Many hybridoma and recombinant myeloma cell lines have been successfully adapted to growth in protein-free media. Compared with serum-supplemented media, use of protein-free media promotes superior cell growth and protein expression and facilitates downstream purification of the expressed product. Owing to its sterol auxotrophy, the NS0 myeloma is normally grown in either a serum-supplemented medium or a serum-free medium supplemented with an animal-derived lipoprotein. CD Hybridoma Medium (a protein-free, chemically defined formulation) grows many cell lines that do not exhibit lipid dependence, but this medium does not support growth of NS0 cells without further lipid supplementation. We tested several commercially available lipid supplements in CD Hybridoma Medium, including bovine EX-CYTE VLE. None of the tested supplements supported long-term growth of NS0 cells in CD Hybridoma Medium. Sustained long-term growth of NS0 cells was achieved in CD Hybridoma Medium supplemented with various animal- or plant-derived lipids complexed with cyclodextrin. NS0 cells adapted to CD Hybridoma Medium supplemented with cyclodextrin-lipid complex reached peak cell densities that were more than double those observed in serum-supplemented medium and were cultured for more than 15 passages. These cultures were also successfully cryopreserved and recovered in this defined medium. Through the use of cyclodextrin-based additives to CD Hybridoma Medium, it is possible to solubilize significant quantities of sterols and other lipids and to maintain a protein-free, chemically defined cultivation environment for NS0 cells. The culture system can be kept entirely free of animal-derived components if the supplement is made with plant-derived or synthetic lipids.

With the current shortage in fetal bovine serum, researchers are turning to a variety of options for cell and tissue culture.

Technical limitations and evolution of therapeuticapplications for cell culture-derived products haveaccelerated elimination of animal-derived constituentsto minimize inadvertent introduction of adventitiousviral or prion agents. Practical considerationsdemand adequate emphasis both on design of theserum-free/protein-free culture environment and onnutrient media manufacturing process controls. Protein components may be acceptable, given adequateattention to synthetic process, sourcing (e.g.,geographic location and endemicity, species andtissue/organ) and validated treatment method. Variousoptions exist for re-engineering of traditionalserum-free formulations (containing insulin,transferrin and other protein factors) withnon-protein substitutes. Caution must also beexercised with sourcing of non-protein additives,particularly amino acids and lipids, to avoidintroducing adventitious contaminants. Simpleguidelines facilitate adaptation, cryopreservation andrecovery of many cell types within a protein-freeculture environment. Scrupulous maintenance offacility and equipment and monitoring of processwater, air handling systems and technical personnelare required to ensure that approved raw materials arecorrectly formulated and dispensed. Validatedsanitization processes provide additional assuranceagainst cross-contamination from previous batches ina multi-use facility.

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The mouse fibroblastic cell line LM(TK-) is unable to grow at external K+ concentrations below a threshold value of 0.4 mM. At subthreshold K+ concentrations, LM(TK-) cells rapidly lose intracellular K+ and eventually lyse. We have analyzed the pathwayprimarily responsible for K+ efflux under these experimental conditions and report its specific inhibition by two diuretics, furosemide and bumetanide. Bumetanide, an analog of furosemide, was a more potent inhibitor (by several orders of magnitude) than was furosemide itself. The effects of ouabain and bumetanide were additive, suggesting independence of diureticsensitive K+ efflux from Na+/K+ pump-mediated fluxes. Characterization of K+ efflux in LTK-5, a mutant derived from LM(TK-) and selected for its ability to grow at 0.2 mM K+, indicated that the mutant had lost the diuretic-sensitive K+ efflux pathway. Net Mammalian cells maintain a high steady-state K+ concentration and a low steady-state Na+ concentration relative to their surrounding physiological fluids. The transmembrane electrochemical gradients established by these two cations have been implicated in several physiological responses, including the regulation of cell volume (1, 2), nutrient cotransport (3-6), and membrane excitability (7-10). Because the steady-state K+ concentration reflects the sum of influx and efflux components, both active and passive, an alteration in the activity of one component must be accompanied by a compensating alteration in another if the cationic steady state is to be maintained (11). Should a particular flux become altered so that antagonistic fluxes are unable to compensate, the resulting change in intracellular cation concentration may be lethal to the cell. Such considerations were the basis for the selection of two K+ transport mutants that gained the ability to grow at a K+ concentration below that minimally required for parental cell growth (12). Both mutants proved capable of maintaining high intracellular K+ concentrations at reduced external K+ through changes in ouabain-insensitive K+ transport.One of the mutants, LTK-5, exhibited altered activity of a Na+-K+ cotransport system (12, 13) similar to that described in human (14, 15) and avian (16) erythrocytes and in Ehrlich cells (17-21) and murine fibroblasts (22,23). Specifically, in characterizing the mutant relative to its parent strain under conditions of K+ depletion and high external K+, Gargus and Slayman (13) noted that LTK-5 demonstrated enhanced furosemidesensitive, Na+-dependent K+ influx. For two reasons, however, it seemed unlikely that the increase in K+ influx was causally related to the ability of the mutant to survive selection. (i) When furosemide-sensitive K+ influx was measured as a function of the extracellular K+ concentration in both parent and mutant cells, it reached a half-maximal rate at 6 mM K+, considerably higher than the Km for the ouabain-sensitive Na+/K+ pump (1.3 mM). At 0.2 mM K+, the concentration used in the selection, furosemide-sensitive influx was barely detecta...

Lipids are critical nutrients for high density eukaryotic cell cultivation, but inclusion of lipid components into dry-form media has been technically challenging. Lipid supplements are usually supplied for separate addition after powder reconstitution and filtration, which increases manipulation and risk in a biopharmaceutical manufacturing facility. Advanced Granulation Technology (AGT) is a novel dry-form media format designed for large-scale biopharmaceutical manufacture. All components of a complex formulation are homogeneously incorporated into a single granulated medium that simply requires water addition to yield a complete reconstituted 1X medium. We investigated whether cyclodextrin technology could be combined with the AGT process to deliver usable lipid in a dry medium format. The test lipids were cholesterol and several fatty acids supplied either as an aseptic supplement to liquid media or as part of a complete AGT formulation. The test system utilized a cholesterol auxotroph, NS0, and a protein-free basal formulation (CD Hybridoma Medium), with or without supplemental lipid. Post-filtration recovery of cholesterol from AGT-processed materials compared favorably with media supplemented with liquid lipid concentrates. Cell growth and viability and expression of recombinant protein were equivalent in all test media.

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