Serum-free culturing of mammalian cells--adaptation to and cryopreservation in fully defined media.

Long term storage of living cells is a central issue in cell biology and medicine. In addition to the cryoprotectant dimethyl sulphoxide (DMSO), foetal bovine serum (FBS) is often added to the freezing medium for the cryoconservation of serum dependent cell lines. FBS, with its high protein content, protects cells against shear forces and gives the medium a desirable osmotic environment with a physiological viscosity. However, the harvesting of FBS is painful for the foetus and should be avoided for ethical reasons. In this work we describe the adaptation of several commonly used cell lines to serum- and protein-free media; however, such cell lines should not be frozen in a conservation medium containing serum. We tested the synthetic surfactant ''Pluronic F68'', known to protect mammalian cells grown in serum-free bioreactors (Papoutsakis, 1991), as an active cryoprotectant. In samples containing 0.1 to 1% Pluronic F68, we found a significant increase in viable cells after thawing. Values up to 115% of starting cell number indicate that the cells proliferate within the first 24 hours after thawing, a property which was not observed in cryoconservation media without Pluronic F68.

Production of antibodies to canine IL-1beta and canine TNF to assess the role of proinflammatory cytokines.

IL-1 and TNF are important proinflammatory cytokines implicated in both antimicrobial host defense and pathogenesis of diseases with an immune-mediated and/or inflammatory component. Respective studies in the dog have been hampered by the unavailability of reagents allowing the specific measurement of canine cytokine proteins and the effect of canine cytokine neutralization by Ab. Starting with recombinant canine (rcan) IL-1beta and rcanTNF, four polyclonal antisera and 22 mAb specific for rcanIL-1beta and rcanTNF were generated. Their usefulness in neutralization assays was determined. Using cytokine-containing supernatants of canine cells in bioassays, polyclonal antisera neutralized either canine IL-1beta or TNF. TNF was also neutralized by three antibodies developed in this study and one commercial mAb. The usefulness of monoclonal and polyclonal Ab in canine cytokine-specific Ab capture ELISA's was assessed. This resulted in the identification of a commercial mAb combination and one pair developed in this study allowing low levels of TNF to be detected by antibody capture ELISA. The detection limit was 141 pg/ml rcanTNF for both combinations. Using rcanIL-1beta as an antigen allowed the detection of lower concentrations of rcanIL-1beta (20 pg/ml, on the average) by a pair of polyclonal antisera than when monoclonals were used. By using such IL-1beta-specific and TNF-specific ELISA's, the respective cytokines were detected in supernatants of canine PBMC stimulated with LPS or heat-killed Listeria monocytogenes and interferon-gamma combined. Thus, monoclonal and polyclonal reagents were identified allowing the quantitation of canine IL-1beta and TNF production in vitro, and the neutralization of these cytokines.

[Monoclonal antibodies: Comparative methods for in vitro production]

The growth of antibody producing hybridoma in the abdominal cavity of the mouse with subsequent harvesting of the ascites fluid is a simple method for the production of monoclonal antibodies. However, due to the massive painful stress of the animal, this method is no longer tolerated by the legislator. In search of alternative methods the scientist has the choice between different techniques: Static tissue culture, spin- or roller-cultures as well as the production in bioreactors (automated cell culture devices). The presented work investigates the different methods and describes their applicability in a research lab. The results clearly show that for any desired amount of antibodies a production technique is available which allows the omission of the use of the "ascites-mouse".