration (Croughan et ai, 1989 Papoutsakis, 1991a Lakhotia Papoutsakis, 1992). Dextran (Sigma) has been used to increase the medium viscosity as discussed below. This is a purely physical mechanism of protection. 

---