title: Large-scale transfection of mammalian cells.
abstract: The large-scale transfection of mammalian cells allows moderate (milligram to gram) amounts of recombinant proteins (r-proteins) to be obtained for fundamental or clinical research. In this article, we describe a one-liter transfection using polyethyleneimine (PEI) for DNA delivery into human embryonic kidney (HEK-293) cells cultivated in serum-free suspension to produce a recombinant human monoclonal antibody that yields up to about 1 g/L in a 10-day process. The method is based on a DNA delivery step performed at high cell density (20×10(6) cells/mL) by direct addition of DNA and PEI to the culture. Subsequently, the cells are diluted 20-fold for the 10-day production phase in the presence of valproic acid (VPA), a histone deacetylase inhibitor. The methods for plasmid purification, antibody quantification by enzyme-linked immunosorbent assay (ELISA), and affinity purification with protein A are also described.
authors: Laboratory of Cellular Biotechnology, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland. lucia.baldi@epfl.ch
authors: Lucia
authors: L
authors: Baldi
authors: David L
authors: DL
authors: Hacker
authors: Carine
authors: C
authors: Meerschman
authors: Florian M
authors: FM
authors: Wurm
mesh_terms: D000818:Animals; D000906:Antibodies / Q000302:isolation & purification / Q000378:metabolism; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D057809:HEK293 Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification; D011094:Polyethyleneimine / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013205:Staphylococcal Protein A / Q000096:biosynthesis / Q000302:isolation & purification; D014162:Transfection / Q000379:methods*
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D011094:Polyethyleneimine; D004247:DNA