September 15, 2015 (Vol. 35, No. 16)

Daniel Bruecher Ph.D. Product Manager Infors HT

Novel Product Introductions Offer Advantages by Increasing DNA and Protein Production

Before and for a time after the research career of Louis Pasteur, microbiology essentially remained a static task. Glass bottles would rest on shelves until the contents were analyzed. This all changed in 1949, when the Nobel Prize was awarded for the development of the antibiotic streptomycin.

When the developers of streptomycin published a journal article to herald their accomplishment, they took the opportunity to share their frustration about the slow growth of the actinobacterium, Streptomyces, in static culture. They pointed out that by employing a new invention—a shaker—cell culture reactions would speed up.

For a culture vessel, the scientists simply chose what was available in the lab: it happened to be an Erlenmeyer flask. Nobel Prizes always attract much attention, so the demand for commercially manufactured shaking machines, that is, Erlenmeyer flasks, for microbiology purposes grew rapidly.
If we inoculate a static culture with bacteria or eukaryotic cells, the cells will invariably sink to the bottom and start to consume nutrients and oxygen. Nutrients are readily available from the medium, but the oxygen is quickly depleted. More oxygen is available in the gas phase above the medium, but the oxygen needs to pass through the liquid and reach the cells. This takes time and results in a gradient: oxygen is available on top, but there are no cells in this layer. Oxygen is scarce at the bottom, where it is actually needed.


Shake Culture

What can be done to resolve this gradient? Simple. Shake the culture. At the same time, we suspend the cells and increase the liquid surface area, which results in improved gas transfer. The Fernbach-style culture flask (invented around 1900) provides a larger liquid surface and hence a better oxygen transfer, albeit at the price of needing more space for the same amount of liquid.

In addition to the surface area of the liquid, there are other factors that influence the oxygen transfer rate, such as the surface-to-volume ratio.

If we fill up an Erlenmeyer flask, the liquid surface gets smaller the more liquid is underneath. This inverse correlation severely impedes oxygen transfer if the flask is filled too high. In many labs, you will see Erlenmeyer flasks used for shake cultures filled to 50% of the nominal volume, which is not a good idea, considering that most cultures are supposed to be aerobic ones.

As a general recommendation for Erlenmeyer flasks, a 30% filling volume should never be exceeded. If maximum oxygen transfer is required, the filling volume should be reduced to as little as 10%, and the shaking speed should be increased as much as the shear stress resistance of the culture (or the mechanical limits of the shaker) permit.

The downside is, of course, that you severely reduce your capacity in terms of liquid volume when you follow these recommendations, and this is why most people just don’t do it.


Figure 1. Production of recombinant proteins derived from cultures of Escherichia coli in Optimum Growth flasks

Boosting Culture Results without Reducing Volume Capacity

If you consider how Erlenmeyer (and Fernbach) flasks came to be used as shake flasks for culturing bacteria and fungi, as well as eukaryotic cells, it is obvious that they cannot constitute an ideal system. Until recently, nobody really thought about challenging their right to be the unquestioned system of choice.

Designing a shake flask cannot be as complex as rocket science, but then, common wisdom has it that the devil is in the details. The secret of how to improve the performance of such a simple item as a shake flask is to consider every aspect of the design.

Common benchmarks for an effective design are a wide neck, with large and efficient sterile filters on top; an optimized shape for maximizing the surface-to-volume ratio; and baffles in the bottom for excellent mixing of the culture liquid.

Thomson Instrument Company claims that cell culture results can be increased up to 600% by using their newly developed flasks (Ultra Yield Flasks™ for bacterial culture, and Optimum Growth Flasks™ for cell culture) instead of the traditional flasks. A range of Thomson Optimum Growth Transfer Caps (patented) is available for their flasks, with connector types that cover all possibilities.

Once connected, they provide a fully contained transfer system, enabling researchers to perform the transfer of the culture from the shake flask into a larger bioreactor—wherever you prefer to do it. Gravity or a pump can be used as a driving force. Gravity is the most gentle method, leading to only minimal losses of cell viability.

Citing a study that was published by Pfizer, Thomson notes that an increase of 610% was reported in the production of recombinant proteins derived from cultures of Escherichia coli in Optimum Growth flasks (Figure 1). Another study, this one conducted with Ultra Yield flasks and Enhanced AirOtop™ seals,  shows that an improved oxygen supply to bacterial cultures can reduce the production of secondary proteins, as shown in the gels depicted in Figure 2.


Figure 2. Gel results demonstrating that an improved oxygen supply to bacterial cultures, using Ultra Yield flasks and Enhanced AirOtop seals, can reduce the production of secondary proteins

Although it is well known that bacteria can live without oxygen, the lack of oxygen will trigger a stress response that results in expression of a number of different kinds of proteins.

A gel that is full of secondary proteins will make the purification of a target protein a challenge, while the gel shown on right-hand side of Figure 2 is what researchers look for.

Finally, the New York Structural Genomics Research Consortium published an increased protein titer of up to 436%, culturing High Five cells in 5 L Optimum Growth flasks (Figure 3).

To achieve the results just shown, it will be necessary to employ higher shaking speeds than most people work with. Supplementation of the culture medium might in some cases be required as well. So, simply replacing traditional flasks by Thomson flasks may not be enough to enjoy the full benefit. The whole culture protocol should be checked and adjusted where necessary.


Figure 3. The New York Structural Genomics Research Consortium published an increased protein titer of up to 436%, culturing High Five cells in 5 L Optimum Growth flasks



























Daniel Bruecher, Ph.D. (d.bruecher@infors-ht.com), is product manager at Infors HT. The company is the exclusive importer of Thomson culture vessels for Europe, except for the U.K.