The therapeutic promise of stem cells is becoming a reality. Because stem cells are a precious reagent, we want to take the best possible care of them – not only to preserve them, but also to keep their responses healthy and nonvariable, supporting consistent experimental results. But with so many types of stem cells, and many kinds of media even for one type of stem cell, it can be overwhelming to figure out which media you require.
Key considerations for selecting the appropriate stem cell media include your intended application with the stem cells, the media components your cells require, and the reliability and consistent quality of the media. “In most cases, media are designed based on the origin of the stem cell: for example, embryonic, neural, or hematopoietic,” says Joy Aho, manager of stem cell department at R&D Systems. “When choosing media, it is important that it is able to allow expansion of your cells in their stem cell state without compromising their ability to differentiate. If you have multiple media that are able to do this, then you can look at ease-of-use and level of definition in the media.” Here are some important points to ponder as you peruse the current stem cell media offerings.
The basics: defined and serum-free stem cell media
For the most reliable media with no surprises, your best bet is defined media. Usually serum-free, defined media contain recombinant (rather than purified) ingredients, so you know exactly what’s going into the brew. Defined media have the least amount of variation, unlike serum-containing media that may have significant lot-to-lot variation. This results in fewer variables and perhaps more consistent experimental results.
That being said, there aren’t a lot of generalities to make about stem cell media – these are special cells with particular, and peculiarly special, needs. As such, there isn’t one type of medium that is ideal for, say, expansion, while another type is better for cloning. Instead, if any generalities can be made, it’s better to look at what media work best for what cell type, and go from there. “It's not often that a medium that works for one application with one particular cell type will work for another application or cell type,” says Matthew Singer, manager of scientific development at Stemgent. “The simplest general answer to choosing a stem cell culture medium is whether the medium maintains your stem cells with regards to, for example, identity, function, and proliferation. While there are no known specific differences in the culture requirements of iPS versus ES cells (of the same species), different ingredients key to different cell types will depend upon the specific requirements (such as cytokines or growth factors) of each particular cell type.”
Stemgent offers stem cell media for three specific applications and cell types. Their Pluriton medium is designed to increase the generation human iPSCs. Their NutriStem XF/FF is a xeno-free and serum-free medium for hPSC culture. “NutriStem is unique because it contains very low levels of essential growth factors (only a few nanograms per milliliter),” says Singer, “much closer to true physiological levels than those found in other available media.” In addition, Stemgent’s WIT medium allows the researcher to maintain primary human mammary epithelial cells without loss of identity or the ability to proliferate for many passages.
Life Technologies’ Essential 8™ Medium (originally developed in the laboratory of James Thomson, a professor at the University of Wisconsin), contains only the eight components most needed to maintain pluripotent stem cells. Life released this year their Vitronectin (VTN-N) substrate, which together with Essential 8™ Medium make an effective feeder-free culture system for pluripotent stem cells. “The majority of feeder-free pluripotent stem cell culture is performed using a standard basement membrane extract (BME) which is undefined and variable,” says David Welch, senior marketing development manager for Life Technologies. “Vitronectin (VTN-N), designed in the Thomson lab for use with Essential 8™ Medium, has been shown to support human pluripotent stem cell attachment and survival better than wild-type vitronectin.” If you do choose to use a feeder-cell layer, however, Singer recommends their KnockOut™ Serum Replacement, saying that it results in less variability than the usual fetal bovine serum.
One of the most important tasks a stem cell lab can do is maintain cells in a kind of holding pattern, while retaining their pluripotent nature. “Since the primary focus of stem cell research is the derivation, propagation and manipulation of the ‘ground state,’ our existing inhibitor-based products and some of our pipeline products are specifically targeted to those applications,” says Stewart Craig, senior VP for development & operations at StemCells. StemCells offers media, such as their SC Proven iSTEM®, GS1-R®, and GS2-M® products, that contain small molecule inhibitors whose purpose is to keep pluripotent stem cells naïve, in a ground state. Yet these cells retain an innate ability to renew themselves. “This is in contrast to other media that are only applicable to the more “primed,” post-implantation state,” says Craig, “in which the pluripotent stem cell is dependent upon extrinsic instruction from inductive, multifactorial agents for self-replication.”
StemCell Technologies just released StemSpan™ ACF, a new medium for expansion of hematopoietic stem cells. StemSpan ACF is a chemically-defined complete medium that is free of all animal-derived components, with only recombinant and synthetic factors. Among other applications, it supports CD34+ expansion, and monocyte to dendritic cell differentiation. Their other StemSpan media include StemSpan™ SFEM, StemSpan H3000, and StemSpan™ SFEM.
Specialized stem cell media
So at what point should you change from your defined, maintenance media to a specialized media? Being experimentally conservative is a common trait among scientists, who want to know the effect of each and every change. And when it comes to culturing stem cells, most say that if there isn’t a good reason to change the media conditions, then don’t rock the boat. “When working with stem cells, we recommend making as few changes to the culture environment as possible,” says Welch. “For example, PSCs can be expanded in Essential 8™ Medium and cryopreserved in Essential 8™ medium plus DMSO. You can then thaw directly into Essential 8™ Medium and continue with expansion. If the use of DMSO is not a concern, there is really no need to change to a different product for cryopreservation.”
On the other hand, it depends on what you are asking of your cells. If you need them to do something as extraordinary as replicating themselves, then you will probably have to give them a little something extra – or more often, a combination of factors that support such a physiological feat. “It is always preferable to use media optimized for a certain application (such as cells expanding or differentiating),” says Veit Bergendahl, head of research and development cell culture technology at Miltenyi Biotec. “It is impossible to say that one particular ingredient over another is important to have, or not have, in a given medium. It is the combination of ingredients, their concentrations, osmolarity, and pH that determines the overall performance of the media for a particular cell type or application.” Miltenyi Biotech uses cell-based screening, including flow cytometry and high-content analysis, to evaluate performance when developing new specialized media. “[This helps] to enhance our options for improving media performance, and to avoid problematic ingredients (such as serum-derived animal components and non-specific agents),” says Bergendahl.
Life Technologies offers specialized media for the expansion and differentiation of hematopoietic, neural, and mesenchymal stem cell types, sometimes together with particular substrates as well. “The choice of media for differentiation depends upon the target lineage or cell type,” says Welch. “We provide three kits, the StemPro® Adipogenesis, Chondrogenesis, and Osteogenesis Differentiation Kits, for differentiation of mesenchymal stem cells to bone, cartilage, and fat cells, respectively.”
Stem cell media for specific applications
When considering the varieties of culture media available, plan ahead so that you can try to predict what your cells’ downstream requirements might be. “The goals of a scientist’s experiments with stem cells dictate their choice of stem cell media,” says Welch. “A scientist modeling a developmental pathway or a specific disease with stem cells may not need the same media system as a scientist moving their research towards the clinic.”
What you plan to do with the stem cells greatly affects the best choice of media. For general culture media, you’ll need to decide how defined it must be. “For freezing, the level of definition is important (for example, requiring serum-free), but the level of recovery after thaw will be the ultimate read out of a good freezing media,” says Aho. “Freezing media requires cryoprotectants such as DMSO to allow cells to survive in liquid nitrogen, and growth media for pluripotent stem cells generally contain some level of FGFbasic, an important growth factor for these cells.”
If you are planning to differentiate your stem cells downstream, make sure the media don’t interfere with future differentiation. “On a chemical level, the ingredients are going to be optimized to affect whatever pathways are important in each cell type at each stage of culture,” says Aho. R&D Systems offers new serum-free mesenchymal stem cell expansion media StemXVivo™ which performs well in the fold expansion of cells in culture, and the expression levels of mesenchymal stem cell markers. The BD Mosaic™ Mesenchymal Stem Cell Serum-Free media from BD Biosciences is a defined expansion media for human mesenchymal stem cells that can be used for stem cells destined for either research or stem cell therapy.
Conclusion
There is no doubt that detailed research into the mysterious lives of stem cells is inextricably linked with more advanced media in which to culture them. “Forward progress in stem cell culture requires expert knowledge of the biological signaling pathways that control self-renewal, differentiation and function,” says Craig. “Elucidation of key signaling and control pathways and molecules will be critical in defining and refining the next generation of cell culture reagents.”