Lindell, J.Girard, P.Muller, N.Jordan, M.Wurm, F.2007-06-052007-06-052007-06-05200410.1016/j.bbaexp.2003.11.016https://infoscience.epfl.ch/handle/20.500.14299/7624WOS:00018860610000514746910We have developed a novel method called Calfection for gene delivery to and protein expression from suspension-cultivated mammalian cells. Plasmid DNA was simply diluted into a calcium chloride solution and then added to the cell culture for transfection. We evaluated and optimized this approach using suspension-adapted HEK293 cells grown in 12-well plates that were shaken on an orbital shaker. Highest expression levels were obtained when cells were transfected at a density of 5x10(5) cells/ml in the presence of 9 mM calcium and 5 microg/ml of plasmid DNA while maintaining a culture pH of 7.6 at the time of transfection. Suspension-adapted BHK 21 and CHO DG 44 cells could also be transfected using this method. Calfection differs from the widely known calcium phosphate coprecipitation technique. The physico-chemical composition of the DNA interacting complexes is not yet known. The transfection cocktail, DNA in a calcium chloride solution, remained highly efficient during long-term storage at temperatures ranging from room temperature to -80 degrees C. In contrast, calcium phosphate-DNA cocktails are only efficient for gene transfer when prepared fresh. Furthermore, passing the calcium-plasmid DNA mixture through a 0.2-microm filter did not compromise protein expression, whereas calcium phosphate-DNA coprecipitates were retained by the filter. High protein expression levels, a limited number of manipulations and the possibility to filter the cocktail make the Calfection approach suitable for both large-scale transfection in bioreactors and for high-throughput transfection experiments in microtiter plates.AnimalsBioreactorsCHO Cells*Calcium ChlorideCattleCell Culture Techniques/*methodsCell LineCricetinaeDose-Response RelationshipDrug*Gene Transfer TechniquesGreen Fluorescent ProteinsHumansHydrogen-Ion ConcentrationLuminescent Proteins/analysis/biosynthesis/geneticsPlasmids/pharmacologySolutionsTemperatureTime Factorstext::journal::journal article::research article