Characterization of hydrodynamic forces and interfacial phenomena in cell culture processes

by Hu, Weiwei

Abstract (Summary)
The maturity of biotechnology as a significant, commercial enterprise has lead to the large scale production of products from a variety of cell types, including bacterial, fungal, mammalian, and microalgae. While both large scale (>10,000 L) mammalian cell culture and microalgae culture are currently used for commercial purposes, there still exist significant engineering challenges. One of these challenges is to quantify, and minimize, the effect of hydrodynamic forces on cells, which is in direct conflict with the ever pressing need to increase gas mass transfer capability and mixing performance of large scale bioreactors to improve productivity. In this dissertation, a microfluidic channel was applied to quantitatively evaluate the effect of hydrodynamic forces on heterotrophic dinoflagellate Crypthecodinium cohnii, a naturally high producer of DHA. In the transient experiments, the lysis of C. cohnii cells was not observed even at high energy dissipation rate (EDR) of 5.8×10e7 W/m3, while a significant sub-lethal effect, the loss of flagellate, was identified at EDR higher than 1.6×10e7 W/m3. The flagellate can be regenerated approximately 45 minutes after exposure. During the “recovery process”, the algae cells began to spin first, and then move in a straight direction. The presence of C. cohnii cells in bubble film and foam layer was also verified by microscopic observation. In the long-term experiments, the microfluidic channel was connected with a small stirred tank bioreactor, which facilitated repeated exposures of cells to high EDR. It was found that the growth of C. cohnii cells was not inhibited until an EDR of 5.9×10e6 W/m3 was achieved. This level was significantly higher than that in shaken flask experiments, where the shear stress was claimed to cause a negative effect on dinoflagellate proliferation by numerous published papers. Consequently, the limitation on oxygen mass transfer was identified to be a key issue in shaken flask cultures. As has been well documented in the literature, bubble rupture typically creates significantly higher hydrodynamic forces than the forces created from impeller agitation. In this dissertation, two approaches were taken to minimize bubble-associate cell damage from gas sparging. The first approach consisted of mass screening surfactants to alleviate cell-bubble attachment. It was found that octyl-, nonyl-, and decyl-maltopyranoside were less toxic to Chinese hamster ovary (CHO) cells among twelve small molecule surfactants tested, and had no negative effect on cell growth in a chemical defined medium until a level around 0.2 times their critical micelle concentration (CMC) was achieved. Trends in the performance of these surfactants were explored based on the chemical structure as well as the dynamic surface tension. Finally, nonyl-maltopyranoside (NM) was identified as the most promising candidate, which can efficiently reduce the cell enrichment in the foam layer to a similar level to Pluronic F-68; a commonly used large molecule protective surfactant. The second approach to minimize bubble-associated cell damage was to develop an optimization strategy for aeration. A novel dual-sparger system was proposed based on theoretical calculations. These calculations suggest the use of microbubbles of oxygen and large bubbles of air to uncouple oxygen supplement and dissolved carbon dioxide removal, which has the potential to minimize the required aeration rates for mass transfer as well as cell damage by bubble rupture and foam problems in large scale bioreactors.
Bibliographical Information:


School:The Ohio State University

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:mammalian cell culture microalgae hydrodynamic forces surfactants mass transfer


Date of Publication:01/01/2007

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