Basic pluripotent stem cell culture protocols

Stem cell research is a rapidly expanding field with the potential to develop therapeutic agents to treat diseases as well as study disease development from early stages. The culture of human pluripotent stem cells shares many of the same protocols as standard mammalian cell culture. However, the successful culture and maintenance of human pluripotent stem cells (hPSCs) in an undifferentiated state requires additional considerations to ensure that cells maintain their key characteristics of self-renewal and pluripotency. There are several basic techniques needed for the culturing of mammalian cells, including thawing frozen stocks, plating cells in culture vessels, changing media, passaging and cryopreservation. The protocols in this document represent a subset of the standard operating procedures used to maintain and culture stem cells at the Massachusetts Human Stem Cell Bank, and have been thoroughly testing and verified. A Stem cell culture considerations Stem cell research is a rapidly expanding field with the potential to develop therapeutic agents to treat diseases as well as study disease development from early stages. However, to fulfill this promise, researchers need to have access to standardized protocols for the development, maintenance and differentiation of these unique cells. Such “best practices” will allow comparisons of different studies and hasten the refinement of these techniques. Such standardization can be driven by resources such as StemBook and by stem cell banks. In addition to standardization of these best practices, stem cell banks also serve as valuable resources of properly identified, quality controlled and characterized cell lines as well as helping to navigate the legal and IP issues that are common to working with stem cells. The protocols in this document represent a subset of the standard operating procedures used to maintain and culture stem cells at the Massachusetts Human Stem Cell Bank, and have been thoroughly tested and verified. A1 Successful stem cell culture The culture of human pluripotent stem cells shares many of the same protocols as standard mammalian cell culture. However, the successful culture and maintenance of human pluripotent stem cells (hPSCs) in an undifferentiated state requires additional considerations to ensure that cells maintain their key characteristics of self-renewal and pluripotency. Successful hPSC culture requires the recreation of the in vivo stem cell microenvironment, or “niche”, which includes growth factors, cell-to-cell interactions and cell to matrix adhesions. Unlike many cell types, hPSCs are Last revised March 28, 2012. Published June 10, 2012. This chapter should be cited as: Borowski M., Giovino-Doherty M., Ji L., Shi M.-J., Smith K.P., and Laning J. Basic pluripotent stem cell culture protocols (June 10, 2012), StemBook, ed. The Stem Cell Research Community, StemBook, doi/10.3824/stembook.1.63.1, http://www.stembook.org. Copyright: C © 2012 Maria Borowski, Maria Giovino-Doherty, Lan Ji, Meng-Jiao Shi, Kelly P. Smith and Joseph Laning. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ∗To whom correspondence should be addressed. E-mail: maria.borowski@umassmed.edu