Cell culture is one of the more complex yet commonly used techniques in life science research. Its applications extend to everything from basic biological studies and specific pathway elucidation to therapeutic production and clinical treatment. Yet optimizing cell culture to keep cells healthy and growing still is somewhat subjective and lab-dependent.
While mammalian cell culture has progressed with certain best practices that enable successful cell growth, stem cell culture is still a work in progress. As we learn more about these unique cell types and how to keep them undifferentiated to study their behavior or differentiated to apply them to a particular disease, the more success we will have with new investigations and their further application.
Powerful but sensitive tools
For studies trying to mimic health or disease states, stem cell cultures can help pinpoint in vivo biological processes. “Unlike immortalized mammalian cells, stem cells are genetically very similar to their individual donor animal. As stem cells both renew and differentiate in vivo, cultured stem cell populations also have these properties,” explains Helen Hardiman, bioengineering product manager at STEMCELL Technologies.
Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are currently the standard for stem cell research with the ability to renew indefinitely, differentiate to most tissues and terminal cell types in the body, and generate tissue-specific progenitors. For more targeted and potentially personalized investigations, adult stem cells can be derived from tissues in the body for the purpose of reintroducing a patient's own cells for therapies and reduce the risk of rejection by the immune system.
“Since in vitro culture exists outside of the native stem cell niche, which is very difficult to replicate in the lab, maintenance of stem cells in the stem cell state requires the culture conditions to be tightly defined,” adds Hardiman. Stem cells are more sensitive to culture conditions than immortalized mammalian cell lines, so specialized medium optimized for each type of stem cell is required. “For example, mesenchymal stromal cells (MSCs) cannot be cultured in the same culture conditions as hematopoietic stem and progenitor cells (HSPCs), and vice versa,” she adds.
John Bracht, assistant professor at American University, has been working with adipose-derived stem cells (ADSCs) as a model system to investigate adipose differentiation in vitro. While ADSCs can easily be purified from subcutaneous fat and differentiated into chondrocytes, adipocytes, and osteoblasts by adding the appropriate mixture of hormones to the culture media, Bracht and his team found that adipose differentiation always appears incomplete in a subpopulation of the cells. While Bracht took the opportunity to learn more about ADSC differentiation and how the subsequent undifferentiated subpopulation behaves, his work also underscores the unpredictability in stem cell growth and differentiation.
Make it or break it: Challenges in growing stem cells
Standard stem cell culture combines growth factor-enriched media with support cells that provide necessary intercellular interactions and extracellular scaffolding. “The goal is to provide robust and stable stem cell culture conditions in an attempt at recreating the in vivo stem cell microenvironment and to ensure that cells maintain their key characteristics of self-renewal and pluripotency,” explains Anne Sloan, staff scientist at Cell Sciences. Recombinant cytokines and growth factors along with other considerations help ensure cell culture medium homeostasis. For example, oxygen pressure can be adjusted to obtain a lower pressure similar to in vivo environments and encourage consistent cell metabolism. Physical constraints of the stem cell microenvironment also influence stem cell fate, making nanotopography as important as medium formulation in optimizing stem cell culture conditions.
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However, stem cells are very sensitive and exposure to many common reagents used for mammalian cell culture produces toxicity or causes them to differentiate and undergo cellular senescence, adds Daniel Boesch, head of life science reagents and kits at MilliporeSigma. Furthermore, some stem cells such as iPSCs metabolize media additives quickly, requiring daily and weekend feeding. Specialized media options like MilliporeSigma’s PluriSTEM™ Human ES/iPS Cell Medium, a defined small molecule-based serum-free medium, enables feeder-free culture of human ES/iPS cells and allows for media exchanges every other day without compromising the morphology or long-term functionality of human pluripotent stem cells.
“Ensuring that cells are differentiated to the appropriate level of lineage-committed progenitor while maintaining stem cell properties is also a big issue. In fact, research has been ongoing to understand and replicate in culture the developmental signals that cause this lineage commitment,” says Hardiman. Directed differentiation protocols and culture requirements will differ depending on the desired progenitor cells. Optimized, commercially available formulations can be best, such as the STEMdiff™ family from STEMCELL Technologies that provides a range of specialized formulations to help easily and reproducibly generate PSC-derived progenitor cells from a wide variety of PSC cell lines. Other stem cell type-specific media are also available, like STEMCELL’s StemSpan™ Serum-Free Expansion Medium (SFEM) and SFEM II for HSPCs.
In Dr. Bracht’s investigations of ADSC differentiation, for example, he observed a population of cells that retain stem cell-like qualities—the ability to propagate, express two known stem cell markers, and maintain the capacity for trilineage differentiation into chondrocytes, adipocytes, and osteoblasts—after adipose differentiation. However, their gene expression analysis showed an overall expression profile similar to that of adipocytes, essentially defining a differentiation-resistant stem-like multipotent cell population. Further research will include testing the role of naturally occurring signaling molecules like hormones (estrogen, testosterone, cortisol) and other peptide-based signaling molecules. “Our work can help broaden our understanding of cellular multipotency, and ultimately could be particularly relevant to obesity-associated metabolic disorders,” explains Bracht.
Unwanted spontaneous differentiation of cells can also occur when passaging human pluripotent stem cells. Differentiated cells proliferate quickly and eventually diminish the overall quality of the stem cell culture. EZ-LiFT™ Stem Cell Passaging Reagent ,developed by MilliporeSigma, is an enzyme-free and chemically defined stem cell dissociation reagent that selectively passages only undifferentiated pluripotent stem cells. The reagent eliminates the need for manual colony selection or cell scraping, produces high cell viability, and can rescue highly differentiated iPS cell cultures.
Best practices for a healthy culture
“Cell authentication is an important aspect for all applications to ensure that cells have the expected genetic fingerprint, are free of various contaminations including viral, mycoplasma, different species, and are not contaminated with another cell line,” offers Boesch. This includes routine testing of the functional attributes of the cell line(s) to confirm that the cells have not undergone genetic drift or have spontaneously differentiated toward an undesired lineage. MilliporeSigma partners with the European Bank of iPS Cells (EBiSC), established to standardize human iPS cell lines for research, allowing access to nearly 800 patient-derived human iPS cell lines for disease modeling, extending their collaboration with Public Health England as a distributor of its European Collection of Authenticated Cell Cultures portfolio.
Hardiman agrees that the most important consideration is to ensure cell quality. High yields won’t be as useful in downstream applications such as regenerative medicine or tissue engineering if cells have lost potency or have suffered genetic changes. Thus, in addition to using an appropriate, optimized cell culture medium, monitoring the quality of the cultures at frequent time points becomes increasingly significant.
“Having key quality attributes by which to measure the cells, like growth rates, viability, expression of key surface markers, genomic integrity, and/or multi-lineage differentiation capacity, will act as a way to ensure that you know the quality of your cells, before you use them in any downstream assays,” Hardiman comments. For example, morphology is a sensitive indicator of quality for PSCs, including uniform, densely packed colonies with dense centers and defined borders. These types of visual cues often precede changes in marker expression.
The culture medium, extracellular matrix, serum, and growth factors required for growth and differentiation of stem cells can also be a source of variability that could have unintended effects and may impact reproducibility. Stringent quality control of cell culture components is critical to the success of any application. For example, when expanding primitive cord blood HSPCs it is important to balance cell proliferation with maintenance of self-renewal. Serum-free culture conditions, using medium like SFEM or SFEM II, decrease variability in vitro and allow more control over cell growth, while still allowing the flexibility to add cytokines and small molecules of interest.
An additional challenge when using adult stem cells is that some may not retain their stem cell nature over long-term culture—losing their phenotype or the ability to differentiate efficiently after a certain number of passages. Newer 3D culture systems such as organoids can help culture adult stem cells for much longer periods while retaining their stem cell properties. Just like 2D cell cultures, cell culture medium for organoids can have a profound impact on reducing experimental variability.
For intestinal organoids, for example, using an optimized medium such as IntestiCult™ from STEMCELL Technologies helps maintain adult stem cells that can both differentiate and expand efficiently. Dr. Bracht also recommends separating single cells, clonally expanding them, and using those homogenous populations for experiments. The stem cells used in his lab are a rich mixture, with many cells isolated from a single lipoaspirate from a patient. Even though this approach takes a significant number of passages, the lab often found the 'mixed' primary stem cell population to be too heterogeneous and noisy to see specific effects in some cases.
Reducing toxicity—Endotoxin-free growth factors in defined media
“When using defined serum-free media containing defined recombinant proteins instead of 10-20% fetal bovine serum, the advantages are consistent and reproducible experimental results. Stem cells cultured in a serum-free environment are extremely sensitive with regard to morphology, cell density, proliferation rates, and sensitivity to mechanical and chemical stresses,” says Sloan.
Recent research has shown that endotoxin contamination in E. coli produced growth factors can affect cell culture, reducing reproducibility of experiments. Small fluctuations in these endotoxin contaminants between lots of even the same growth factor, will have varying, unpredictable effects. This includes standard culture protocols targeted for clinical use that typically use a variety of animal products and can pose a risk of exposure to retroviruses and other pathogens from the culture environment. Cell Sciences recommends plant-derived recombinant proteins such as the Hordeum vulgare (barley) expression of recombinant products, which includes endotoxin-free growth factors and cytokines, to increase precision and reproducibility of cultures.
New developments in enhancing stem cell culture conditions, from media choice to 3D culture systems, are extending the life of stem cell cultures, allowing us to obtain a better understanding of how these cell types function and how they can be applied to further investigations.