Cell suspension stability

Terranova, B. E., and Bums, M. A., Continuous Cell Suspension Processing Using Magnetically Stabilized Fluidized BedsBiotechnol. Bioeng., 37 110... 

The presence of foreign protein in the medium of plant cultures does not necessarily mean that all or even most of the product can be recovered from the medium. In many expression systems where an appropriate signal sequence has been used, considerable amounts of foreign protein remain within the plant cells and/or tissues. For example, in a comparison of IgG antibody production in tobacco cell suspension and hairy root cultures, a maximum of 72% of the total antibody was found in the medium of the suspension cultures whereas only 26% was found in the medium of the hairy root cultures [17]. This result could indicate that secretion and/or transport across the cell wall was slower in the hairy roots alternatively, it could indicate poorer stability of the secreted protein in the hairy root medium. If foreign proteins are to be purified from the medium, improved secretion and extracellular product stability are desirable. 

Clancy, R. M., Miyazaki, Y., and Cannon, P. J. (1990). Use of thionitrobenzoic acid to characterize the stability of nitric oxide in aqueous solutions and in porcine aortic endothelial cell suspensions. Anal. Biochem. 191, 138-143. 

As noted earlier, plasma from blood samples must be promptly stabilized and, if necessary, the acidified samples may be stored frozen at — 65°C. Because of the existence of oxyhemoglobin in whole blood or red cell suspensions, some consideration must be given to inactivate oxyhemoglobin or use an assay for total ascorbic acid content. With respect to tissue analysis, some discretion must be considered as to the degree of blood contamination. 

In contrast to the stability of endogenous ABA-GE, applied ABA-GE is rapidly hydrolyzed. For example, in Ricinus leaves, applied ABA-GE was hydrolyzed prior to uptake and translocation of free ABA in the phloem [119]. In wheat seedlings, ABA-GE was split by glucosidases rather than esterases [127], When radioactive ABA-GE was applied to cell suspension cultures of Lycopersicon, free ABA appeared in the medium and cells within 20 minutes, but the radioactive conjugate was not detected in the cells until much later [125]. Hydrolysis presumably took place at the plasma membrane, the free acid then entered the cell and was conjugated again inside the cell. These observations indicate that ABA-GE as such cannot enter cells. 

The reactor was loaded with 75 ml granular carrier material [14], and finally, the entire reactor system, including tubing and recirculation reservoir, was autoclaved at 120°C for 30 min. Before use, the reactor system was gassed for 15 min with N2/CO2 (4 1) to ensure anaerobic conditions and filled with BA medium with an initial xylose concentration of 10 g/1. The reactor was started up in batch mode by inoculation with 80 ml of cell suspension with an optical density (OD578) of 0.9-1. The batch mode of operation was maintained for 24 h to allow cells to attach and to immobilize on the carrier matrix. After the batch run, the system was switched to continuous mode, applying a hydraulic retention time (HRT the volume of the reactor divided by the influent flowrate) of 8 h and up-flow velocity of 1 m/h. To achieve operational stability, the reactor was run for 7 days under... 

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