Orbital shaker technology for the cultivation of mammalian cells in suspension

Abstract For large‐scale applications in biotechnology, cultivation of mammalian cells in suspension is an essential prerequisite. Typically, suspension cultures are grown in glass spinner flasks filled to less than 50% of the nominal volume. We propose a superior system for suspension cultures of mammalian cells based on orbital shaker technology. We found that “square‐shaped” bottles (square bottles) provide an inexpensive but efficient means to grow HEK‐293 EBNA and CHO‐DG44 cells to high density. Cultures in agitated 1‐L square bottles exceeded the performance of cultures in spinner flasks, reaching densities up to 7 × 10 6 cells/mL for HEK‐293 EBNA cells and 5 × 10 6 cells/mL for CHO‐DG44 cells in comparison to (2.5–4) × 10 6 cells/mL for cultures of the same cells grown in spinner flasks. For 1‐L square bottles, optimal cell growth and viability were observed with a filling volume of 30–40% of the nominal volume and an agitation speed of 130 rpm at a rotational diameter of 2.5 cm. Transient reporter gene expression following gene delivery by calcium phosphate–DNA co‐precipitation was the same or slightly better for HEK‐293 EBNA cells grown in square bottles as compared to spinner flasks. Reductions in cost, simplified handling, and better performance in cell growth and viability make the agitated square bottle a new and very promising tool for the cultivation of mammalian cells in suspension. © 2004 Wiley Periodicals, Inc.