Culture medium for cell growth and transfection

Abstract

The present invention is directed generally to cell culture media (particularly serum free, non animal derived, and/or chemically defined media) which are useful for introducing macromolecules and compounds (e.g., nucleic acid molecules) into cells (e.g., eukaryotic cells). According to the invention, such introduction can take place in the presence of said medium. Cells containing such introduced materials can then be cultured in the medium and the effect of the introduced materials on the cells can be measured or determined. In particular, the invention allows introduction of nucleic acid molecules (e.g., vectors) into cells (particularly eukaryotic cells) and expression of proteins encoded by the nucleic acid molecules in the cells. The invention obviates the need to change the cell culture medium each time a different procedure is performed with the cells (e.g., culturing cells vs. transfecting cells). The invention thus provides efficient and high throughput methods to transform/transfect culture and cells avoiding the need for multiple manipulations and transfers of cells during transfection and expression studies. The invention also relates to compositions and kits useful for culturing and transforming/transfecting cells.

1 . A cell culture medium, wherein said medium supports introduction of one or more macromolecules into at least one eukaryotic cell in culture and supports cultivation of said at least one cell subsequent to said introduction, 
 wherein it is not necessary to remove medium used during said introduction from the presence of said at least one cell to support growth of said at least one cell, and/or    wherein after said introduction growth can be accomplished in cultivation in a volume of medium that is about the same volume up to no more than about 10 times the volume of the medium in which said introduction occurred.    
     
     
         2 . The cell culture medium of  claim 1 , wherein said medium also can support cultivation of said at least one cell prior to said introduction.  
     
     
         3 . The cell culture medium of  claim 1 , wherein replenishing, replacing or supplementing said medium with fresh medium after said introduction does not improve said cultivation, relative to cultivation in said medium without said replenishing, replacing or supplementing.  
     
     
         4 . The cell culture medium of  claim 1 , wherein said culture is a suspension culture.  
     
     
         5 . The cell culture medium of  claim 1 , wherein said one or more macromolecules includes at least one nucleic acid molecule.  
     
     
         6 . The cell culture medium of  claim 1 , wherein said cultivation includes the production of at least one protein product.  
     
     
         7 . The cell culture medium of  claim 1 , said medium comprising at least one compound selected from the group consisting of a polyol, a hydroxypyridine derivative, 1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene, ethylenedi-amine-N,N′-tetramethylenephosphonic acid, trisuccin, an acidic saccharide (e.g. ferrous gluconate), a glycosaminoglycan, diethylenetriaminepentaacetic acid, nitrilotriacetic acid mono-, bis-, or tris-substituted 2,2′-bipyridine, a hydroxamate derivative (e.g. acetohydroxamic acid), an amino acid, deferoxamine, ferrioxamine, iron basic porphine, porphyrin and derivatives thereof, DOTA-lysine, a texaphyrin, a sapphyrin, a polyaminocarboxylic acid, an α-hydroxycarboxylic acid, a polyethylenecarbamate, picolinic acid, 4-pyridoxic acid, 3-hydroxy-2-pyridinemaltol, ethyl maltol, Ustilago ferrichrome, nicotinic acid-N-oxide, IRC011, 2-hydroxypyridine-N-oxide, 3-hydroxy-4-pyrone, 3-hydroxypypyrid-2-one, 3-hydroxypyrid-2-one, 3-hydroxypyrid-4-one, 1-hydroxypyrid-2-one, 1,2-dimethyl-3-hydroxypyrid-4-one, 1-methyl-3-hydroxypyrid-2-one, 3-hydroxy-2(1H)-pyridinone, pyridoxal isonicotinyl hydrazone, nicotinic acid-N-oxide, and 2-hydroxy-nicotinic acid.  
     
     
         8 . The cell culture medium of  claim 1 , wherein said eukaryotic cell is selected from the group consisting of a 293 cell, a PER-C6 cell, a CHO cell, a COS cell, a BHK cell, a HeLa cell, and a Sp2/0 cell.  
     
     
         9 . The cell culture medium of  claim 1 , wherein said medium substantially lacks or does not contain transferrin.  
     
     
         10 . The cell culture medium of  claim 1 , wherein said medium substantially lacks or does not contain insulin.  
     
     
         11 . The cell culture medium of  claim 1 , wherein said medium substantially lacks or does not contain any polyanionic compounds.  
     
     
         12 . The cell culture medium of  claim 1 , wherein said medium substantially lacks or does not contain dextran sulfate.  
     
     
         13 . The medium of  claim 1 , wherein a cationic lipid is admixed to said medium for said introduction.  
     
     
         14 . A method of making a medium comprising admixing water and at least one ingredient selected from the group consisting of an amino acid, a sugar, a fatty acid, a vitamin, a pH buffer, a surfactant, a trace metal or salt or hydrate thereof, an amine compound, a growth factor, an agent to control osmolarity and/or ionic strength and/or maintain membrane potential, a flavin, a compound that participates in or is a product of the glycolytic pathway, an alcoholamine, a cyclic alcohol, a phospholipid or portion thereof, a salt of selenious acid, a divalent or trivalent cation, a pteridine derivative, a valeric acid, and a coenzyme, 
 wherein said medium supports introduction of one or more macromolecules into at least one eukaryotic cell in culture and supports cultivation of said at least one cell subsequent to said introduction,    wherein it is not necessary to remove medium used during said introduction from the presence of said at least one cell to support growth of said at least one cell, and/or    wherein after said introduction growth is accomplished in cultivation in a volume of medium that is about the same volume up to no more than about 10 times the volume of the medium in which said introduction occurred.    
     
     
         15 . A method of cultivating eukaryotic cells comprising: 
 (a) contacting said cells with the cell culture medium of  claim 1;  and    (b) maintaining said cells under conditions suitable to support cultivation of said cells in culture.    
     
     
         16 . A method for introducing one or more macromolecules into at least one eukaryotic cell in culture, said method comprising: 
 (a) culturing at least one eukaryotic cell in said medium of  claim 1  in culture;    (b) introducing at least one macromolecule into said culture under conditions sufficient to cause one or more of said at least one macromolecule to be introduced in said at least one cell; and    (c) cultivating said at least one cell in said medium to produce a product whose production is controlled by said at least one molecule,    wherein growth of said at least one cell continues in said medium in the absence of said medium being with fresh medium, wherein it is not necessary to remove medium used during said introducing from the presence of said at least one cell to support growth of said at least one cell, and/or    wherein after said introducing growth is accomplished in cultivation in a volume of medium that is about the same volume up to no more than about 10 times the volume of the medium in which said introducing occurred.    
     
     
         17 . The method of  claim 16 , wherein said medium is not during said production for at least twelve hours subsequent to said introducing.  
     
     
         18 . The method of  claim 16 , wherein said medium is not during said production for at least twenty-four hours subsequent to said introducing.  
     
     
         19 . A kit for the cultivation and transfection of cells in vitro, said kit comprising the cell culture medium of  claim 1 .  
     
     
         20 . A composition comprising the cell culture medium of  claim 1  and at least one component selected from the group consisting of at least one eukaryotic cell, one or more agents for the introduction of at least one nucleic acid molecule into at least one cell, and one or more nucleic acid molecules.