The Spin on Rotary Culture

Image: Courtesy of Leoncio A. Vergara, UTMB, Marguerite Sognier & Nasa/JSC Musculoskeletal Tissue Engineering Lab SPACE-AGE CELL CULTURE: This 3-D human rhabdomyosarcoma cell aggregate was grown in a disposable High Aspect-Ratio Vessel (HARV) in Synthecon's Rotating Cell Culture System. Biotechnology advances at a furious pace, yet for the most part, cell culture remains fixed in the past. Over the last decade, however, a new technology has emerged that models the microgravity of space--and it's allowing some researchers to go where no researchers have gone before. The Rotary Cell Culture System™ (RCCS), invented at NASA and introduced commercially by Houston-based Synthecon in 1990, uses motion to launch cells into a three-dimensional (3-D) orbital dance and a near- weightless environment in which they tend to interact, differentiate, and grow. Fragile cell and tissue cultures that won't grow in other culture systems tend to bloom into complex, large-scale, 3-D constructs. "If you recognize that cells exist in three dimensions in tissues, why would it not make sense to grow them that way?" posits Synthecon's chief scientist Stephen S. Navran, who first saw the NASA prototype during his tenure at the Baylor College of Medicine in Houston. "The difficulty had been to find a system that would do that easily. People have tried embedding cells in collagen and all kinds of things, but the architecture just doesn't effectively model what goes on in the body. While it is impossible to exactly reproduce every structure in human tissue, this technique comes closer to [doing that] than any on the market." Navran flips a switch on a system set up in the company's conference room. As the device's rotating cylinder begins to spin, the cells inside move effortlessly in the medium. "The particles are actually in a free fall environment ... with no selective pressure constraints that can disrupt the growth process," explains C.D. 'Andy' Anderson, Synthecon cofounder and president. "Everything is in this fluid orbit relative to other cells, so cells can make contact without being pulled apart like they are in a spinner, and they assemble in three dimensions with oxygen, nutrients, and wastes being exchanged with minimal stress," expounds Navran. Researchers around the world are now beginning to use this system to: produce bioproducts such as enzymes, hormones, antibodies, and antigens; grow human tissue for testing drugs and vaccines, as replacement tissue, or to study pathogenesis of disease; and study intercellular communication, differentiation, and function. Yet despite years of research and development and the integral role RCCS has played in some important recent discoveries, convincing the greater biological research community "to fall through the inert substrates and into this three-dimensional wonderland," as Anderson puts it, has turned out to be something of a bumpy mission. Image: Courtesy of Baylor College of Medicine GREATER THAN THE SUM OF THE PARTS: Dissociated guinea pig HIT-ß and mouse aTC6 cells as they spontaneously self-assemble into functional islets in the RCCS (left). A cross section of a pancreatic islet (right). OPENING UP THE THIRD DIMENSION The RCCS was born of a NASA project patterned in many ways after the Lockheed 'skunk works' projects that developed the SR-71 Blackbird and the U2 spy plane: A small, fiercely dedicated team was chosen and tasked with a specific goal--in this case, finding a way to study cell cultures in space to evaluate health risks to astronauts. The requirements were daunting: a system free of air bubbles that could operate vertically or horizontally, withstand all the variables of the launch (including delays, blastoff, and reentry), and be able to function in the weightlessness of space for seven days or more. In other words, NASA wanted a life-support system or synthetic physiology for the tissue. The space agency turned to Anderson, who had recently retired from NASA after a 20-year career to work for Houston-based KRUG Life Sciences, an agency contractor. As manager of the project based at the Johnson Space Center (JSC), he brought in KRUG engineer Ray Schwarz, NASA engineer-physician David Wolf, and KRUG medical technician Tinh Trinh as the design team. "With the first several devices, we calculated cell death, not cell growth," he remembers with a pained chuckle. Then one day in May 1986, Anderson walked into the lab and saw Trinh with a speed drill in his hands. "Attached to it was a syringe with fish flakes, the little particles we use for flow battery analyses with perfused vessels," he continues. "Tinh slowly pulled the trigger back and the syringe started spinning, and these little particles went into this beautiful orbital path. Everybody just said: 'Eureka!'" The following spring, Anderson took a biological leap of faith when Thomas J. Goodwin, the KRUG scientist brought in to establish a laboratory to utilize the new technology, approached him with an idea. "Tom held up one of the bioreactors and said: 'I think if I mix two cell types together in this thing they might construct something that looks like human tissue right here on the ground,'" Anderson recounts. "It seemed logical to me. 'Sure. Go ahead,' I said. We ended up with the colon cancer polyps you see in all the publications now. That was the first culture of differentiated tissue that ever occurred in this device, and Tom and Dave [Wolf] went on to develop the technique."1,2 THE NEW WORLD IN BIOLOGY By 1990, NASA had secured a patent on the Rotating Wall Vessel3 and transferred the technology to Anderson and Schwarz, who then founded Synthecon. Wolf, who had been inducted into the astronaut corps, Goodwin, who would accept a fulltime NASA position in the biotechnology division, and Trinh continued work at the JSC. Anderson and Schwarz soon found out that it was one thing to gain license to produce the technology, and quite another to get the scientific community to take notice. "This technology didn't borrow on anything," reminds Bill Anderson, Andy's son and a vice president at Synthecon. "We had just recreated the whole playing field." Disbelief was not an uncommon reaction. Synthecon submitted one of its first grants to the National Institutes of Health through the Stehlin Foundation for Cancer Research, to grow 3-D cancer tumor tissues for testing chemotherapy agents. "On a scrawled note, we were informed: 'Such technology does not exist. Do not resubmit,'" recalls Andy Anderson. "They were very blunt in their response." So Synthecon hit the road, demonstrating the now-renamed technology, the Rotating Cell Culture System, at conventions, annual meetings, and to private-sector companies. While Goodwin, Schwarz, and Wolf continued publishing2,4 and giving presentations on the technology, the space agency, determined to get a bioreactor laboratory established at the nation's foremost biological research institution, reached out to the NIH. "NASA saw this as the new world in biology," recalls Joshua Zimmerberg, of the National Institute of Child Health and Human Development (NICHHD). The timing was fortuitous. Zimmerberg, who was researching membrane fusion during viral entry, had been wanting to work "at a higher level of complexity, and if possible ... with human tissues." Two years before, he had formed an alliance with Russian researcher Leonid Margolis, who had developed complex histocultures. So when the space agency knocked, Zimmerberg answered. An interagency agreement between NASA and the NIH enabled him to establish the Center for Three-Dimensional Tissue Culture at the NIH in 1994. Receptive to testing any systems that promote 3-D growth, the Center gave both ideology and the new technologies a much-needed boost. Image: Courtesy of Leoncio A. Vergara, UTMB, Marguerite Sognier & Nasa/JSC Musculoskeletal Tissue Engineering Lab HOLD ON LOOSELY: A co-culture spheroid of fibroblast/epithelial cells grown in the RCCS, with a single cell still attached to a bead from which the aggregate originated. RESEARCHING IN THE 3-D WORLD To date, more than 40 investigators have availed themselves of the Center's facilities in a wide variety of studies. Zimmerberg says that he, Margolis (now heading the intercellular interactions unit at the NICHHD's laboratory of cellular and molecular biophysics), and colleagues have had impressive results with the RCCS. For one study on HIV pathogenesis, they grew the virus in human tissue "with its native architecture" and studied lymphocyte trafficking, "something that we couldn't do in another system," he notes. In addition, they showed "the robust replication of HIV" in the system, demonstrating "its utility for testing hypotheses for pathogenesis."5 Beyond that, they have also used the system to cultivate precancerous cells in a state of metaplasia.6 Zimmerberg points out, "we have only seen this [latter result] in these biopsy samples that we maintained in the rotary cell system." As the work at NIH progressed and other early adapters took up the technology, Synthecon continued to make improvements in the hardware, developing several new 'spins' on the original design and building customized systems on request. When R&D magazine awarded Synthecon for introducing "one of the 100 best new technology products" in 1997, the phones finally started to ring. NASA, which plans to fly a new generation of RCCS systems in coming years on the International Space Station, moved to further commercialize the technology by licensing another range of rights last year to StelSys, a small, private biotech company based in Baltimore, Md. There, researchers are using the device to grow liver cells for a liver-assist device, and kidney cells to create specific metabolites (such as vitamin D), as well as to study infectious disease, according to CEO Paul M. Silber. For now, the research possibilities seem endless, something that has never been lost on Synthecon. With the positive results from a NASA-funded Small Business Innovative Research program to test protein productivity, the start-up company acquired additional investment, opened the doors to its own laboratory last year, and promptly acquired the exclusive license to produce recombinant proteins with human cells in its proprietary system. "Our findings indicate that not only can we make proteins more efficiently because the environment is much less harsh, but we can also tailor the cell to the protein," says Navran. He won't say which proteins the company now has in development, but he does say that the end goal is "to scale up to a commercial-size system ... to show that we can do this." Not every researcher, of course, needs the advantages that come with 3-D culturing. "But there are a number of people who couldn't do what they are doing except with this device," Zimmerberg says. Silber agrees, adding: "We know, for example, that when we put liver cells into traditional cell culture, within a very few days they stop behaving like liver cells. They de-differentiate. If we want to study differentiated function ... we need a differentiated liver cell model. So here it's not a question of 'well, this works a little better.' It's black and white." Image: Courtesy of Leoncio A. Vergara, UTMB, Marguerite Sognier & Nasa/JSC Musculoskeletal Tissue Engineering Lab HT29 COLONIC CANCER CELLS growing on microcarrier beads. 3-D BIOLOGY HERE TO STAY After 12 years of struggling, Synthecon seems finally to be entering the scientific mainstream. The company has sold more than 1,000 systems, and some 200 peer-reviewed papers bear witness to the technology's efficacy. "The weight of publications coming through on ... the system is starting to stir a lot of interest within the academic community," notes Kevin Shakesheff, professor in pharmaceutical sciences, University of Nottingham, who uses the RCCS. He says that Lisa Freed's work with growing cartilage at Massachusetts Institute of Technology7 served as "probably the most important factor" in his decision to employ the system for tissue engineering applications. The most alluring aspect of the technology comes from its most novel aspect. "As soon as you have the ability to open the door to a whole new avenue of investigation on a disease that hadn't had an in vitro model system before, you open the door to developing therapeutics or vaccines for that disease. It's that simple," says Zimmerberg. "This is a platform technology, like PCR or DNA sequencing, that can be--and will be--broadly applied," adds Silber, "to solve problems or generate models for services, to create new products." Acceptance is growing, but it may yet be awhile before the RCCS becomes standard equipment in every biology laboratory. The cost--$2,300 (US) for a single unit system--is a significant barrier. Nevertheless, Andy Anderson remains optimistic: "We are now about where the laser was 30 years ago," he says. "People are beginning to realize that this is not only a dandy laboratory device, but it's one with some pretty fantastic commercial applications." A.J.S. Rayl is a contributing editor. References 1. T.J. Goodwin et al., "In vitro three-dimensional tissue modeling," Research and Technology Annual Report 1988, NASA Technical Memorandum 100473, 1988. 2. T.J. Goodwin et al., "Morphological differentiation of colon carcinoma cell lines HT-29 and HT-29KM in rotating-wall vessels," In Vitro Cellular and Developmental Biology, 28A:47-60, 1992. 3. R.P. Schwarz et al., Rotating bioreactor cell culture apparatus. US Patent 4,988,623, 1991. 4. R.P. Schwarz et al., "Cell culture for three-dimensional modeling in rotating-wall vessels: An application of microgravity," Journal of Tissue Culture Methods, 14:51-8, 1992. 5. L. Margolis et al., "Lymphocyte trafficking and HIV infection of human lymphoid tissue in a rotating wall vessel bioreactor," AIDS Research and Human Retroviruses, 13:1411-20, 1997. 6. L. Margolis et al., "Long term organ culture of human prostate tissue in a NASA-designed rotating wall bioreactor," Journal of Urology, 161:290-7, 1999. 7. L.E. Freed et al., "Cultivation of cell-polymer tissue constructs in simulated microgravity," Biotechnology & Bioengineering, 46:306-13, 1995.

The Spin on Rotary Culture

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Image: Courtesy of Leoncio A. Vergara, UTMB, Marguerite Sognier & Nasa/JSC Musculoskeletal Tissue Engineering Lab
SPACE-AGE CELL CULTURE: This 3-D human rhabdomyosarcoma cell aggregate was grown in a disposable High Aspect-Ratio Vessel (HARV) in Synthecon's Rotating Cell Culture System.