Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Cell lines have been a staple of medical research at least since Henrietta Lacks’ ovarian tumor became HeLa cells more than half a century ago. Today, culturing continuous, immortalized cells is relatively routine—just take a bottle of medium off the shelf, throw in some serum (if it’s not already in there), add it to a flask and keep it cozy in a CO2 incubator.
Primary cells and stem cells, on the other hand, are fussier. They have a limited life span, need a special blend of factors to grow and are susceptible to other factors that can take them down an undesired differentiation pathway. Here we look at some of the media researchers use to keep such finicky cells happy.
The definition of media
Over the years, many “classical” media have been formulated—such as Minimal Eagle’s Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM) and Roswell Park Memorial Institute medium (RPMI)—which vary from each other in the identity and quantity of salts and nutrients. Growth factors and other requirements typically are supplied in the form of 10% to 20% fetal calf serum (FCS), aka fetal bovine serum (FBS), enabling a variety of cell lines to thrive. “Serum is complex and undefined, as such it is covers a lot of ‘sins’ of media, such as limited nutrients” notes Kevin Grady, product line business manager for the American Type Culture Collection (ATCC).
Because serum typically comes from pools of animals fed a nonstandardized diet, exposed to different environmental conditions and sacrificed at different times of year, each lot is likely to be different in some subtle or not so subtle ways. To avoid the associated variability in their experiments, researchers screen serum from several vendors, and even different lots from the same vendor, and stock up on a single lot.
There are further challenges in culturing primary cells—“those have very specific nutritive requirements, and you generally can’t just use a classical media with serum supplementation. You have to add a lot of other factors to get primary cells to grow,” Grady explains. Serum itself is liable to both sins of omission and sins of commission: Besides lacking the necessary factors to sustain growth, there may be “an unknown component in your serum that will cause the primary cells to differentiate, for example.”
And so “with the advent of primary-cell culture, people started to try and move away from serum,” Grady notes. “Most primary-cell media are going to be serum-free or at the very least contain very low serum.”
In lieu of purchasing ready-made media, some researchers opt for serum replacements that may be directly substituted for FBS in traditional recipes. These may contain discrete proteins, such as bovine serum albumin, or they may be protein-free but have animal, plant or yeast hydrolysates. Or they may be “chemically defined and animal-component free,” like Essential Pharmaceutical’s Cell-Ess, the company’s product director D. G. Ferneyhough says. These latter “are the same, lot after lot, year after year, which means an immense level of standardization compared to using serum.”
No size fits all
Different cells require different components. Growth factors used to maintain one cell type may be a differentiating factor for another, for example, and a balance of components is needed to maintain the cells close to the physiological state from which they were isolated, says Theresa D’Souza, cell biology R&D section manager for Lonza’s primary-cell portfolio. “So we start with a classic media recipe and tailor it and add supplements, which vary depending on cell type. Some may require transferrin and a little more glucose and fatty acids. We tinker around in the concentrations of the different amino acids. We do a lot of DOE [design of experiment] to help build the formulation of the media for that particular cell type.” For example, endothelial cells require some growth factors that epithelial cells don’t; fibroblasts tend to take over cultures, so components may be added that discourage their growth; and keratinocyte media should be low in calcium to keep them from differentiating.
Although one type of media may not be appropriate for many different cell types, often the basal formulation is a common starting point. Thus vendors such as Lonza market their endothelial growth medium, for example, as a bottle of Endothelial Basal Medium-2 plus the EGMTM-2 SingleQuots™ Kit which is composed of separate vials of “growth factors, cytokines, and supplements.” The combined package of both, is offered as the EGMTM-2 BulletKitTM. So customers have the flexibility to control the concentration of supplement, depending on their experiments, Dsouza says.
Other vendors help researchers reduce shipping and storage costs and eliminate the inconvenience of needing separate kits by offering ready-to-use media formulations. “Our goal is to use media, preferably as 1X,” says Mohan Vemuri, R&D leader, cell biology, at Thermo Fisher Scientific. “If it has a supplement, it would need different shipping conditions and storage specifications—whereas with 1x media, we know for sure, it is a simplified one and the shelf life is at least one year.”
Stepping back, moving forward
Sometimes rather than making sure mesenchymal stromal cells remain as mesenchymal stromal cells, say, researchers want them to turn into something different. Some media, such as STEMCELL Technologies’ TeSR™-E7™ Medium for Reprogramming, are formulated to support the reprogramming of more differentiated cells back to create an embryonic stem cell-like cell (called an induced pluripotent stem cell, or iPSC) when used with reprogramming factors.
Media such as Gibco Essential 8™ from Thermo Fisher Scientific are designed to allow for the maintenance and expansion of embryonic stem cells (ESCs) and iPSCs in culture with minimal differentiation. Using this medium, “one can grow these cells for an indefinite period, but also can create master cell banks,” says Vemuri. “And from this master bank, one can differentiate the cells to different lineages of endoderm, mesoderm and ectoderm, to make any type of tissue as seen in the human body.”
Not surprisingly, there are a host of media available to support the differentiation of ESCs and iPSCs, such as the new Definitive Endoderm Differntiation Media by Gibco from Thermo Fisher Scientific: “One can drive the cells toward, say, a hepatic or pancreatic or thymic, lung or intestine lineage—in order to get to this kind of endodermal lineage first, one should get a starter population of definitive endoderm cells,” Vemuri says. “Our media enables that kind of definitive endoderm [cell] to be generated from iPSCs within three days.”
There are also a host of media to support adult stem cells’ journey down various lineages—for example, Lonza offers media BulletKits that allow for the differentiation of adipocytes, chondrocytes or osteocytes from mesenchymal stem cells.
Another dimension
There is a big push in cell culture toward three dimensions, in which cells are grown on scaffolds or in hollow fibers, or in co-cultures with multiple cell types. If you have the right medium formulation, “you can actually get endothelial cells to form tubes with the smooth muscle cells aligning themselves on the outside of those structures and the fibroblasts growing between those structures,” says Grady. “So you sort of get a 3D model of what’s going on in the body.” These types of co-culture require the development of new media that can support all the cells simultaneously.
Even getting a two-dimensional culture to stick to a plate requires certain adhesion factors—like laminin, fibronectin or vitronectin—that are found in serum and must be added to serum-free media. (In the absence of added adhesion factors, plating the cells at higher density may do the trick, Ferneyhough says.)
The proliferation of cell-culture media—especially since the advent of primary and stem cell culture—is a reflection of increased research efforts on the part of vendors and academics alike to ascertain which components (and what concentrations of them) can best derive, maintain, re-program and differentiate the cornucopia of cell types being studied and manipulated in the laboratory. Chances are, at least one of these will work for the cells and your research needs.
Image: Shutterstock