This chapter describes a detailed protocol on human pluripotent stem cells (hPSCs) cultivation as matrix-free cell-only aggregates in defined and xeno-free culture medium in stirred tank bioreactors (STBRs). Starting with a frozen stock pre-expanded on conventional culture dishes (2D), the ultimate process is performed in 150 mL culture scale in stirred tank bioreactors (3D) and is designed to produce up to 500 million pluripotent hPSC within 7 days. The culture strategy includes perfusion-based cell feeding facilitating process control, automation, and higher cell yields. Ultimately, this detailed protocol describes an important step for generating a defined starting cell population for directed lineage differentiation and subsequently fueling human cell-based assays and regenerative medicine approaches.
Keywords: Automation; Human pluripotent stem cells (hPSCs); Perfusion feeding; Process development; Stirred tank bioreactors (STBRs); Suspension culture.