Production and modification of cyanobacterial gas vesicles and their application as oxygen carriers in animal cell culture

Oxygen is conventionally supplied to cells in microbial fermentations through vigorous agitation and aeration. This has been found to be unsuitable for mammalian cell culture systems because of the shear sensitivity of animal cells. Oxygen transfer to the culture is, hence, a major growth limiting factor. The presence of oxygen limitations results in low product titers. This has led to attempts at supplying oxygen by non-conventional means. This research involved the production and modification of cyanobacterial gas vesicles and their application as oxygen microcarriers in animal cell culture.;Gas vesicles were isolated from cultures of the filamentous cyanobacterium Anabaena flos-aquae, which were harvested by natural flotation. To optimize gas vesicle production, the growth and flotation characteristics of these cultures were studied. By examining changes in cell properties along cell growth, maximum buoyancy was found to correspond with the maximum specific gas vesicle content and minimum filament density. A cell harvesting efficiency of 75-80% and an overall gas vesicle volumetric productivity of 20-25 ;Glutaraldehyde treatment to cross-link the proteinaceous surface of gas vesicles was seen to protect them from the protein-stripping action of detergents like SDS and urea. The relative oxygen permeability (with respect to water) of the gas vesicle was experimentally measured to be 29.09. This implied that the oxygen permeation rate through the gas vesicle is much quicker than through water, with the gas vesicle wall offering some finite resistance.;A medium supplemented with 1.8% (v/v) gas vesicles was perfused through a bioreactor comprising of a packed bed of microcarrier beads with attached Vero cells. This medium was found to be capable of supporting a 20-25% higher viable cell concentration than the one without gas vesicles under oxygen limiting conditions in the reactor, indicating the improved oxygen carrying capacity of gas vesicle-containing media.;The novelty of this approach of using gas vesicles as oxygen carriers lies in the development and application of a naturally occurring product of biological origin. There are no problems of chemical compatibility and toxicity that exist in some other approaches. The inherent properties of gas vesicles simplify bioreactor design and operation.