Chapter 3 - Culture of Human Pluripotent Stem Cells in Feeder-Free Conditions

Abstract

There are multiple methods for culturing hPSCs under feeder-free conditions. Here we describe a system using StemPro medium and Geltrex or Matrigel substrata with Accutase passaging. In our hands hPSCs cultured this way remain pluripotent and karyotypically normal for more than 40 passages. The advantage of Accutase passaging is that hPSC cultures are dissociated into single cell suspensions; this allows scale-up and applications such as transfection, FACS sorting, and cloning. Here we explain the appropriate hPSC feeder-free culture technique, recommend cell seeding densities, and show examples of colonies with good and bad morphology. Alternative feeder-free culture methods are discussed, such as alternative media, matrices, and passaging methods. We also provide a protocol for adapting hPSC cultures from feeder layers to feeder-free conditions, which can sometimes be a roadblock for many laboratories. Finally, we describe a method for the successful cryo-preservation of hPSCs cultured under feeder-free conditions.