Traditional cell culture models flatten the three-dimensional landscape of the human body into a one-cell-thick slab. NASA's bioreactor has given a new depth to these cultures and provided insights into HIV infection and Lyme disease.
Doctors practicing evidence-based medicine - the judicious use of external evidence to help determine treatment - often look to studies based on cells in culture as strategic guides. After all, when dealing with infectious agents like HIV or autoimmune illnesses like arthritis, it is often impossible to peer into the human body itself. Without outside evidence, how can practitioners know with any precision what is really going on? Yet the journey from theory to practice has been slippery, largely because the culture studies often cited, even the most rigorous of them, are flawed. The reason: A vast difference between tissue inside the body and tissue in the lab. In the body, complex genetic instructions and a host of messages from the environment cause cells to differentiate into specific organs and tissue types. But in the petri dish, the fundamental law of bench biology known as "contact inhibition" rules. In this unyielding phenomenon, replicating cells stop dividing once they sense neighbors, resulting in tissue just one layer thick.
|Scientists had been unable to culture cells in 3-D.|
Because of contact inhibition, medical science has been unable to culture human tissue to the mature states of differentiation found in the body. While physicians often look to studies based on these cultures to guide their treatment decisions, they cannot really know if the data is relevant and to what degree.
But now all that may change. Through one of the most important technologies to trickle down from NASA since the inception of the space program, biologists can conduct experiments with three-dimensional tissue right here on Earth. NASA's great enabler is the bioreactor, a cylindrical chamber that bathes tissue in microgravity - the same near-weightless condition astronauts experience during their walks in space. Rotating slowly at the speed of a long-playing record album (33 RPM), the bioreactor keeps cells suspended relative to each other, as if the force of gravity were almost zero, allowing cells to grow in three dimensions (as happens in life) instead of just two dimensions.
|Traditional cell cultures are limited.|
The epicenter of this work, planetside, is the laboratory of Joshua Zimmerberg, chief of the Laboratory of Cellular and Molecular Biophysics at the National Institute of Child Health and Human Development, National Institutes of Health, in Bethesda, Maryland. A number of years back, hoping to drill down into the process that ensues when HIV infects lymphoid tissue, Zimmerberg found himself limited by experimental design. When growing cells in laboratory culture that was flat as a pancake, he knew his findings would be of just limited use. "For instance, we knew that human tissue contained literally 100 times as many immune cells inside than at the periphery," Zimmerberg notes. Yet in the petri dish, "periphery" was all he had.
The problems were underlined by Leonid Margolis, Zimmerberg's colleague and chief of the Unit on Intercellular Interactions in his lab. Margolis had spent his career showing that the biology of any given cell depends upon its complex interaction with the tissue surrounding it and vice versa. Depending upon a series of adhesions and attachments, both invading cells and tissues change their genetic expression and physiological form over time. It is through such interaction, Margolis showed, that the pathology of any given disease is defined.
|Ultimately, we want to understand cell behavior.|
"As biologists, we do reductionist science in the test tube," Zimmerberg says, "and through that means, we learn a lot. But ultimately, if we want to understand cell behavior, if we want to know the relevance of our findings, we've got to find a situation that approximates the living organism. There is a relationship between the disease state and the pathology of the tissue, but the cultures we'd been using were not up to the job."
Zimmerberg was pondering these problems in 1995, he says, "when someone handed me a proposal from NASA." The space agency was seeking a group to study biological complexity using its new microgravity chamber. The synergy could not have been more appropriate, and a partnership was born. One of the most important advances to emerge from that partnership is a tissue model of AIDS, one that closely resembles pathology in a living host. "AIDS is the first pandemic to start in the era of molecular biology," says Margolis. "As a result, we know much about the molecules encoded by the viral genome. In contrast, however, we know relatively little about the mechanisms of viral pathogenesis. AIDS is a complex disease, in part because HIV infects the cells that fight infection and disrupts multiple, little understood cell interactions in the lymphoid system. Moreover, the virus evolves rapidly and continuously over many years in the body under as-yet-unidentified selective pressures, and its properties can be strikingly different early in infection compared with later, when severe immunodeficiency occurs." In short, HIV infection involves a complex interplay between both infected and noninfected cells of the human immune system - a situation that is simply impossible to study with tissue one cell layer thick.
|Impossible in the petri plate, doable in the bioreactor.|
But what was impossible in the petri dish became doable in the bioreactor, enabling researchers to resolve some long-standing mysteries with ease. One issue confounding AIDS researchers, for instance, was the observation in standard culture of a phenomenon known as syncytia, in which viral particles fuse together, forming aggregate "cells" with many nuclei instead of one. Despite the fact that syncytia had never been observed in tissue, scientists thought this trait so important they used it to classify HIV strains.
Margolis and Zimmerberg were the first researchers to prove that syncytia do indeed occur in living tissue when they examined HIV-infected tissue in the bioreactor, but there was a catch. In the body, the syncytia in lymphoid tissue are destroyed almost instantly by the body's cells. The classification system based on observations in flat-dish cultures is not particularly relevant, after all.
|Researchers replaced a vague HIV classification system.|
As a result of this and other studies, a vague and controversial classification system based on the complex interaction of HIV with particular cells has now been replaced by one built on the firmer basis of viral molecular characteristics. "This new classification may have a profound impact on HIV research, similar to the impact of Mendeleev's periodic table on chemistry," says Margolis. The task ahead, he adds, is using molecular biology to understand how HIV impairs living tissue and causes AIDS.
Toward that end, current work focuses on the transmission of AIDS during the earliest days of infection. To marshal the power of simulated microgravity, the scientists use a special, dual-chamber bioreactor divided by a lifelike membrane wall; uninfected tissue is cultured on one side of the divide and tissue biopsies from patients with known disease on the other. One goal, says Zimmerberg, is to see which strains of HIV are transmitted most efficiently. Another goal is the determination of conditions least and most conducive to transmission of the disease. Says Zimmerberg: "We plan to use this model of transmission to test methods of preventing vaginal and rectal transmission at the point of entry."
|How does Borrelia burgdorferi "adapt" to different human tissues?|
Working with Paul Duray, Division of Clinical Sciences Laboratory of Pathology Branch, National Cancer Institute, National Institutes of Health, Zimmerberg is also using the bioreactor to study the pathology of Lyme disease, a multisystemic illness caused by infection with the spirochete Borrelia burgdorferi (Bb) and the most common vector-borne infection in the United States. "The Bb spirochete is able to persistently infect humans and animals for months or years in the presence of an active immune response and is able to 'adapt' to distant human tissue such as the brain, liver, and joint," Zimmerberg explains. But how do the spirochetes do it? The recent isolation of p100, a glycoprotein expressed in the spirochetes upon invasion of human brain tissue, supports his theory that Bb undergoes genetic alteration based on contact with specific tissues. The genetic changes not only cause expression of new proteins, he surmises, but also facilitate attraction to and invasion of the tissue that sparked the change in the first place. Using the bioreactor, Zimmerberg hopes to isolate the genes and proteins expressed at different points during the course of infection.
Though the molecular pathways have yet to be unraveled, the bioreactor has already revealed an important piece of new information. Scientists culturing the Bb spirochete in the past have puzzled over what seemed to be a contradiction - while infected tissue grown in standard culture could sustain just small numbers of spirochetes, the severity of patients' symptoms were often off the charts. What could be making these people so sick if the bacterial load is so small? To scientists working with "pancake cultures" and other, less direct means of detection, an autoimmune reaction seemed a likely answer. But Zimmerberg and his team have found that when grown in the bioreactor, tissues present with "exponentially more Borrelia," probably because, as in AIDS, the immune system of the infected tissue is repressed. While Margolis's and Zimmerberg's teams continue to look for any evidence of an autoimmune reaction in their bioreactor work, they now know that many symptoms can be explained by the persistence of the disease.
|Microgravity aids the study of many more diseases.|
In addition to HIV and Lyme disease, Zimmerberg's team has been studying prostate disease, cyclospora (an intestinal parasite), herpes, diabetes, rheumatoid arthritis, squamous metaphasia (thought to be a precursor to skin cancer), and skin cancer. In work with NASA, they are also studying the immune system under conditions of microgravity to see whether astronauts on extended space missions might be at greater risk.
The team is also using the microgravity chamber to bioengineer tissue for the next generation of work. "We're using this technology to prepare better models of human colon, prostate, breast, and ovarian tumors," Zimmerberg states. "While cells grown in conventional culture systems may not differentiate to form a tumor typical of the cancer of origin, tumors that form in the bioreactor resemble the original tumor." Similar results have been observed with normal human tissues, including cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver cells, and kidney cells, to name a few. The ultimate goal is creation of what Zimmerberg calls "a universal pathogen culture," nothing less than a "multitissue equivalent that creates the necessary microenvironment" for most forms of human disease.
|Can cells sense small changes in gravity?|
The work also has raised some fascinating possibilities about the nature of cells in particular and life writ large. For instance, the experiments hint at the possibility that cells may be able to sense small changes in gravity. "If cells have sensors for gravity," notes Zimmerberg, "we'd have to rethink our models, because we have missed something major. Cells that could sense gravity would represent a paradigm shift in the way life works."
The next step for Zimmerberg and Margolis is conducting bioreactor experiments in space, where gravitational forces will be truly lower, reducing stress enough to grow larger tissue samples that live longer and approximate conditions within the body more precisely than is possible now. Zimmerberg thinks that better culturing conditions could change the way life science is done. "The culturing of cells on solid substrate is the most popular technique in the entire biomedical enterprise," he says. "Just as the culturing of bacteria led to nineteenth century advances, the culturing of animal cells has fueled the tremendous progress of the twentieth century. But, despite a hundred-year history for cell culture, there are still limitations. Cells in tissues within patients do not respond to therapeutics like cell lines selected for quick growth on the inner surfaces of flat flasks. Even in primary culture, we lose the location of a cell within the logic of the tissue structure. There are disease states whose pathology cannot be reproduced merely by growing the right cells. Rather, a complex interplay of cell-cell interactions on a matrix of extracellular material, bathed in the body's biochemicals, dominate diseases like HIV, diabetes, and Lyme. We simply do not have model systems for these and many other diseases we would like to study in vitro. Yet our tremendous ability to fish out individual molecules and learn their activities makes us yearn to understand their significance in the pathology of the disease."
Pamela Weintraub is a former staff writer at Discover, former editor in chief of Omni, and the author of 15 books on health and science.
Cary Barnhard grew up in New Jersey, where his senior class voted him "most unique." He maintains that honor is a polite way of being voted "most likely to need therapy." After a few misadventures in the music industry, he started pretending to be a graphic artist. Eventually it became the truth.
Synthecon Incorporated - makers of the commercially available rotary cell culture system (i.e., NASA's bioreactor), provides information about the technology and its applications.
Microgravity Science Primer - an introductory article covering such topics as protein crystal growth and cell culture.
Microgravity - the Web site for NASA's program, provides research summaries, news, and more.
New Insights into Factors that Affect HIV-1 Transmission - a brief update based on one researcher's laboratory's work, which includes an organ culture model. From Drug Discovery Today, 2000, 5:6:219-220. Full text available from BioMedNet.
Borrelia Pathogenesis Research in the Post-Genomic and Post-Vaccine Era - a review of recent findings. From Current Opinion in Microbiology, 2000, 3:86-92. Full text available from BioMedNet.
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