Single-use systems have become an important tool in the development and scale up of biotechnology processes. Single-use systems save space, increase flexibility in scale and space planning and, to a large extent, eliminate cleaning costs in development and change over. However, single-use systems come with their own challenges, including complete integration, scalability, and most of all with extractables and leachables. A comprehensive technical report has been published to guide potential new users through the process of implementing single-use technologies throughout their bioprocesses.[1]  Upstream processing has most fully embraced single-use technologies. From the thaw of the vial to the harvest of the bioreactor, it is possible to construct a fully disposable process stream for a cell culture process. However, limitations in heat and oxygen transfer have excluded bacterial and yeast processes. In this article, we review the available technologies and advances that have been made. The first steps of a cell culture biotechnology process are typically the thaw of a seed bank vial and the expansion of the cells in a series of shake flasks. In principle, these steps have alway...