Fukoku Co. Ltd 1 article published in JoVE Bioengineering An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates Ikki Horiguchi1,2, Ikumi Suzuki3, Takashi Morimura3, Yasuyuki Sakai1,4 1Department of Chemical System Engineering, University of Tokyo, 2Department of Biotechnology, Osaka University, 3Biotech Business Unit, Fukoku Co. Ltd, 4Institute of Industrial Science, University of Tokyo Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.