Endowed with the powers of self-renewal and multipotency or pluripotency, stem cells will probably never be simple to culture, and with good reason. These are enigmatic cells with complex requirements meant to preserve a powerful characteristic: the ability to become an entirely different type of cell in response to signaling cues from their environment. This article offers expert advice for optimizing the growth and maintenance of stem cells.
Maintaining stem cell pluripotency
Because stem cells are sensitive to their environment, protocols need to take factors such as media, atmosphere, substrate, and cellular interactions into consideration. Horizon Discovery created a defined cell culture workflow for quality and consistency in cultures. “Our stem cells are grown in fully defined growth media and extracellular matrices, with cells being visually inspected daily for any signs of spontaneous differentiation, which, if encountered, is removed using aspiration,” says Yasmin Paterson, Senior Scientist at Horizon Discovery, a Revvity company. “We also conduct routine quality checks, including but not limited to the assessment of genomic integrity through screening of recurrent chromosomal abnormalities, assessment of differentiation potential such as through tri-lineage differentiation in pluripotent stem cells, as well as gene and cell surface marker assessment.”
Paterson notes that it’s important to maintain a balance between stem cell self-renewal and differentiation. “The stability of this stem state is essential not only to ensure the maintenance of phenotypically well-defined, karyotypically stable cells, but also to ensure that pluripotency or multipotency is maintained for downstream applications,” she says. “As such, careful control of the cellular environment, as well as continuous monitoring and refinement of stem cell culture protocols using the latest technological developments within the field, are vital.”
Other cues can also provide evidence of healthy stem cell cultures. “Researchers should look for a high nucleus-to-cytoplasm ratio and high expression of pluripotency markers,” says Omar Farah, Scientist in Field Applications at Thermo Fisher Scientific. He recommends looking for tight colonies and refractive edges in monolayer stem cell cultures, and in suspension-based cultures “you should look for round, uniform spheroids no more than 400 um in size to avoid a necrotic core or hypoxic conditions.”
Different culture approaches
Stem cells—including the commonly used adult stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs)—retain their self-renewing ability using distinct signaling mechanisms, which scientists attempt to recreate in vitro with precise culture conditions. The combination of media, growth substrate, atmosphere, and cellular interactions together form a complex signaling network experienced by the stem cells. “When attempting to culture one type of stem cell versus another, we have to consider the factors needed to maintain the transcriptional and proteomic phenotype of that particular cell type,” says Farah. He notes, for example, that pluripotent stem cells require essential factors to maintain pluripotency in a chemically defined medium, while more differentiated mesenchymal stem cells require specific factors and matrices to be properly maintained in vitro.
In another example, embryonic stem cells grow in culture media that usually contains basic fibroblast growth factor, a component that other stem cell types don’t often require. “Each stem cell type requires its own cocktail of cytokines and growth factors,” adds Paterson. “Some require altered cell culture coatings or extracellular matrices to support growth, three-dimensionality, differing topographical cues, or alterations in atmospheric pressure in order to maintain potency status.”
Different culture approaches can also alter cells, such as growing cells in 2D compared to 3D conditions. When MilliporeSigma’s ReNcell® human neural stem cells are grown in 3D cultures (but not 2D monolayers) while expressing Alzheimer’s-related genes, they can form an in vitro model system for Alzheimer’s disease, including observed brain pathology such as amyloid-beta plaques and filamentous tau proteins. MilliporeSigma offers products and protocols to support this “Alzheimer’s In A Dish™” model system.
Farah also notes that when culturing PSCs as 3-dimensional spheroids, “it’s important to consider how well the media composition and the protocols associated with keeping cells as spheroids can support PSC spheroid aggregation and expansion while maintaining the pluripotency of cells.” Thermo Fisher Scientific offers a range of media to support stem cell culture, including Thermo Fisher’s Essential 8 Medium, StemFlex Medium, StemScale PSC Suspension Medium, and a host of Cell Therapy System (CTS) products designed for clinical cell therapy applications.
Media and supplements
Using the appropriate media and supplements is crucial to successfully culturing healthy stem cells. “Specialized culture media, supplements, and tissue culture plate coatings are required to support not only cell maintenance and expansion but also downstream differentiation and functional assays,” says Paterson, who recommends media suppliers as a knowledgeable source of information on selecting the best culture media and supplements.
Choosing media often depends upon cell type. Nick Asbrock, Global Product Manager at the Life Science Business of Merck KGaA in Germany (operates as MilliporeSigma in the U.S. and Canada), recommends reagents that are recombinant, pre-qualified, and animal-free to prevent contamination. “Each stem cell type requires a unique set of both media and environmental components that must be consistent from lot-to-lot,” he says.
MilliporeSigma offers pre-qualified media and reagents to ensure proper stem cell expansion or differentiation. They also optimized formation of organoids from iPSCs from colon and lung, and sell these along with organoid tools. “We offer both cryopreserved organoids/progenitors and serum-free expansion/differentiation media used to differentiate into both colon and lung organoids,” says Asbrock.
Other new tools are helping researchers to culture stem cells that have previously proven notoriously difficult. “Historically human iPSCs have been grown in feeder-free conditions on mouse tumor derived basement membrane extract, which is cumbersome to use,” says Asbrock. MilliporeSigma’s ECMatrix™-511 E8 Laminin Substrates, a recombinant laminin protein fragment, enhances iPSC attachment and growth without laborious precoating of plasticware. “This product has been very useful for bioprocess customers who are expanding a large quantity of iPSCs for potential future cell therapies because of the added ease-of-use and time savings,” he says.
Developing stem cell differentiation protocols remains challenging, in part because we don’t fully understand the important signaling events that occur prior to and during differentiation. “There is still a need to learn more about generating many iPSC-derived cell types that properly resemble fully differentiated somatic cell types,” says Farah. Today’s tools are helping researchers understand more about the nature of stem cell culture and differentiation, and to develop better tools for further study.