February 15, 2015 (Vol. 35, No. 4)

Balance Differentiation and Proliferation to Make Stem Cell Culture Commercially Routine

“Stem cells always balance between self-renewal and differentiation”—so began a notable review of stem cell culture optimization. The review, which appeared in the Journal of Cellular Biochemistry in 2010, proffered many observations that remain timely. For example, it noted that “stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments.”

By the time the review appeared, it was already recognized that cell culture conditions not only support cell growth but are also “instructive.” In addition, experts were already replacing ill-defined ingredients such as serum and feeder cells with cytokines and growth factors, and reducing oxygen concentrations inside incubators to levels that exist normally in tissues.

Accordingly, the review’s authors, scientists at the Grenoble Institute for Neurosciences led by Didier Wion, Ph.D., paid due attention to the future of stem cell culture. These scientists emphasized that “research in many different fields including stem cell biology, nanotechnology, and bioengineering” indicated that culturing cells in Petri dish or flasks would “soon be as outdated as flying across the Atlantic Ocean in Lindbergh’s plane.”

That was five years ago. Today, how close are we to “soon,” which we might characterize as the jet age of stem cell culture?

Upstream, Downstream Quality Issues

The goals of any cell culture are to achieve appropriate growth levels and to maintain viability and utility. The ability of pluripotent stem cells to differentiate throws a wrench in the turbine blades, however.

“While they’re growing you don’t want them to differentiate,” says Uma Lakshmipathy, Ph.D., director and principal scientist for cell biology at Thermo Fisher Scientific. “You want them to remain in their pristine, undifferentiated state.” This concern adds a layer of complexity not found with common cell or microbial cultures. “You have to provide the right microenvironment.”

In that regard, adult stem cells are somewhat easier to handle than embryonic or induced pluripotent stem cells. The latter do not grow as single cell monolayers on coated tissue culture plastic, but as clusters within a matrix to which they adhere.

Minimizing the likelihood of differentiation for pluripotent stem cells requires a good deal of hands-on activity, for example, daily media changes to remove metabolites and replenish crucial growth factors and small molecule reagents that inhibit differentiation. “If you’re working with pluripotent stem cells,” notes Dr. Lakshmipathy, “you’re working seven days a week.”

Laboratories can minimize human involvement to some extent, but implementing, say, automation requires the utmost care. Automated media and nutrient changes are emerging but are not yet mainstream.

“That would be wonderful,” remarks Dr. Lakshmipathy, “but you must validate any new system you introduce, to assure it does not alter the cells’ genomic stability or, more importantly, that when the time comes for cells to differentiate, they go in the intended direction. You don’t want to affect downstream cell functionality.”

Stem cell culture experts dream of culturing under “ideal” conditions where their cells grow like other cells, in suspension as single cells, but stem cells do not thrive if not in contact with other cells. Labs have traditionally employed a feeder layer of cells to which embryonic stem cells could adhere and grow. Feeder-dependent stem cell culture is becoming somewhat less common with the emergence of products such as Gibco® Essential 8™ media, which allows feeder-free conditions.

Adherence dependence also has implications for counting cells. Cell counting in traditional suspension cultures is relatively easy because cells are free-floating. With stem cells, one can only estimate based on the number of plates and removal of an aliquot. But unless one is comparing the benefits of one media/nutrient system with another, cell number is not that important.

Stem cell scientists must always keep an eye on downstream products, the hepatocytes or neurons that are the ultimate product of the culture. For this reason, absolute growth rate is not always an obviously good or bad thing (as it often is in CHO or Escherichia coli fermentations).

“Just because they’re growing fast doesn’t mean you’ve found the ideal media system,” Dr. Lakshmipathy emphasizes. “If they’re growing faster, you may have to worry about genomic integrity, that cells have gained an extra chromosome that confers a growth advantage. If they’re growing slowly, you may have to worry about differentiation.

“[What is critical] is not growth rate, but [whether the] cells maintain their normal karyotype and remain undifferentiated. Stem cell culture requires a lot more characterization than just revving up cells and making a whole bunch of them.”

Human induced pluripotent stem cell cultured in media containing KnockOut™ Serum Replacement on mouse feeder cells (left panel) or in feeder-free media, that is, in Essential 8™ media on Vitronectin-coated plates (right panel). Pluripotent stem cells with the typical morphology of tight, clustered cells with defined edges are stained positively for the self-renewal marker, Alkaline Phosphatase Live Stain, and feeder cells are stained positively with the fibroblast marker CD44-PE-Cy5® antibody.

How It’s Different

Contrasting stem cell culture with the production of therapeutic proteins through cell culture, Ohad Karnieli, Ph.D., vice president, technology and manufacturing, Pluristem, says that “the main difference is that in stem cell-based cell therapy, the cells are the product.” Noting that “every minor change in the culturing conditions might affect the stem cells,” Dr. Karnieli echos the now seldom-heard bioprocess adage, “the product is the process.”

“Since the cells are the product, achieving high-population doublings without effecting the phenotype is challenging,” he adds. For Dr. Karnieli, optimization of nutrients, media ingredients, and growth factors is the key to successful stem cell cultures. Thus, what has been true for mammalian production cultures comes to pass for stem cells.

“Since you cannot get high stem cell density, the surface-to-volume ratio in adherent cells is critical to efficiency and cost effectiveness,” explains Dr. Karnieli. “Adding growth factors [to increase proliferation] and controlling pH and oxygen levels can be helpful, depending on the cell type used. But in the case of progenitor cells, care must be taken to eliminate differentiation initiated by cytokine combinations or even additional feeder layer cells.”

On the Shoulders of Giants

Emer Clarke, Ph.D., CSO at contract research firm ReachBio, echoes Dr. Lakshmipathy’s position on stem culture proliferation. “If you get overproliferation, you may get differentiation as well.”

ReachBio uses standard techniques and materials, including the protein HoxB4, to maintain expanding stem cell populations in an undifferentiated state. Conditions include a low-oxygen carbon dioxide atmosphere in a humidified incubator at 37°C. “We should be thankful to scientists of the past who worked for so many years defining conditions that enable cells to survive,” Dr. Clarke affirms.

The company specializes in stem cells, progenitor cells, and mature cells of hematopoietic lineages (erythrocytes, lymphocytes, platelets) used in drug development. Unlike most chemotherapy drugs in use today, which kill all rapidly dividing cells, agents currently under development are also toxic but more specific.

It is of great interest in terms of patient safety and dosing to determine which lineages are most affected, and precisely where in the development of stem cells to progenitor cells to mature cells the toxic effects occur. If later on, say on mature cells or even possibly on progenitors, hematopoietic capacity may be sustained throughout treatment.

Drug developers have an obvious interest in learning early if their compounds will completely wipe out the capability to form new blood cells by killing stem cells. “The assays we employ help us to examine cells at different stages of development,” asserts Dr. Clark, who adds that the assays can also suggest how drugs may affect the cells. 

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