The Five Heresies of Cell Culture Debunk Conventional Wisdom
Efficiency. Consistency. Repeatability.
This is the promising future of cell culture and the key to translating more therapeutics from the lab and clinic to patients at more reasonable costs.
The bioprocessing and biotechnology industries are on the cusp of a new cell culture reality where automated processes and closed-loop systems are able to create ideal cell growth conditions and enable seamless, highly productive, and cost-effective scale up from the smallest form factor to the largest bioreactor.
This future is attainable in the near term. However, the cell culture field is struggling to move the needle, and the fundamental element of good experimentation, that it must be reproducible when performed with the same methods under the same conditions is seemingly being left out in the cold. In 2016, Nature surveyed 1,576 scientists, of which ~70% indicated that they had tried and failed to reproduce the experiments of other scientists and ~50% could not reproduce their own experiments. In that same survey, 52% of respondents believe there was a significant reproducibility crisis and selective reporting is identified as the top-ranked factor contributing to irreproducible research.
So Why The Crisis?
Change is difficult and takes time. And bucking conventional wisdom is often seen as heretical until it becomes the norm.
The fact is that automation, real-time culture monitoring, and closed-loop systems technology exist to ameliorate this crisis. Cell culture scientists and bioprocessing engineers simply need to understand more about the tools available and get comfortable adopting them. This, however, is no small task.
“At the heart of this crisis is that the research industry hasn’t fully evolved to use modern tools like optical sensors. The production world has evolved; GE and Sartorius have sold over $1B of equipment enabled by SBI’s technology, but the researchers are not yet widely adopting these tools. For the industry to move forward we need to put these tools into the hands of researchers. This is our responsibility as the technology manufacturers,” stated John Moore, President of Scientific Bioprocessing, Inc.
“We need to move cell culture scientists from using a gross control like incubator gas to utilizing a precise control that uses optical sensors at the pericellular level,” he added.
Cell culture and bioprocessing conventional wisdom remains a hurdle for the wider adoption of more precise tools and technologies such as those offered by SBI. The more the bioprocessing field becomes familiar with these newer technologies and experiences their vast benefits across the development process, the more adoption will increase and what was once heretical and radical, will simply become a new, better, and different way to do this important work.
At SBI, we’ve pulled together a list of five heresies of cell culture that have been published in peer-reviewed journal articles to illustrate the required transition from conventional cell culture wisdom to this new and better way forward for researchers.
The incubator chamber oxygen concentration is a proxy for what the cells experience at the pericellular level.
Cell Culture Heresy #1:
Continuous non-invasive monitoring of pericellular liquid phase partial pressure oxygen (pO2) shows there is a significant difference between assumed and observed liquid phase pO2.
The carbon dioxide cell culture incubator was introduced in 1965. Conventional wisdom held that regulating carbon dioxide would help researchers control the pH in buffered cell culture media. This was the established process for years: that incubator gas control was an accurate predictor of what cells experience at the pericellular level.
In 2011, Dr. Linda Bambrick, currently the Program Director at the Division of Neuroscience in Extramural Programs at National Institute of Neurological Disorders and Stroke, led a team of researchers exploring the correlation between incubator gas control and partial pressure oxygen (pO2) at the pericellular level. Dr. Bambrick and her team deployed SBI’s optical sensors to continuously monitor what the cells were experiencing within the leading hypoxia chamber on the market, giving her the best available gas controls.
The research team set the oxygen gas control at 5% in the hypoxia chamber. However, SBI’s sensors reported that the cells initially read 3% and then went into anoxia.
Based on Dr. Bambrick’s work there is no clear correlation between controlling the gas phase and predicting oxygen levels at the pericellular level, so this begs the question: If you can use optical sensors to monitor at the pericellular level in real time, why should researchers try to control the gas phase in the incubator?
Essentially, since the cellular oxygen uptake rate exceeds the oxygen diffusion rate, the real-time, direct pericellular dissolved oxygen (DO) monitoring delivered by SBI’s optical sensors is essential for scientists to target the right oxygen levels for their applications and to reproduce experimental results. The deployment of non-invasive sensors that provide real-time data feedback will also help researchers better understand the relationship between gas phase oxygen controls and the pericellular oxygen level cells experience.
Adequate oxygen is provided for microbial cell growth in a vigorously agitated shake flask.
Cell Culture Heresy #2:
Since no dissolved oxygen monitoring solution for shake flasks has been available until now, this is an assumption.
The technology to accurately monitor oxygen levels in small form factors like shake flasks hasn’t existed until recently. Electrochemical probes are simply too large and invasive for shake flasks, so this piece of conventional cell culture wisdom is really an assumption of convenience, not a data-backed and verified process.
Now, with optical sensors providing real-time monitoring in shake flasks, researchers know that oxygen-limited conditions can persist over time, which is detrimental to the cells. Three papers, including Dr. Leah Tolosa et al. 2002, Dr. Atul Gupta and Dr. Govind Rao, 2003, and Dr. Govind Rao and Dr. Xudong Ge et al. 2012, discuss this topic in great detail.
Today, researchers can make small adjustments to the shake speed or pH to move cells from anaerobic to aerobic states and regulate media conditions using the real-time, actionable data on DO and pH obtained from the shake flask via optical sensor technology.
In the future, cell scientists will be able to set their target oxygen levels, and closed-loop control systems in shake flasks will help them maintain the conditions and nutrient levels that they are seeking. This will make their lives easier and help them get better and more repeatable results.
For mammalian cells, the surface to volume ratio of a T-flask is such that cells receive adequate oxygen by diffusion.
Cell Culture Heresy #3:
The T-flask is overwhelmingly kLa or mass transfer limited, and growing cells in a T-flask depletes liquid phase oxygen and barely any of the gas phase oxygen. Diffusion from the gas to liquid phase is far too slow to adequately meet the oxygen needs of most cells.
The T-flask is the most efficient cell culture vessel for the mass transfer of oxygen. However, research published by Dr. Lisa Randers-Eichhorn et al. 1996 and Dr. Jose R. Vallejos et al. 2012 illustrate that the widely adopted practice of setting incubator gas levels at target levels for cells cultured in T-flasks is flawed. Researchers need to use dynamically rocked T-flasks with closed loop controls to set pO2 levels to physiologically-relevant normoxic levels for each cell type.
Vast amounts of cells are currently grown in cell stacks that lack real-time monitoring and control of oxygen levels and therefore many of these cells go anoxic. Because researchers are not getting out more of what they put in, they are in turn getting something that’s changed due to anoxic conditions, and that’s a really big problem.
It is necessary and desirable to maintain incubator CO2 gas levels throughout the cell expansion phase.
Cell Culture Heresy #4:
An excess of CO2 becomes cytotoxic and needs to be constantly controlled and observed. Dissolved CO2 concentration over a certain level inhibits cell growth and affects cell morphology, proliferation, and metabolism.
Heresy #4 is similar to Heresy #1, as it begs the question, “Why measure incubator CO2 gas when researchers now have the ability to directly monitor pH at the pericellular level?”
Papers by Dr. Longan Shang et al. 2003, Dr. Bastion Blombach et al. 2015, Dr. Chatterjee et al 2014 and Dr. Viki R. Chopda 2019, all reinforce the importance of real-time pericellular monitoring as a key to creating an ideal culture environment for optimal cell growth within the design parameters of a given experiment. In addition, these papers illustrate that surface aeration/disruption and the removal of heavier than air CO2 from the liquid phase leads to improved cell growth and recombinant protein production.
SBI’s optical sensors empower researchers to create and sustain optimal cell culture conditions. Using these sensors, researchers can monitor culture conditions in 10 second intervals across long periods of time, enabling them to make condition adjustments in real time and intervene should adverse conditions arise.
By utilizing pH buffered media and a CO2 incubator, cells are provided a protective environment for growth.
Cell Culture Heresy #5:
There is a widely held misconception that pH buffered media has an inherent ability to regulate the pH of a solution to a predefined level. The lack of environmental conditions disclosure is one of the reasons that 70% of researchers surveyed by Nature reported they had tried and failed to reproduce the experiments of other scientists.
Dr. Pawel Swietach, an SBI collaborator and Professor of Physiology at the University of Oxford, believes that the lack of real-time, pH monitoring and control is at the heart of the reproducibility crisis cited by Nature (Swietach et al. 2012). pH buffered media controls acidity levels for a time, but when these buffers are disturbed, pH levels can swing very quickly and kill off cells fast. This becomes a serious quality control issue that cannot be mitigated by pH buffered media alone.
Conventional wisdom says that if you buy pH buffered media and grow cells in a CO2 incubator, the desired, protective environment for cell growth should be created. But based on the best available information that’s not the case. Unless real-time conditions are being monitored it is nearly impossible to understand what is happening at the pericellular level inside a flask or bioreactor system.
To improve data reproducibility in the field, researchers must monitor and control pH in real-time for all culture systems.
We call these the five heresies of cell culture, and because the stakes for medical research are so high it’s time to take a closer look at the real conditions cells are experiencing.
The cell culture field can no longer rely on the developments of generations past and with the emergence of novel diseases and an increased reliance on biotechnological advancements, the need for high quality, repeatable results is greater than it has ever been.
The original cell culture pioneers adapted the tools of other industries and used them to the best of their abilities. Tools like the Erlenmeyer flask, originally developed for the chemical industry, were what early cell researchers had access to and since they didn’t have any buying power, they took this flask and adopted it to cell culture. It was all they could do at the time, and despite it being less than ideal for their needs it has lasted to the current day.
The cell culture industry is big enough and important enough today that it deserves its own tools. Tools that can help make the work of scientists not only easier, but faster and better. The interface of cell culture and engineering has been neglected for far too long and advancing biotechnology is ready for solutions.