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3R-Project 109-08
Evaluation of lipid fractions for the substitution of serum in cell culture media
Paul Honegger and Marie-Gabrielle Zurich
Department of Physiology, University of Lausanne, CH-1005 Lausanne, Switzerland.
paul.honegger@unil.ch
Keywords: rat; brain; cell cultures: aggregate; cell cultures: organ-specific; cell cultures: serum free; cell cultures: serum free; replacement; standardization
Duration: 2 years End of the Project: 2010
Background and Aim
Despite great efforts during the past 30 years, animal sera are still required for most cell culture applications, in conflict with the principles of the 3Rs. Several recent findings indicate that the indispensable component of serum resides in the lipoprotein fraction. Furthermore, it has become possible to reconstitute lipoproteins from synthetic lipids and recombinant apolipoproteins, exempt from animal sacrifice. Therefore, our aim is to provide either purified or synthetic lipid fractions as substitutes for serum actually used in most cell culture applications, and thus to reduce or abrogate the current animal sacrifice for this purpose.
Method and Results
in progress (present status)
The different lipoprotein fractions of serum (taking both fetal and newborn calf serum as sources) will be prepared by conventional density gradient ultracentrifugation (1). Furthermore, if indicated, phospholipid liposomes loaded with cholesterol (2) and reconstituted lipoproteins will be prepared. All these fractions will be assayed using aggregating brain cell cultures as test models. Aggregating brain cell cultures are primary cell cultures prepared from embryonal rat brain cells, which form three-dimensional histotypic structures (3) and have proved to be useful in brain research3 and neurotoxicology (4). The best fraction(s) will also be tested in human fibroblast cells and in 3T3 cell lines. The latter will be used for long-term experiments over several passages. The potential beneficial effects on the cells of the different lipid fractions will be evaluated by the analysis of cell type-specific enzyme activities and quantitative RT-PCR. Several published methods describe the preparation of reconstituted proteoliposomes (5) and lipoproteins (6-10) from purified apolipoproteins and distinct lipids. Most protocols for the reconstitution of lipoproteins make use of the sodium cholate removal technique6. Methods are now also available to prepare recombinant apolipoproteins, which can then be applied for the reconstitution of lipoproteins (10-13), and thus for the preparation and use of entirely synthetic lipoproteins.
Conclusions and Relevance for 3R
The results expected from this study will broaden the access to serum-free culture methods and therefore contribute to the replacement of animals in biological and medical research. It seems that any researcher would prefer the use of a defined medium over serum-containing medium whenever possible and affordable. As a minimal result, it can be expected that serum lipid fractions will be found that allow the serum-free culture of primary cells and cell lines derived from the nervous system, but the chances are good that they will be suitable for the majority of cell culture models. The latter outcome would lead to a drastic decrease or even a stop of serum use for cell culture work.
The availability of a synthetic substitute for animal serum would stop animal sacrifice for serum preparation, and promote the use of cell culture models for animal replacement, thus helping to reach the ultimate goal of the 3Rs, namely the abolition of animal sacrifice for research purposes.
References
1. Brown M.S. et al. J Biol Chem 249 (1974) 789-796.
2. Dobreva I. et al. Biol Chem 386 (2005) 909-918.
3. Honegger P. and Monnet-Tschudi F. In S. Fedoroff and A. Richardson (eds.) Protocols for Neural Cell Culture, 3rd ed., Humana Press, Totowa, N.J. (2001), pp. 199-218.
4. Zurich et al. Neuroscience 134 (2005) 771-782.
5. Prattes S. et al. J Cell Science 113 (2000) 2977-2989.
6. Matz C.E. and Jonas A. J Biol Chem 257 (1982) 4535-4540.
7. Bonomo E.A and Swaney, J.B. J. Lipid Research 29 (1988) 380-384.
8. Pittman R.C. et al. J Biol Chem 262 (1987) 2435-2442.
9. Davidson W.S. et al. J Biol Chem 270 (1995) 5882-5890.
10. Rye K.-A. et al. J Biol Chem 271 (1996) 4243-4250.
11. Gillotte K.L et al. J Biol Chem 271 (1996) 23792-23798.
12. Gillotte K.L et al. J Biol Chem 277 (2002) 11811-11820.
13. Rye K.-A. et al. J Lipid Research 47 (2006) 1025-1036.
| Dernières modifications: 29.08.2010 |  |