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“Biotechnology” is a term that has only been used for a few decades. However, the fundamentals of biotechnology, including the concepts of shaking, heating or cooling a fermenting mixture, actually have been used for thousands of years. Humans first began experimenting with cell culture by trial and error, utilizing a seemingly “magic” process to yield fermented goods from a carefully treated batch of substances.
Well before the metabolic activity of microbes was understood—and well before microbiota were identified—many industrious peoples took to fermenting food products, such as liquors. They discovered that agitating or handling a fermenting culture in a certain way resulted in variances in output, production time and flavor. Agitating an exposed cell culture essentially encompassed aeration, the idea by which oxygen is introduced to certain actively metabolizing cell cultures as they ferment a substrate.
Shaking incubators offer a controlled, mechanical means to aerate cell cultures by rotating a magnetic or mounted tray back and forth and/ or in a circular motion while maintaining a specific temperature range. In ancient times, cell cultures were manually agitated and were maintained at certain temperature conditions—for example, the constant, low temperature of a hole in the ground, a cave or a cellar. With the evolution of molecular biological research in the past century, the concept of shaking incubation emerged onto the laboratory scene. An increased range of metabolic byproducts were desired from cell cultures, and scientists were now able to prepare large batches of useful molecules such as medicines. For example, in the 1970s, Eli Lilly demanded large-scale shaking, incubating and fermenting for creating batches of insulin from genetically engineered Escherichia coli bacteria.
Very early on, rudimentary shaking mechanisms were placed inside standard incubators with water baths to agitate incubating flasks.
Cell cultures were often stirred, not shaken, introducing the potential of contamination. Modern shaking incubators combine the best of incubation and motion technology along with an increasingly diverse set of parameters to control. The micro-monitored environment within modern shaking incubators eliminates the need for direct contact with the cell culture, optimizing cell growth and output of aerobic and anaerobic metabolic activities.
The first historical account of a commercialized biological shaker dates back to the 1940s, when scientists at Rutgers University teamed with a group at New Brunswick Scientific. An example of one of these pioneering biological shakers travels the country to trade shows and elsewhere as a historical exhibit. Back then, the shaking feature was the most important aspect. With the addition of computerized controls over the past few decades, shaking incubators have been outfitted with a host of features to better control cell growth.
Applications: biotechnology
The viability of a cell culture is largely limited by the substrate, or food source of the cells, the handling of the culture and the maintenance of optimal growth conditions. However, by their simple motion, shaking incubators can optimize cell viability.
Cell cultures actively metabolize a food source, or a substrate, often delivered in commercially prepared media. The motion of the shaking incubator ensures that oxygen, other gases and nourishing compounds from the culture media are actively redistributed. Shaking incubators can be utilized for the culture of bacterial, yeast, fungal and tissue cells. They can also be used to enhance enzymatic activity of cell media in specialized cell cultures, namely, in biofuel ethanol production, or in the production of medicines.
Shaking incubators can be used for growing small to large culture batches. Large-scale fermentation can be expedited by growing a starter culture of microorganisms and substrate in a pilot batch, maintaining that culture within a shaking incubator for a set period of time and then transferring the successful culture to a larger fermenter or bioreactor vessel.
Growing starter cultures for batch fermentation
In a routine small batch fermentation of E.coli cells at 37°C, a shaking incubator increases the oxygen uptake of cells and maintains homogeneity of the culture media. Researchers can analyze the viability and growth rate of the cells within the shaking flask by taking samples and measuring cell culture density with a method such as UV/VIS spectroscopy. From that point, cells can be transferred to a fermenter, where the cell culture is maintained at a constant temperature and grown to a bigger scale. Cell cultures produce metabolic byproducts, some of which are acidic—a large-scale fermentation operation typically involves maintaining a specific pH with chemical treatment and growing over a number of days. However, the shaking incubator is the first step to growing a successful cell culture.
Range of instruments and examples
There are many applications for cell growth, but the concept of shaking incubation is simple: it starts with identifying the type of cell culture to be fermented. Is the cell culture large or small? Is the cell culture supposed to be in very strict conditions, or is there some flexibility? Will it be shaking for long periods of time?
The biggest differences in shaking incubators are found in the range of sizes and in differences across the manufacturers’ drive mechanisms. Most shaking incubators, ranging from benchtop to floor-model sizes, feature a drive mechanism and a motor that moves the platform to make vessels go in a circular orbit. Shaking incubators offer a range of operating rpms and the convenience of temperature control.
Smaller units do not sacrifice performance or features for size
Figure 1 – H1010 Incu-Shaker 10L from Benchmark and Terra Universal.Specialty incubator shakers such as Bibby’s (Staffordshire, U.K.) Microtitre SI505 are benchtop-sized and include specialized motion control for microtiter plates, as well as the option to shake small tubes of sample. Labnet International (Edison, N.J.) offers the Vortemp 56 shaking incubator for simultaneous heating and mixing of small samples. The Benchmark Scientific Incu-Shaker (available from Benchmark [Edison, N.J.] and Terra Universal [Fullerton, Calif.]) (Figure 1) is a mini benchtop shaker that features induction drive with computerized control for continuous shaking of smaller batches.
PHENIX Research (Candler, N.C.) offers the model I-5211DS “Model 211” digital shaking incubator with a space-saving volume of 1.7 cubic ft, a temperature range suitable for growth of common cultures at 30–95 °C, with an rpm range from 20 to 400.
Eppendorf’s (Hauppauge, N.Y.) Excella line features general-purpose benchtop-sized shakers for flasks, offering the capability of long-term shaking because of their heavy-duty counterbalanced drive mechanism that is machined from parts with extremely high tolerance standards, ensuring longevity and performance.
Larger shaking incubators— stackable chests and floor models
Some larger, long-term orbital shaking incubators offer the option of mobility within the lab space with mounted wheels. Examples include the Amerex (Concord, Calif.) GYROMAX 767 (heated) and 767R (refrigerated) shaking incubators, which offer increased insulation and durability. Similar options for shaking and incubating large volumes of cell culture (up to 6 L) include the Eppendorf Innova 43 (heated) and 43R (refrigerated) large-capacity incubator shakers.
With the addition of computerized controls, there are more ways to manipulate the cell culture. INFORS HT’s (Laurel, Md.) stackable Multitron cell incubator shakers feature additional pH control and monitoring along with hygienic, direct steam humidification within their chambers. On the smaller scale, the highly controllable MIDSCI (Valley Park, Mo.) LabDoctor mini incubator shaker features cooling and heating from –15°C to 60°C for ELISA microplates, qPCR setups, immunohistochemistry stained slides, Western blotting and staining and more.
If a lab is trying to simulate plant growth within an incubator shaker, it might consider purchasing an incubator with a photosynthetic light feature. There is also the consideration of time control—a shaking incubator can incorporate a simple timer or a computer control, from a toggle switch to one that can be monitored wirelessly or via mobile device.
Considerations for purchase: performance, longevity, requirements for metabolic activity
Some shaking incubators have a drive system that is designed to run continuously, 24 hours per day, for decades. Any lapse or downtime caused by an incubator shaker that cannot withstand heavy usage can potentially translate to millions of dollars lost.
Does the cell culture need to be carefully confined and controlled, or can it be managed in a basic, open-air shaker that is exposed to the air and grows at ambient temperature? For budget considerations, purchasing the simplest model possible is ideal.
What facility will it be used in? Unlike academia, industry is faced with regulatory rules that govern the use of specific incubator shakers in different protocols, such as pharmaceutical research and development. In developing a new cell culture at the start of an experimental medicinal batch, the performance and quality of the shaker really launches the production process. Having a reliable piece of equipment is extremely important because controlling the cell culture at the first stages translates to better results down the line.
Latest developments, notable facts
Figure 2 – S41i incubator shaker from Eppendorf.Companies such as Eppendorf and INFORS HT are offering an increasing suite of options, including temperature-controlled incubator shakers that allow both cooling and heating options, offering an expanded range of application within one piece of equipment. Eppendorf’s S41i (Figure 2) is an industry-first incubator shaker that integrates control of CO2 and O2 levels, eliminating the need for buying a CO2 incubator and shaker separately and potentially sacrificing performance or yield by retrofitting products to work together.
Regulatory bodies often emphasize the use of certain parameters and controls in shaking incubation procedures, most often seen in the pharmaceutical research and development industry. Certain USA-made and European-made shaking incubators are specifically approved for use—in designing a pilot run for culturing a specific metabolite, it is imperative to review regulatory standards for equipment before proceeding.
Emilia Raszkiewicz is managing editor, American Laboratory; [email protected] American Laboratory would like to thank Daniela Marino, product manager, ULT Freezers and Biological Shakers, at Eppendorf North America, for her input.
Please see our Shaking Incubator / Incubator Shakers section to find manufacturers that sell these products