Advanced Lab Bioreactors Extend Cell And Tissue Culture Capabilities

Capabilities AUTHOR : FRANKLIN HOKE, p.17 With roots in such age-old processes as wine, beer, and soy sauce fermentation, cell-culture technology has been well explored. Especially in the decades following World War II, sophisticated culturing systems--called fermenters or bioreactors-- made their appearance in many laboratories. But advances in just the past few years, especially in the areas of sensors and computerized control, are expanding the efficiency and range of research applications of these systems even further, according to scientists and manufacturers. The following companies are among those manufacturing and distributing bioreactor and fermenter systems or supporting software for these systems: Applikon Inc. 1165 Chess Dr., Ste. G Foster City, Calif. 94404 (415) 578-1396 Fax: (415) 578-8836 Astra Scientific International Inc. 6900 Koll Ctr. Pkwy.Suite 417 Pleasanton, Calif. 94566 (510) 426-6900 Fax: (510) 426-6990 B. Braun Biotech Inc. 999 Postal Rd. Allentown, Pa. 18103 (800) 258-9000 Fax: (215) 266-9319 LH Fermentation 3942 Trust Way Hayward, Calif. 94545 (510) 786-0224 Fax: (510) 786-0229 New Brunswick Scientific Co. Inc. Box 4005 44 Talmadge Rd. Edison, N.J. 08818 (800) 631-5417 Fax: (908) 287-4222 Systems designed to culture cells can be laboratory-scale units with 1- to 20-liter capacities or larger, production-oriented systems. They are, for the most part, computer controlled, with prices beginning at a few thousand dollars and climbing to tens of thousands. Culturing cells--whether bacterial, plant, or animal--is integral to the research of many life scientists, among them cell biologists, immunologists, microbiologists, and virologists. The ability to culture mammalian cells, in particular, is central to many biotechnology processes, such as the production of monoclonal antibodies, recombinant proteins, and other cell products. Culturing mammalian cells, however, presents a set of special problems, according to researchers. "Mammalian cells are delicate," says Julia Cino, fermentation product manager for New Brunswick Scientific Co. Inc., New Brunswick, N.J. "And they're very, very finicky with respect to what they require in terms of nutrients and oxygen demand." Most researchers, Cino says, would probably prefer to clone a gene and insert it into E. coli or another microbial cell that can grow rapidly, is easy to isolate, and from which it is relatively easy to obtain secreted or other cell products. The delicate nature of mammalian cells means that, in cell culture, they are highly susceptible to what is called shear. Shear is the potentially damaging force applied to cells as they contact impeller blades (which circulate the nutrient media), the walls of the culture vessel, the fluid media, or even each other. In culture systems that bubble air, oxygen, or other gases through the media, the surface of the bubbles themselves can cause shear. "If you take a garden hose and point it at your hand, that's shear," explains Glenn Spaulding, the biotechnology program manager at the National Aeronautics and Space Administration (NASA) Johnson Space Center in Houston. "That shear force of the water flowing over your hand causes minute damage to your hand." Essentially, movement of the media is necessary to circulate nutrients to the cells and remove waste products, but turbulence associated with that movement causes shear. Many strategies have been devised to reduce shear and to be able to carefully balance shear against nutrient-delivery capacity. Unfortunately, shear and nutrient availability are inversely related, in most cases-- more media flow means higher nutrient levels and, thus, higher growth rates, but it also means higher damage levels. Many mammalian cells also cannot grow in suspension--that is, floating in the media. They require some sort of surface upon which to grow, something to which they can adhere. To provide such surfaces, a number of systems have been developed using beads or other supports packed into a subcontainer within the bioreactor vessel. The media are then circulated gently through the supports. Cell-culture systems go by different names, either fermenters or bioreactors, and the terms are often used interchangeably. But there is a difference, at least in custom of usage if not in hard definition, according to those who work with them. "The term `bioreactor,' in the generic sense, is anything you grow a living cell in," says Cino, "so it covers both fermenters and cell-culture devices. But it's becoming common in industry today to use the word `fermenter' for anything you grow microbial cells in and `bioreactor' for anything you grow mammalian cells in." "People don't like to use the word `fermenter' in cell-culture applications," says John Chory, president of Applikon Inc., a manufacturer of cell-culture systems located in Foster City, Calif. "When you start dealing with, let's say, mammalian cells, then they'll usually start calling it a bioreactor. But the two terms are, for the most part, interchangeable." One general definition of a bioreactor, according to Chory, is "a controlled environment for some sort of microorganism or cell to grow in." The key to successful cell growth for research or production purposes is reliable, reproducible control. "[Bioreactors] control the process parameters," says Kenneth Clapp, fermentation product manager for B. Braun Biotech Inc., an Allentown, Pa.-based manufacturer of fermenters and bioreactors. "The basic five are temperature, pH, dissolved oxygen, agitation, and foam." Nutrient levels are also controlled in a cell-culture system, as are the levels of oxygen and other gases. "Anything that you can measure, you can control," says E. Terry Papoutsakis, a professor of chemical engineering Northwestern University, Evanston, Ill. "Typically, what [microbiologists] do is they allow one of the parameters, say the pH, the glucose, or another nutrient--phosphate, iron, you name it--to limit the culture. This is how people have found out what various nutrient limitations mean for the cell: How does the cell adjust its biosynthetic machinery in response to [changes in] this particular parameter?" Another goal of such studies is to find the shortest possible doubling time for the organism or cell, to maximize its reproduction, either for research uses or for the production of diagnostic or therapeutic cell products. Sensors and software systems that have only recently become available are combining to make it easier to more precisely control the bioreactor environment. "With some of the sensors that have come out, like cell-density probes, which are very reliable now," says Clapp, "there is the ability to do online glucose analysis or to look into other parts of the metabolic cycle. So, if a researcher knows that the glucose concentration is critical to the growth of a glycoprotein that forms an antigen on the surface of some tumor cell, now he has the feedback; he can make sure that he keeps the concentration high enough." Chory says that advances in cell-culture sensors and controls have followed the same pattern as has been seen in other high- technology production processes, such as those in the chemical industry. "If you were to look at bioreactors 10 years ago," Chory says, "you would see huge analog controllers that were quite limited in what they could do. Today, it's not unusual to see direct digital control systems performing a variety of functions." Besides the improvements in sensors and software, there have also been some important innovations in hardware. Several years ago, for instance, magnetically coupled impellers appeared, improving the reliability of the systems considerably. Magnetically coupled impellers do not use a shaft to connect the drive motor to the impeller blades, but use strong magnets instead. "You have to have agitation in your system," explains Chory. "But with mechanical stirrers you can have a problem with bearing failure or seal failure, allowing contamination of the fermenter. Or, in situations where you're working with pathogens, you don't want them getting out of the fermenter. Magnetic stirrer assemblies pretty much eliminate that problem." The variations among basic design architectures of bioreactors derive, in large part, from efforts to control damaging shear forces. The type that has been in use longest, and is still the most common, is called a stirred-tank, or agitated, bioreactor. Other types include the airlift bioreactor, which uses oxygen or other gases rising through the media to promote circulation, and bioreactors using a hollow, permeable fiber technology to bring nutrients to the cells, which are immobilized between the fibers. Stirred-tank systems have an advantage over other systems in that culture protocols developed at the research level can often be reliably translated into production-level efforts. But the problem is that stirred-tank bioreactors usually use an impeller to circulate the medium. Impellers are effective, but they tend to cause shear in mammalian cell culture. "You get maximum shear at the tip of that impeller," says Cino, "and every time the impeller goes around, it kills a couple of million cells." To adjust for shear potential, impellers vary in blade pitch and shape. In systems in which cells are cultured on beads or other supports held in a subcontainer inside the bioreactor, the cells are protected from direct contact with the impeller. The nutrient media, circulated by the impeller, flows gently over the immobilized cells. The primary benefit of both the airlift and hollow-fiber bioreactors is that they produce lower shear environments than the stirred-tank systems. They can be difficult, however, to scale up. Spaulding, at the Johnson Space Center, says that a bioreactor variant developed by NASA researchers may answer the problem of reducing shear while keeping nutrient supplies high. The device is called the rotating wall vessel, or rotating wall bioreactor. The basic aim of the NASA bioreactor is to simulate a near- weightless condition. The cells being cultured are suspended in the media within the cylindrical bioreactor, which is oriented sideways, so that its rotational axis is perpendicular to the gravitational vector. The cylinder is then rotated at 12 to 40 rpm. The direction of gravity's pull on the cells is thus constantly changing. "Just as the cells start falling down, you rotate a few degrees," explains Spaulding. "Now they're falling in a different direction, and then a different direction, and on, and on. They end up not falling anywhere." What NASA has discovered, he says, is that the cells will stay suspended in the media, growing for long periods. The cells experience no shear and are perfused with nutrients in three dimensions. According to Spaulding, there are a number of unexpected benefits to this arrangement. "When two cells of dissimilar size or kind touch one another, there's no shear to dislodge them from contact," he says. "So, they stay in place, in contact with one another, and are allowed to put down proteoglycans and collagens and matrix. They then adhere to [the matrix], divide, and grow outward in three dimensions. You have this synergy between cells, just like it happens in the body. They grow larger and longer." In cultures of normal human tissue, he says, researchers have seen small sinuses developing and the first signs of capillary formation, akin to what they might see in normal development. According to Spaulding, there are a number of other markers of normal development--biochemical markers--that appear in tissues cultured in the rotating wall bioreactor but that do not appear in other cell- and tissue-culture systems. He speculates that, in the low-shear environment, the tissues are able to shift resources away from cellular repair and toward more normal growth. "We can bring it up to the first trimester in development," Spaulding says. "And then they become big enough that, like in a centrifuge, they spin out of solution, start hitting the wall, and doing damaging things [to themselves]." The solution is to take the new bioreactor into space, where the effects of gravity will be even further reduced. For now, the space shuttle is the only available platform, which will limit experiments to the amount of time that the shuttle craft is in orbit, usually a matter of days. The space station, however--if it is ever built--would allow much longer- term experiments. "On the shuttle, we'll look at cancer, which grows much faster," Spaulding says. "On the space station, we'll look at normal tissues." Spaulding anticipates the new bioreactor will have important uses, including, perhaps, the culturing of tissues for transplantation, for example. The rotating-wall bioreactor also may advance investigations of many cancers and viruses that cannot currently be cultured adequately. He notes that some viruses grow only in fully developed human tissues--that is, the living body--making them difficult to study. "You typically do not think of gravity as a variable," Spaulding says. "And gravity affects a lot of things we take for granted." He adds: "When we dropped the shear force an order of magnitude on the ground by rotating and emulating the microgravity environment, suddenly these tissues started to form. Now, in space, we'll drop it by another order of magnitude--or two. We don't know what's going to happen at that point. "Perhaps it's like the microscope story," he says. "When the microscope was first invented, everybody said, `Who cares?' But every time we see a new tool, we see a tremendous deluge of new science."

Advanced Lab Bioreactors Extend Cell And Tissue Culture Capabilities

Interested in reading more?

Become a Member of