Reduction in laboratory animal use by factorial design

Factorial experimental designs (FEDs) can be used to study the effects of controllable variables, such as an experimental treatment, sex, strain, age, diet and prior treatment of animals, on some defined response. Such designs have been widely used in optimising manufacturing processes, but have rarely been used in optimising animal experiments in drug discovery. FEDs generally provide more information than the alternative "one-variable-at-a-time" approach, because each animal contributes information on the effect of every factor, and because such designs can highlight any interactions among the variables. Although FEDs can have any number of factors and levels of each factor, where many factors are to be explored, it is common to do an initial experiment using two levels of each factor, and in some cases fractional factorial designs can be used to reduce the total number of treatment combinations to manageable levels. These designs have been used successfully at AstraZeneca in the optimisation of in vivo drug screening experiments, where their use has effectively reduced the numbers of animals used in some routine screens.     

Use of flow cytometry to rapidly optimize the transfection of animal cells

Plasmid transfection is the first step in the generation of stably transformed animal cells and is also a useful tool for analyzing transient gene expression. Maximizing the transfection efficiency and expression level from the introduced plasmid is critical to the success of these processes. By means of lipid-mediated transfection, a plasmid vector expressing the green fluorescence reporter protein has been coupled with flow cytometry to conveniently investigate those parameters that impact the efficacy of transfection of lepidopteran insect cells. The key feature of this technique is the rapid and simultaneous quantification of transfection efficiency and heterologous protein expression level per cell. Using this technique, we developed an optimized transfection protocol for insect cells by investigating the following parameters: lipid incubation time, lipid/DNA mixture incubation time, lipid and DNA concentration, incubation vessel and transfection duration. Following optimization, transfection efficiencies of 37%-40% were obtained for Bombyx mori Bm5 and Spodoptera frugiperda Sf-21 cells.     

Opportunities and strategies to further reduce animal use for Leptospira vaccine potency testing

Hamsters are routinely infected with virulent Leptospira for two purposes in the regulation of biologics: the performance of Codified potency tests and maintenance of challenge culture for the Codified potency tests. Options for reducing animal use in these processes were explored in a plenary lecture at the “International Workshop on Alternative Methods for Leptospira Vaccine Potency Testing: State of the Science and the Way Forward” held at the Center for Veterinary Biologics in September 2012. The use of validated in vitro potency assays such as those developed by the U.S. Department of Agriculture for Leptospira (L.) canicola, Leptospira grippotyphosa, Leptospira pomona, and Leptospira icterohaemorrhagiae rather than the Codified hamster vaccination–challenge assay was encouraged. Alternatives such as reduced animal numbers in the hamster vaccination–challenge testing were considered for problematic situations. Specifically, the merits of sharing challenge controls, reducing group sizes, and eliminating animals for concurrent challenge dose titration were assessed. Options for maintaining virulent, stable cultures without serial passage through hamsters or with decreased hamster use were also discussed. The maintenance of virulent Leptospira without the use of live animals is especially difficult since a reliable means to maintain virulence after multiple in vitro passages has not yet been identified.     

Systematic use of the incomplete factorial approach in the design of protein crystallization experiments

We have used the Incomplete Factorial Approach (Carter, C. W., and Carter, C. W., Jr. (1979) J. Biol. Chem. 254, 12219-12223) in conjunction with the program Cristal (Roussel, A., Serre, L., Frey M., and Fontecilla-Camps, J. (1990) J. Crystal Growth 106, 405-409) to crystallize six different proteins. We were able to obtain crystals and to identify the critical factors for crystallization for each of these six proteins. In some of the cases, we succeeded on the first try while using only minute amounts of protein. This study proves that the Incomplete Factorial Approach is a powerful tool in identifying the factors that need to be varied to achieve crystallization. Single crystals of adequate size were obtained for all the proteins reported here, although some did not diffract well enough to be studied by x-ray diffraction methods; the asymmetric units of these latter crystals contain a large metric units of these latter crystals contain a large number of molecules, which is most likely due to the presence of significant amounts of carbohydrate in the proteins.     

Evolutionary operation (EVOP) to optimize three-dimensional biological experiments

Evolutionary operation (EVOP) is used as an efficient technique for optimization of three variable experimental parameters using two-level factorial designs with center points. For metabolic function like enzyme synthesis by microorganisms, small amounts of metal ions, surfactants, and vitamins act as inducers. The desired quantities of these chemicals are, however, species dependent and have to be found out or each microorganism. By employing the EVOP technique, the requirements of these chemicals have been optimized for the production of protease by Rhizopus oryzae (RO, IIT KGP). (c) 1993 John Wiley & Sons, Inc.     

Use and misuse of an imprecise concept: alternative methods in animal experiments

The concept of inhumanity of Russell & Burch in 1959 with proposals for human procedures in experiments on animals by way of replacement, reduction and refinement (the 3 R's) and the revival of these by Smyth (1978) as alternatives to animal experiments are presented. Since then under the name 'alternative methods', replacement of in vivo by in vitro methods has found great public attention and promotion. It is argued that alternative methods are a fallacy, because in the progress of research a continual control and reinvestigation of findings on each system level is required until knowledge is complete, which makes in vivo experiments irreplaceable. True alternatives exist only for refinement within a system level. On the level of the organism, refinement would be in the care of animals and experimental procedure through alleviation of stress. The possibilities which refinement offers for animal welfare have been nearly forgotten and need promotion among researchers, animal technicians and attendants.