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 x 10(6) cells/mL for HEK-293 EBNA cells and 5 x 10(6) cells/mL for CHO-DG44 cells in comparison to (2.5-4) x 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.

Production of lentiviral vectors by large‐scale transient transfection of suspension cultures and affinity chromatography purification

“…This may be due to the fact that transient transfection is still commonly regarded as a nonscaleable technology. However, it should be noted that in recent years transient transfection of suspension-growing cells has emerged as a powerful technology for the large-scale production of biopharmaceuticals (Durocher et al, 2002;Geisse and Henke 2005;Muller et al, 2005;Pham et al, 2003Pham et al, , 2005Rosser et al, 2005;Sun et al, 2006). Additionally, this technology has recently shown great potential for the large-scale manufacturing of adeno-associated viral vectors (Park et al, 2006;Reed et al, 2006).…”

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection

“…More recently proto-oncogenes, cell cycle control genes, growth factor genes and anti-apoptotic genes have been used to improve production hosts or transient expression (Wurm 2004;Backliwal et al 2008). Finally alternative cultivation methods for suspension cells are applied such as orbital shaker technology working under agitation within an incubator (Muller et al 2005) which allow cell densities up to 5 9 10 6 cells mL -1 while our cells reached densities of 1-2 9 10 6 cells mL -1 only.…”

Suspension-adapted Chinese hamster ovary-derived cells expressing green fluorescent protein as a screening tool for biomaterials

“…Suspension-adapted Chinese hamster ovary cells (CHO DG44) were cultivated in ProCHO5 medium (Lonza Verviers SPRL) as previously described (De Jesus et al 2003;Muller et al 2005). …”