Design of experiment, a statistical tool, has been used to determine an optimal cell culture formula by balancing growth factors and accounting for their interactions. [California Institute for Regenerative Medicine]
Design of experiment, a statistical tool, has been used to determine an optimal cell culture formula by balancing growth factors and accounting for their interactions. [California Institute for Regenerative Medicine]

Just as more or less pleasing music can emerge from a sound studio’s mixing board, more or less robust stem cells may crop up, depending on a lab’s stem cell culture formulation. Yet, to date, labs have lacked anything like a music producer’s ear. That may be changing, thanks to the growing appreciation of design of experiment (DOE) techniques in biology.

DOE is often used in scientific endeavors to measure and account for variations in data. For example, engineers use DOE to optimize physical structure design, and medicinal chemists use DOE to optimize drug formulation. And now, according to scientists at the University of California, San Diego, DOE is being explored as a way to optimize human pluripotent stem cell (hPSC) culture conditions.

These scientists, led by Alysson Muotri, Ph.D., described their DOE approach to medium formulation May 4 in Scientific Reports, in an article entitled, “Systematic optimization of human pluripotent stem cells media using Design of Experiments.”

“Using pluripotency and cell growth as read-outs, we determined the optimal concentration of both basic fibroblast growth factor (bFGF) and neuregulin−1 beta 1 (NRG1β1),” wrote the authors. “The resulting formulation, named iDEAL, improved the maintenance and passage of hPSC in both normal and stressful conditions, and affected trimethylated histone 3 lysine 27 (H3K27me3) epigenetic status after genetic reprogramming.”

Dr. Muotri emphasized how her group was sensitive to an oven-overlooked issue in culture formulation: the possibility that different culture components may interact in unpredictable ways. Obscure interactions not only make it hard to define optimal conditions, they also challenge labs to maintain batch-to-batch consistency, to say of nothing of producing results that other labs can replicate.

“If you ask a biology student what is the ideal temperature and pH for an enzyme, he/she will try to determine the best temperature in one experiment and the best pH in another experiment. Then, the student will erroneously conclude that these represent the optimal temperature and pH,” said Dr. Muotri. “What is missing is the interaction between temperature and pH. The best working temperature may not be the most optimal pH condition. DOE takes into account positive, negative or neutral interactions between multiple factors at the same time.”

Building upon earlier work, which had analyzed hundreds of other factors in hPSC media, the researchers determined the best formulations for bFGF and NRG1β1. They noted, however, that their findings are not fixed. “If science discovers a new factor that affects hPSC proliferation, we can add it into our DOE matrix to quickly test and re-formulate the media,” noted Dr. Muotri.

The researchers hope their findings will lead to a new standard for hPSC cultures. “Any lab in the world can have access to the same formulation, with no variability,” explained Dr. Muotri. “We also think this method could be applied towards the development of culture conditions during differentiation of human stem cells. Ideally, we want to create transition media formulations that subtly change during cell type specialization, mimicking the human embryo.”








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