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Scaling-Up from Spinners, T-Flasks & Shakers: A versatile bioreactor for mammalian and microbial cells

By Chris Julien; Reprinted with permission from American Biotechnology Laboratory.

Scaling up from spinners, T-flasks or shakers to a bioreactor system enables the researcher to reap the benefits of saving time. As an example of the typical savings one could expect, a single 5-L bioreactor has been shown to produce the equivalent amount of hybridoma cells as 150 250-mL T-flasks1. Moreover, a bioreactor requires only one laboratory technician and less than 2 ft of bench space for operation. The most significant decision when scaling up rests in selecting the equipment best suited to the users needs. Several factors must be considered, including the amount of product required (milligram or gram quantities); whether the end goal is the biomass itself or the products produced as a result of the culture; and whether the process uses an animal or microbial cell line, since the culture requirements and therefore the reactor design are inherently different for each.

This article focuses on the third factor, providing an overview of the most important differences between microbial culture and mammalian cell culture requirements, as well as describing a single reactor system capable of handling the varied needs of both.

Mammalian vs. Microbial Culture
Six basic areas in which culture requirements differ are growth rate, temperature, pH, dissolved oxygen, shear sensitivity, and foam. The following sections will discuss each of these areas in more detail.

Growth Rate - Growth rate, or the amount of time needed for cells to double, can be counted in minutes for microbes and upwards of a day for animal cells. A typical microbial culture may run just a few days to a week, and is therefore often run in batch mode. Because mammalian cultures will likely last several weeks, these systems are of ten operated in perfusion mode, allowing for continuous addition of fresh nutrients with continuous harvesting of secreted products. However, the longer the run, the greater the risk of contamination. Therefore, mammalian cell culture bioreactors require added contamination safeguards, such as use of magnetic-coupled bearing housings and threadless welded ports among others.

Temperature - Microbes have a wide range of temperature tolerances, with strains found in the sub-freezing environs of the North Pole, as well as in ocean vents at temperatures in excess of 100C. Mammalian cells, meanwhile, tolerate only a very narrow temperature range, typically between 37C and 42C. Microbial fermentors, therefore, usually control temperature to within 1C, while mammalian cell bioreactors require regulation capabilities within 0.1C.

pH - The acidity level of the culture medium is expressed by pH, with microbes thriving in conditions with a typical range of 2 - 10 pH depending on the microorganism, and mammalian cells requiring a pH level of 6.8 - 7.2. The method and responsiveness of the control significantly influence the process.

Acid and base addition is the method of choice for pH control of microbial cultures. Typically, a pH sensor linked with the fermentors internal controller signals it to activate a peristaltic acid or base pump. Not all fermentors handle the pH setpoint and deadband control in the same manner. An improper deadband setting can cause an action-reaction scenario, in which the pH overshoots the maximum limit activating one pump after another, leading to oscillation and unstable pH control. Since cells derive their nutrients from the transport of culture medium components through their walls, if the medium fluctuates between acidic and alkaline conditions, the cells will stop taking-up nutrients and eventually starve to death. The fermentor controller should therefore be configurable to allow smooth control of pH without oscillation.

In mammalian culture, direct addition of acid creates too harsh an environment. A better method of control is through addition of gasses. Sparging carbon dioxide (CO2) into the liquid solution lowers the overall pH, while sparging nitrogen (N2) neutralizes the effect of CO2 to raise pH. However, the amount of gas that can be physically dissolved into the medium is governed by Henrys Law. The level of dissolved oxygen (D.O.) will be influenced by the amount of sparged CO2 and N2. As a result, an interactive four-gas controller is recommended to enable proper balance of each gas component to assure proper pH and D.O. control.

Dissolved Oxygen - Most microbial and mammalian cells require oxygen to support their metabolism. Air, the most commonly used source for oxygen, may be added to the culture medium either by direct sparging, indirect gassing or gas overlay. Microbial fermentors are rated for addition of up to 2 volumes of gas per volume of liquid per minute (VVM), while mammalian bioreactors are usually rated up to 0.5 VVM. Consequently, the airflow control capabilities of the mammalian cell culture bioreactor must be more refined than those for microbial culture.

Shear Sensitivity - Microbes have very robust cell walls ( Figure 1 ), allowing direct gas sparging without fear of reduced cell viability due to shear forces exerted by contact with gas bubbles. Agitation rates in microbial fermentors are often set to 800 rpm or more, providing both ample oxygenation and homogenous mixing of viscous cultures. The preferred microbial impellers are radial type, such as the Rushton turbine impeller.

Mammalian cells are extremely shear-sensitive and can not survive high agitation rates ( Figure 2 ). Agitation i n mammalian culture rarely exceeds 150 rpm. Additionally, specialized low-shear impellers, such as marine blade, pitched blade, or Cell LiftTM (New Brunswick Scientific, Edison, NJ) impellers are required. The patented Cell Lift impeller allows for direct sparging of large amounts of gas into the medium through the impellers cell-free aeration cage. Cells and microcarriers are too large to pass through the pores of the screen and are therefore protected from the hazards of the gas bubbles shear forces. This impeller supports high K La (mass transfer coefficient) values and is ideal for microcarrier cultures.

Foam - Direct sparging with gas and a high protein content of the medium are the two main culprits in foam production. The addition of antifoam agents via a peristaltic pump controlled by a foam sensor in the culture medium is common for microbial cultures, but is usually not considered in mammalian cell culture. The Cell Lift impeller also features a foam-elimination chamber and an optional Air Wash SystemTM to minimize foaming.

Combination Fermentor and Bioreactor

Given the major differences between microbial and mammalian culture needs, careful attention should be given to planning for future operations, because the cell type being used today may not be the one needed next year. To provide for changing requirements within the laboratory, it may be advantageous to consider a reactor designed for use both as a fermentor and as a dedicated cell culture system. The BioFlo 3000 fermentor (New Brunswick Scientific) provides the versatility necessary to accommodate the diverse requirements of virtually any microbial, yeast, animal, mammalian, insect or plant cell line. The benchtop reactor includes a built-in, interactive four -gas supplementation system with selectable assignable pumps for acid, base, nutrient addition or harvesting as well as foam control, and is PC -compatible for simplified data logging, monitoring and bioprocess control. For production of secreted products from mammalian cells, the patented immobilized-cell basket accessory ( Figure 3 ) enables production rates of 1 gram of MAb/day in as little as 5 L of culture medium2. Researchers facing the challenges of scaling up should be aware that introductory information is available to ease their transition, including papers on fermentor and bioreactor selection and operation.3-5

1. G. Wang, W. Zhang, Y. Chen, C. Jacklin, H. Song, and D. Freedman. Design and Performance of a Packed-Bed Bioreactor for the Production of Recombinant Protein Using Serum-Free Medium. Edison, NJ: New Brunswick Scientific, 1994.

2. J. Doyle, L. Johnstone and L. Cottis. Gram Quantities of MAb Produced with Simple Bioreactor in Serum-Free Perfusion Culture Replacing Ascitic Fluid Production. AGEN Biomedical Ltd., Queensland, Australia. Published in September 1996 Biomass News edition of New Brunswick Scientific newsletter.

3. J. Cino PhD and S. Frey. 20 Tips for Purchasing Research Fermentors and Bioreactors. Biopharm Sept 1996 & Feb 1997 (reprints available from New Brunswick Scientific.

4. Fundamentals of Fermentation Techniques for Benchtop Fermentors. Edison, NJ: New Brunswick Scientific, 1995.

5. Selected References to Scientific Research in Fermentation and Cell Culture. Edison, NJ: New Brunswick Scientific, 1998.

Mr. Julien is Fermentation and Cell Culture Product Manager, New Brunswick Scientific Co., Inc., 44 Talmadge Rd., P.O. Box 4005, Edison, NJ 08818-4005, USA; Tel.: 800-631-5417, 732 -287-1200; F ax: 732-287-4222; E-mail:



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