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Oct 15, 2009 (Vol. 29, No. 18)

Enhancement of Cell Culture Techniques

Emerging Insights on Metabolomics, Media, and 3-D Structures Could Lead to Improvements

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    Mouse embryonic stem cells grown in a 3-D nanostructured bioreactor at SUNY Albany. (Yubing Xie, Ph.D.)

    The CHI conference “Optimizing Cell Culture Technology,” which was held in Boston recently, featured several speakers whose work directly affects the biopharmaceutical industry.

    Yubing Xie, Ph.D., assistant professor at the College of Nanoscale Science and Engineering at SUNY Albany, discussed nanoparticle methods for delivering genes to cells and a hydrogel technique for creating 3-D tissue cultures whose microenvironments replicate complex tissue physiology. Applications include tissue regeneration, artificial organs, and drug screening.

    “Cells in our bodies are in a 3-D environment,” noted Dr. Xie. “The idea here is to use nanotechnology and nanoengineering to mimic that environment, including extracellular factors such as the tissue matrix.”

    Dr. Xie’s mouse embryonic stem cell  model can be programmed to take on the characteristics of tissue-specific cells for organ reconstruction. Similarly, they can be differentiated into hormone-secreting cells to replace malfunctioning organs, for example, in diabetes or into cytokine-secreting cells for treating cancer and other diseases.

    The key is the microenvironment, or niche, that stem cell experts say is required (along with the right chemical stimulus) to coax cells into differentiating into their final forms. Dr. Xie hopes to replicate these niches through nanoengineering.

    3-D microstructure also plays a role in how well cell cultures perform during drug screens. Drug discovery scientists spend billions of dollars per year on animal models because plated cells, which exist in flat or 2-D configurations, lack the form and function of living tissue. Where test drugs bathe cells cultured in monolayers in Petri dishes or microtiter plates, they must penetrate into living tissues. 3-D structures replicate the difficulties of penetration more closely and “can greatly reduce usage of test animals, which is always welcome,” Dr. Xie added.

    Zhaohui Geng, Ph.D., principal scientist in culture process development at Pfizer, described her work on analysis of intracellular and extracellular metabolites that could predict the quality of proteins expressed in cell culture.

    Dr. Geng first noticed that, in 1 L bioreactors, elevated pH caused lactate concentration to rise in CHO cell cultures. In an effort to understand the relationships between pH, lactate, and energy-production pathways, she submitted samples from cultures with elevated pH and control cultures, for metabolomic analysis to Metabolon.

    The study uncovered significant differences in concentrations of metabolites related to energy production, particularly lipids. For example, glycerophosphorocholine, which is released from cells to combat osmotic stress, was particularly elevated in high pH cultures. This raised an interesting question: was pH or high osmolality to blame for lactate accumulation and concomitant changes in glucose and amino acid metabolism?

    The metabolomic study quantified hundreds of metabolites, globally, unbiased, and without any pathway focus. Some, like pH, are measured daily in cell cultures, but many others are not. “We wondered if there were other trends, in measurements we did not take daily, that we should know about,” said Dr. Geng.

    After plotting all the variables, she found that pH rose during the entire culture time, but the metabolic changes did not occur until the osmolality rose. “The metabolic change curve matched the osmolality change rather than the pH change.” She confirmed this finding by raising osmolality without pH changes by addition of sodium chloride, and, sure enough, found a salt concentration-related rise in lactate. 

    “I think the osmolality-related metabolic changes are universal across cell lines and cultures,” Dr. Geng explained. “But the more interesting question is whether metabolic changes are related to changes in product quality. This question is currently unanswerable by looking only at daily reaction monitoring data.”

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