Given the preliminary success with CDM HD, I jumped straight into developing a protocol to adenovirally transduce cells and then collect a secreted, recombinant protein of interest in 10% CDM. It worked like a charm. CDM HD serves the purpose, and I am very pleased about that.
Too few cells in relation to volume of media can result in lag phase. In the hollow fiber cartridge lag phase can be defined by the cells not consuming glucose but still show significant viability via the trypan exclusion assay. Always seed the cartridge with the recommended cell density. If this is not possible, adjust the volume of media down to keep the cells in culture from being too dilute.If the cells do go into lag phase you can either:
Hybridoma cells growing at high density in the C2011 cartridge using CDM-HD medium
Simply perform your harvest as usual. One of the issues that many researchers have when using our system is that they are afraid to harvest out too many cells. The cartridge can be overgrown if you don’t remove cells. After centrifugation your cell pellet should be between 1-3 mL or more (with the C5011 as much as 5 mL). Simply decant off the supernatant and re-suspend the cell pellet in 2 pellet volumes of fresh medium that has been diluted 10% with sterile distilled water. Leave it overnight at room temperature. The cells will consume the medium and also the hypotonicity will slightly squeeze the cells causing them to release any intracellularly sequestered antibody. This will get you an extra few milligrams every time you harvest.
Various components of serum. The major component, BSA binds many substances non-specifically and can be an overwhelming protein burden in some purification schemes, immunoglobulins can interfere with specific antibody purifications and the “other” can have many undefined effects. None of these characteristics will be reproducible from lot to lot.