Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Most cell culturists grow cell cultures in dishes, flasks or tubes. But if you need your cells to produce large amounts of, say, a particular secreted protein, all those dishes can be limiting (not to mention cumbersome). At that point, it makes sense to scale up your culture conditions—growing a lot more cells to make a lot more protein. This is best performed in a larger vessel, called a bioreactor, which can be used for a range of applications, including: fermentation; stem cell development; strain selection and cell-line optimization; bioprocess development; early cell screening and media selection; and producing monoclonal antibodies, vaccines and recombinant proteins.
But the transition from the familiar small-scale cell-culture setup to a new bioreactor protocol can be daunting. Never fear—there are tools out there to help. Tips for making the transition to a bioreactor are discussed here.
Making the transition
A couple of the first things to consider are the volume of cells you plan to grow in your bioreactor and how to scale the cells’ current experience in culture to their projected conditions in a bioreactor. “Most important when transferring cultures from dish or shake flask to bioreactors is a good understanding of the process,” says Karl Rix, vice president of sales and support bioprocess at Eppendorf’s Bioprocess Center. “The influence of different process parameters on the cultures’ behavior or product formation is essential.”
According to Davy De Wilde, director of marketing in fermentation technologies at Sartorius Stedim Biotech, ensuring a smooth transition requires more than simply using larger cell-culture vessels. “It is important that throughout scale-up the same environment is created for the cell culture,” he says. “This is realized by having the same gassing strategies, spargers, impellers, but especially also by having constant vessel ratios. For the latter especially, height to diameter of the vessel, and impeller diameter vs. vessel diameter [are] essential.”
Bioreactors come in reusable or single-use formats, with the latter increasingly available. For example, Sartorius offers systems for single-use bioreactors, such as the smaller AMBR systems or the larger BIOSTAT system, that facilitate scaling up from conventional cell cultures. Barney Zoro, product manager at TAP Biosystems (part of Sartorius Stedim Biotech), notes a small-scale bioreactor such as the 15-ml AMBR15 microbioreactor system “is the ideal tool to enable a smooth transition from small-scale shaken systems into a true bioreactor environment.”
For researchers who do plan to scale up their culture volumes, bioreactor systems use many parameters to help you keep tabs on the health of the cultures. “Typical scale-up parameters such as power input per volume, kLa [(the mass transfer coefficient, which determines the oxygen transfer rate)], tip speed and mixing times are defined, and the data [are] available,” says De Wilde. “This will support the user when setting up … experiments.” Sartorius also uses consistent sensor technology in its BioPAT® Sensor Toolbox across all bioreactor sizes and culture scales, which De Wilde says results in more consistent data and less batch-to-batch variability.
Monitoring bioreactor performance
Indeed, sensors are integrated into most bioreactors to provide the researcher with as much information as possible. “The more information you have about the characteristics of the clone in the small-scale system, the easier the transition,” says Eva Lindskog, upstream marketing leader in bioprocess at GE Healthcare Life Sciences. “This is true, for example, in terms of pH requirements, start cell density and what gas conditions to use.” GE’s Xcellerex™ and WAVE Bioreactor™ systems also include sensors controlled by bioreactor software for researchers to monitor cell conditions closely.
The ability to keep track of what is going on inside the bioreactor via sensors, which then “talk” to computer software, makes for some sophisticated monitoring systems. This means that your software can tell you if conditions inside the bioreactor start to take a dive—before you lose your entire culture, and perhaps your sanity. Most monitoring systems are able to make compensatory adjustments in inputs to the bioreactor, such as gas flow rates, fluid pump speed or temperature controls.
“Eppendorf offers monitoring and control of all critical process parameters, such as pH, dissolved oxygen (DO), temperature, liquid feed additions, with our parallel- and single-bioreactor controllers,” says Rix. GE’s newest bioreactor, ReadyToProcess WAVE™ 25, is designed to be more hands-off than other models. “[It] uses advanced software based on extensive characterization to optimize the bioreactor performance and minimize the hands-on time required from the user,” says Lindskog.
To accomplish this, the sensors of most bioreactors are integrated with software that can run optimization analyses. For example, Sartorius’ Process Analytical Technology (PAT), within its BioPAT® Sensor Toolbox, is designed for optimizing conditions. “The Sartorius BioPAT Sensor Toolbox includes technologies like inline, single-use biomass and online, single-use glucose-lactate measurement for automated monitoring and control of cell-culture processes,” says De Wilde. The Toolbox also includes sensors for pH; DO; pressure and flow; media and buffer preparation; virus inactivation; and crossflow- , ultra- and diafiltration, according to De Wilde.
Infors offers bioreactors with sensors that can be informed by a type of computer analysis called computational fluid dynamics (CFD). “Next to the traditional methods for scale-up from small-scale to larger bioreactors using kLa, stirrer-tip speed or power input, we offer our customers data based on modern methods like CFD,” says Tony Allman, product manager in fermentation at Infors. “The data can be used to identify ideal operating conditions for bioreactors and for shakers with shake flasks or single-use shaker bags.”
Starting with small-scale bioreactors
When making the transition from conventional dish-based cell culture to bioreactors, it can be less daunting to start with smaller vessels and scale up if needed, after conditions are optimized. A number of benchtop mini- or micro-bioreactors are available that incorporate sensors and use software to optimize conditions.
For example, the company m2p-labs offers the BioLector® and BioLector® Pro microbioreactors, which are high-throughput fermentation systems that can be used for microbial, plant and anaerobic cells. Using 32 or 48 parallel cultures, the microbioreactors measure important parameters, such as biomass concentration, pH and DO, and they also can measure levels of fluorescent molecules.
“In addition, the BioLector® Pro uses unique microtiter plates with a microfluidic chip,” says Frank Kensy, managing director at m2p-labs. “This way, the system controls continuously the pH value of each culture individually as well as the feeding for realizing fed-batch cultivations.” Other vendors also offer smaller-scale bioreactors, such as Sartorius/TAP Biosystems’ AMBR15 and AMBR25; Eppendorf’s DASbox Mini Bioreactor Systems; and Infors’ Minifors, Minifors2, Minitron and MultitronPro/Cell.
Transitioning from conventional cell culture to a small-scale bioreactor may be wise as a first foray into bioreactors, but it also has another advantage. If you choose to scale up to larger bioreactors, you will be one step ahead after optimizing conditions for your small-scale bioreactor.
Zoro sees small-scale bioreactor results as a “tool which is highly predictive of results in larger bioreactors.” Working initially with a small-scale bioreactor can “enable high-throughput early studies and allow rapid process scale-up without problems,” Zoro adds. This can provide peace of mind because, he says, “proven scalability enables much large screening and optimization studies to be carried out earlier in development, with confidence in results.”