Barrett, T.A.; (2008) Microwell evaluation of mammalian cell lines for large scale culture. Doctoral thesis , University of London.

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Abstract

Experimentation in shaken microplate formats offers a potential platform technology for the evaluation and optimisation of cell culture conditions. Provided that the results obtained are reliable, and indicative of large scale performance, it should be possible to obtain process design data early and cost effectively. This work describes a detailed engineering characterisation of liquid mixing and gas-liquid mass transfer in microwell systems and their impact on suspension cell cultures. Furthermore, an initial attempt at scaling a microwell culture to shake flasks and a 5-L stirred-tarik reactor is made. For suspension cultures of murine hybridoma cells producing IgGl, 24-well plates have been characterised in terms of power dissipation (P/V) (via CFD). Fluid flow patterns and oxygen transfer rate as a function of shaking frequency and liquid fill volume. Predicted ka values varied between 1.3 and 29 h_1 mixing time, quantified using decolourisation of iodine, varied from 1.7 s to 3.5 h while the P/V ranged from 5 to 35 W m-3. CFD simulations of the shear rate predicted hydrodynamic forces will not be lethal to cells. High shaking speed (> 250 rpm) was shown to be detrimental to cell growth, while a combination of low shaking speed and high well fill volume (120 rpm. 2000 //l) resulted in oxygen limited conditions. Using matched average energy dissipation as a basis for scale translation, cell growth and antibody titre were found to be similar in a 24-well plate. 250 ml shake flask and 5-L stirred-tank reactor. Overall this work has demonstrated that cell culture performed in shaken microwell plates can provide data that is both reproducible and representative of larger-scale cultures. Linked with automation this provides a route towards the high throughput evaluation of robust cell lines under realistic process conditions.

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