Within this article, a detailed protocol for mass production of definitive endoderm (DE) cells from human pluripotent stem cells (hPSCs) in scalable and easy-to-handle suspension culture using a rotating Erlenmeyer flask or a stirred tank bioreactor is described.
This protocol uses two different chemically defined and xeno-free media, and thereby is compatible with good manufacturing practice (GMP), and includes procedures for cryopreservation of obtained cells.
The protocol allows for efficient DE cell production within 3 days and generates DE cells with >92% purity when using the bioreactor.
The hPSC-derived DE cells can be differentiated into various endodermal lineages.
This protocol should facilitate the flexible production of mature DE derivatives for physiologically relevant disease models, high-throughput drug screening, and toxicology testing, as well as cellular therapies.
Production and cryopreservation of definitive endoderm from human pluripotent stem cells under defined and scalable culture conditions
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