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A New Dimension in Modeling Infectious Disease Three-dimensional cell cultures closely mimic in vivo tissues, offering a powerful new means for studying infectious diseases Cheryl A. Nickerson and C. Mark Ott ultured mammalian cells, including primary cultures, organ cultures, and continuous cell lines, have long been used to investigate host-pathogen interactions that lead to infections. While these models continue to contribute to our understanding of infectious diseases, each has inherent limitations, raising questions about their relevance or practicality. Hence, we and other researchers are moving toward developing novel physiologically relevant, three-dimensional (3-D) tissue culture models and away from the decades-old standard that consisted of monolayer cultures of mammalian cells grown in two dimensions on plastic or glass. As compared to standard monolayers, these newer, 3-D cell cultures contain structural and functional properties that more closely approximate conditions that pathogenic microorganisms encounter in the host tissues they infect. One popular option for generating 3-D cells that retain a differentiated phenotype is to embed them in an extracellular matrix of structural proteins such as collagen and laminin. In another approach, we use rotating-wall vessel (RWV) bioreactors, designed by investigators at the National Aeronautics and Space Administration (NASA), to grow 3-D cell cultures that faithfully model structures and behaviors that occur in tissues in vivo. In addition to providing physiologically relevant models of human tissues, this novel approach confers other important advantages when studying tissue-level mechanisms of infectious disease. In particular, these 3-D cell cultures represent a highly controlled and reproducible model that can create extremely high n values per experiment, and C enormous numbers of cells which can be studied by techniques not possible in matrix culture and somewhat limited in monolayer culture. Problems in Using Traditional 2-D Cultures in Infectious Disease Studies Researchers who use primary cell and organ cultures to study infectious diseases have recognized several important limitations associated with these systems. For instance, the mitotic potential of cells growing in such culture systems is limited, thereby restricting experiments to short-term studies, causing reproducibility problems, and significantly increasing costs. Continuous cell lines solve many of the challenges associated with using primary cell and organ cultures to study infectious diseases. However, problems are also associated with using continuous cell lines, including the fact that they are not truly representative of the in vivo tissues from which they derive. In particular, propagating such cell lines as standard, 2-D monolayers on impermeable surfaces prevents cells from differentiating and acquiring polarity. These steps are blocked because cells grown as monolayers on flat plastic or glass surfaces cannot form the cell-cell and cell-matrix interactions as they would in their parental tissues. In the body, tissues are found as well-organized 3-D structures that are critical for establishing and maintaining differentiated form and functions. Accordingly, flat layers of cells being cultured under standard 2-D conditions differ physiologically from their in vivo counterparts. Many of these differences are believed to result when cells dissociate from their native 3-D tissue structure in vivo as they are made to Cheryl A. Nickerson is Associate Professor in the Department of Microbiology and Immunology at the Tulane University Medical Center, New Orleans, La. C. Mark Ott is the Laboratory Manager at the Microbiology Laboratory at the NASA Johnson Space Center, Houston, Tex. This article is based on a presentation during the 103rd ASM General Meeting, Washington, D.C., May, 2003. Volume 70, Number 4, 2004 / ASM News Y 169 optimized technology for growing 3-D cells under conditions that maintain many of the specialized features of in vivo tissues (Fig.1A). The primary advantage of the RWV over either dynamic or static tissue culture systems is that its lowshear environment allows cells to aggregate, grow three-dimensionally, and differentiate. Within RWV bioreactors, cells are maintained in a gentle fluid orbit, enabling them to attach to one another and to form the fragile connections that are required for complex 3-D structures and to attain a more tissue-like phenotype. Thus, unlike cell and tissue cultures grown in standard 2-D monolayers, cells cultured in the RWV are structurally and functionally similar to in vivo tissues. Moreover, because of their lowshear, low-turbulence operation, RWV bioreactors minimize mechanical cell damage and thus largely solve many challenges associated with suspension culture. That is, (A) The operation of the RWV. The cylindrical culture vessel is filled with growth medium and the cells and microcarriers can be suscells are added. All air bubbles are removed from the culture vessel. The vessel is attached to the rotator base and rotated about the horizontal axis (power supply not shown). Cell aggregate pended without turbulence, while particles establish a fluid orbit within the culture medium in the rotating vessel. As 3-D tissues providing adequate nutrition and grow in size, the rotation speed is adjusted to compensate for the increased settling rates of the oxygenation. The low-shear condilarger particles. Oxygen supply and carbon dioxide removal is achieved through a gas-permeable silicone rubber membrane at the back of the bioreactor. (B) Cells cultured in the RWV are tions in RWV bioreactors are maintained in a gentle fluid orbit. Cell aggregates establish a fluid orbit within the culture medium thought to be similar to particular in the RWV. The sedimentation of the cells within the vessel is offset by the rotating fluid, environments in the body, such as becreating a constant free fall of the cells through the culture medium. (C) Depiction of a single collagen-coated microcarrier bead to which cells have attached on the surface. The microcarrier tween the brush border microvilli of beads are porous, which allows for the establishment of an internal compartment (i.e., the lumenal epithelial cells, that are encountered space) in the 3-D cells, which is essential for the function and architecture of epithelial cells in vivo. by numerous bacterial pathogens during infections of the gastrointestinal, respiratory, and urogenital tracts. propagate as 2-D monolayers on impermeable surfaces. The principal design feature of the RWV bioTreating vessel surfaces with matrix products such as collagen or reactor is based on a horizontally rotating vessel growing cells on collagen-coated inserts can enhance cell-cell and that is bubble-free when filled with culture mecell-matrix interactions and preserve some aspects of the differentidium. As it rotates, both the wall and fluid rotate ated phenotype. However, these systems do not fully model the at the same angular rate, as if they formed a solid complex 3-D cell architecture that is important for the function of in body. The sedimentation of the cells within the vivo tissues. Moreover, researchers observe several important differvessel is offset by the rotating fluid, creating a ences between the pathogenesis of microorganisms in human infecconstant free fall of cells through the culture tions and in commonly used cell culture models. medium (Fig. 1B). This constant free fall through the medium facilitates nutrient exchange and localized mixing. Oxygen is proRWV Apparatus To Generate 3-D Cell Cultures vided to the aggregates through a hydrophobic NASA engineers designed the RWV bioreactor in the early 1990s to membrane on the back of the bioreactor. model effects of weightlessness on cells. That bioreactor led to an Ample oxygen and nutrient exchange allow FIGURE 1 170 Y ASM News / Volume 70, Number 4, 2004 these aggregates to grow to a substantial size. Limited shear in the vessel not only reduces cell damage but also facilitates 3-D aggregation based on natural cellular affinities. By manipulating the rotational velocity, the cell aggregates can be maintained in suspension with minimal contact against the walls of the reactor. The result is assembly of 3-D tissue-like aggregates— sometimes called “organoids”—that closely resemble natural tissues. These 3-D tissue cultures are being used in a wide variety of fundamental biomedical applications besides infectious disease research, including studying immune-cell interactions, growing tissues for transplantation, cancer biology, developing and testing novel therapeutic drugs, angiogenesis, toxicology, and producing a variety of important products, such as antibodies, hormones, and vitamins. Researchers have published many peer-reviewed articles about 3-D cell culture in RWV bioreactors and, to date, more than 50 normal and transformed cell types have been grown under such conditions. Applying 3-D Cell Cultures to the Study of Infectious Diseases One advantage of cell culture in the RWV is its simplicity. Cells that are to be cultured in RWV bioreactors are first grown as monolayer cultures. When cells reach an appropriate density, they are removed from their flasks, resuspended in fresh medium, and incubated with dextran microcarrier beads that are coated with collagen or another suitable extracellular matrix protein to allow for cell attachment (Fig. 1C). Cells that are cultured on microcarrier beads represent a dual-chamber system where the porous bead matrix constitutes a second compartment that is absent in standard flat monolayer cultures. This second chamber (i.e., the internal compartment within the porous microcarrier) is important because it is highly likely that ion and solute gradients established by the 3-D cells between these two compartments contribute to the establishment of differentiated phenotypes. (This concept is depicted in an animated supplement to the online version of this article.) Once cell-bead complexes are introduced into the RWV vessel, it is made to rotate. The medium subsequently is changed as necessary, and the rotation speed is increased as aggregates develop to maintain the cells in free-falling suspension. When used in infection studies, the 3-D cells are removed from the bioreactor and distributed evenly in wells of tissue culture plates. In this regard, the 3-D cells are treated in the same manner as monolayers, except care should be taken when removing media and washing the cells because they do not adhere to the wells. The 3-D cells can also be treated to remove them from beads for studies that require homogeneous cell suspensions, such as when flow cytometry analysis is being used. If warranted, studies also may be conducted on cells while they are in the bioreactor. RWV bioreactor-grown cells are being used to study microbial pathogenesis, providing physiologically relevant model systems. For example, we have developed a variety of different 3-D cell culture models of human cells and tissues that recapitulate many aspects of cellular structure, differentiation, and function that occur in vivo, and which are currently being used in infection studies by us and our colleagues. The key component of each of these models is a novel 3-D architecture generated in the RWV bioreactor. In each of these models, the 3-D cells display a phenotype that more closely approximates the parental in vivo tissue than do the same cells grown as standard monolayers. Modeling Salmonella Pathogenesis Our laboratory reported the first use of 3-D cell cultures of human intestinal epithelial cells generated in RWV bioreactors for studying the enteric bacterial pathogen Salmonella enterica serovar Typhimurium. S. enterica serovar Typhimurium is among the most common causes of foodborne infections resulting in gastroenteritis and diarrheal disease in humans worldwide, making it a major public health problem. An essential feature of the pathogenicity of Salmonella is its interaction with host intestinal epithelial cells. However, investigators studying such interactions have relied primarily on animal models and conventional tissue cultures of human cell lines, neither of which accurately duplicates human intestinal mucosa. Thus, we developed 3-D cell culture models of human small intestinal epithelium that approximate the behavior of in vivo tissues, bringing these models closer to the environment encountered by Salmonella during the natural course of infections. Specifically, we used the human in- Volume 70, Number 4, 2004 / ASM News Y 171 FIGURE 2 Transmission electron micrographs of 3-D Int-407 aggregates as compared to Int-407 monolayers. This figure shows TEM images of Int-407 monolayers (A) and 3-D Int-407 cells (B). The monolayers demonstrated inferior tight junction formation as compared to the 3-D aggregates (concave arrows). Note the presence of numerous, well-formed vacuolar-like structures in the 3-D aggregates as compared to their poorly developed counterparts in the monolayers (arrow heads). (C-F) Scanning electron micrographs of uninfected and Salmonella-infected Int-407 monolayers and 3-D Int-407 cell aggregates (2,000X). Uninfected monolayer control (C). At 2 hours postinfection, Int-407 cell monolayers exhibit major loss of structural integrity with numerous membrane alterations including blebs and the formation of pathological lesions, as well as some surface bound bacteria (D). Uninfected 3-D Int-407 control showing dense masses of cells with extracellular secretions (E). At 2 hours postinfection, the surface of the 3-D Int-407 cells is quite irregular and associated with what appear to be numerous surface lesions. (F). Note that the 3-D Int-407 cell aggregates infected with Salmonella do not display as extensive loss of structural integrity as observed for infected monolayers following the same time-course of infection. Few surface-bound bacteria are observed. (Reprinted with permission from ASM, Nickerson et al., Infect. Immun. 69:7106 –7120). testinal epithelial cell line Int-407, which is one of the most extensively studied for this purpose. However, when grown in monolayers, Int-407 cells are poorly differentiated, lack polarity, do not produce mucus, and lack other physiological features associated with their 172 Y ASM News / Volume 70, Number 4, 2004 in vivo counterparts, including tight junctions, desmosomes, and secretory granules. Thus, the Int-407 cell line provided us an excellent opportunity for determining whether the RWV bioreactors would enable such cells to differentiate into 3-D tissue-like masses that more accurately model in vivo human intestinal epithelial tissues. On the basis of immunohistochemical, histological, and morphological analyses, the Int-407 cells that we grew in the RWV bioreactors indeed formed 3-D aggregates that retain many in vivo features. For example, unlike monolayers, these 3-D cells exhibit inherent apical and basolateral polarity, and also form extensive tight junctions and desmosomes, which are important for organizing and maintaining normal epithelial structure (Fig. 2A-B). Moreover, according to immunofluorescence and light microscopy studies, the 3-D cells express numerous markers of epithelial differentiation, including type IV collagen, laminin, cadherin, desmoplakin (Fig. 3A), ZO-1, epithelial-specific antigen, villin, vimentin, fibronectin, the M-cell glycoconjugate sialyl Lewis A antigen, and a variety of cytokeratins. In addition, these 3-D Int-407 cells exhibit well-formed and numerous microvilli at apical cell surfaces, abundant and well-developed vacuoles (Fig. 2AB), and produce mucus. All of these are important physiological features of in vivo tissues which were either absent or not expressed or distributed at physiologically relevant levels in monolayer cultures of the same cells. Moreover, cytokeratins 18 and 19, which are markers for tumor and undifferentiated cells, are down-regulated in 3-D Int-407 aggregates compared to monolayers. This finding is important because the Int-407 small intestinal epithelial cell line also contains HeLa cells that derive from a human cervical adenocarcinoma cell line. 3-D Cultured Cells Outperform 2-D Cells as Infection Models When we infected 3-D Int-407 cells with S. enterica serovar Typhimurium, we noticed im- portant differences and a closer resemFIGURE 3 blance to typical in vivo infections compared to the same infections in monolayer cells, including differences in tissue pathology, adherence, invasion, apoptosis, and expression of cytokines. A representative confocal image of the 3-D Int-407 cells infected with serovar Typhimurium shows the complex, tissue-like arrangement of these cells (Fig. 4D). We also used scanning electron microscopy (SEM) to examine surface interactions and membrane structural alterations during the course of Salmonella infections of 3-D and monolayer Int-407 cells. Following the same time course, the 3-D Int-407 cells display minimal loss of structural integrity and more rapid recovery of cell structure, whereas their monolayer counterparts show a major loss of structural integrity (Fig. 2C-F). The response of the 3-D Int-407 cells following infection is relevant to that observed in vivo, where serovar Typhimurium-induced damage (A) Immunofluorescent image of 3-D Int-407 intestinal cell aggregates stained with a to the small intestinal epithelium is monoclonal antibody against desmoplakin. Note the enrichment of the intercellular junction marker, desmoplakin, a protein component of desmosomes, at cell-cell borders short-lived, followed by rapid repair of (see arrows), especially where multiple cell aggregates express desmoplakins. Magnifidamaged cells along with restoration of cation, x400. Arrowheads point to desmoplakin associated with cell-cell interfaces. The the mucosal surface. 150m carrier beads are delineated by a dotted outline. (B) Immunofluorescent image of 3-D HT-29 colon cell aggregates stained with a monoclonal antibody against villin. HT-29 Additional results from adherence, 3-D aggregates show well-organized staining, and clear junction association for the invasion, and apoptosis studies support cytoskeletal marker villin (an abundant protein in the brush border of intestinal epithelial the tissue pathology observations recells). Arrowheads point to villin associated with cell-cell interfaces. (C) Immunofluorescent image of 3-D A549 lung cell aggregates stained with a monoclonal antibody against garding epithelial damage. Specifically, occludin. The distribution of the tight junction-associated protein occludin in the 3-DSalmonella are less able to adhere to cultured A549 cells shows a concentration of this protein at epithelial junctions and invade 3-D Int-407 cells, compared (arrowheads), which is reflective of stabilization of tight junctions. (D) Immunofluorescent image of 3-D SGHPL-4 placental extravillous cytotrophoblast cell aggregates stained with to infected monolayers. Moreover, an antibody against collagen IV. SGHPL-4 cells were derived from first trimester chorionic there was a rapid onset of apoptosis villous explants from a normal, healthy human placenta. The 3-D aggregates express following Salmonella infection of Intcollagen type IV (see arrowheads), an important extracellular matrix protein involved in cytotrophoblast differentiation. 407 monolayers, with 70 –90% of cells undergoing apoptosis within 90 minutes. In contrast, there was no difference in apoptosis between infected and uningrowth factor-1 (TGF-1) mRNA and prostafected 3-D Int-407 cells during this same glandin E2 (PGE2) as compared to uninfected monolayers. Both of these molecules have the postinfection period. Considering that fewer potential to limit damage to the intestinal epithan 5% of cases of Salmonella-induced gastrothelium following infection with invasive enteric enteritis are reported, it seems unlikely that bebacteria such as Salmonella. TGF-1 frequently tween 70 –90% of human intestinal epithelial serves in an immunosuppressive role. The encells would undergo apoptosis after infection hanced basal level of expression of TGF-1 in with this pathogen. the uninfected 3-D Int-407 cells as compared In further support of these tissue pathology to monolayers resembles conditions found in studies, uninfected 3-D Int-407 cells constituvivo, where the intestinal mucosa constitutively tively express higher levels of transforming Volume 70, Number 4, 2004 / ASM News Y 173 FIGURE 4 (A, B) Monolayers and 3-D-aggregates, respectively, of the human colon cell line HT-29, infected with S. enterica serovar Typhimurium. HT-29 cells were infected with S. typhimurium and incubated for 1hr at 37°C. The actin filaments are stained with phalloidin (red), the cell nucleus with DAPI (blue), and serovar typhimurium with a FITC-conjugated LPS antibody (green). The arrowheads point to actin accumulation near the site of Salmonella invasion. Note the complex tissue-like architecture which is clearly evident in the 3-D HT-29 cells as compared to monolayers of the same cells. (C) Immunofluorescent image of SGHPL-4 human placental 3-D-aggregates infected with human cytomegalovirus (HCMV). SGHPL-4 3-D cells were infected with HCMV and incubated for 48 hours at 37°C. The 3-D cells stain positive for the nuclear HCMV immediate early proteins (IE1/2, red), indicating that the cells are permissive for in vitro HCMV infection. Cell nuclei are stained with DAPI (blue). Magnification, x100. (D) Confocal image of Int-407 3-D cells following infection with serovar Typhimurium. Phalloidin labeling of the actin cytoskeleton of 3-D Int-407 cells (red) at 90 minutes after infection with GFP-labeled serovar Typhimurium (green). Reprinted with permission from Synthecon, Inc. (E-F) SEM analysis of 3-D A549 human lung aggregates before and after infection with Pseudomonas aeruginosa. Panels E–F correspond to uninfected 3-D aggregates (E), and 3-D aggregates 6 hours postinfection with P. aeruginosa (F). Magnification, x2,000. Note the complex tissue-like architecture in these cells as well as the clear difference in tissue morphology between the uninfected 3-D control (E) and 6 hour infected 3-D cells (F). The arrowhead points to a Pseudomonas bacterium on the surface of the 3-D cells. produces high levels of this cytokine. Prostaglandins may protect the mucosa by downregulating proinflammatory cytokines such as interleukin-1 (IL-1). Indeed, we observed that IL-1␣ and IL-1 mRNA expression were upregulated in Int-407 monolayers compared to 3-D aggregates following Salmonella infection. The results from these studies are in agreement with a role for prostaglandins in mediating a protective response against Salmonella-induced damage to the epithelial mucosa. Versatility of 3-D Cells for Modeling Other Infectious Diseases Based on our studies of Salmonella infection of 3-D intestinal epithelial cells, we believe that 174 Y ASM News / Volume 70, Number 4, 2004 RWV bioreactors could be used to generate 3-D aggregates from a variety of other cell types to model many other infectious diseases. Hence, we have been generating other 3-D cell cultures from different human tissues, including the colon, lung, placenta, bladder, and periodontal ligaments that are currently being used in collaborative infectious disease studies. Specifically, the 3-D colon cells are being used to study infections with Salmonella sp., Vibrio sp., and other enteric pathogens. Meanwhile the 3-D lung cells are being used to study infections caused by Pseudomonas aeruginosa and Klebsiella pneumoniae, while the 3-D placental tissues are serving as models of human cytomegalovirus infections during pregnancy. Additionally, the 3-D bladder cells are being used to investigate host-pathogen interactions with uropathogenic Escherichia coli, and the 3-D periodontal ligament cells are serving as useful models to study Porphymonas gingivalis and other pathogens of the oral cavity. On the basis of immunohistochemical profiling, each of these newer 3-D cell cultures is already proving to be representative of their respective in vivo parental tissues (Fig. 3B–D). Indeed, numerous markers appear to be distributed in a more physiologically relevant manner in 3-D cells compared to those grown in monolayer cultures on collagen-coated vessels. We have used fluorescent staining and SEM to study several pathogens infecting these 3-D cell cultures, including serovar Typhimurium in the colon, cytomegalovirus in the placenta, and P. aeruginosa in lung cells (Fig. 4A-C, E-F). We can further modulate 3-D cell culture systems—for instance, by introducing biological signals that mimic what such tissues encounter in vivo. Many different relevant materials could be introduced into the culture system, including extracellular matrix components of the interstitial and basement membranes, bioscaffolding materials, growth factors, hormones, nutritional supplements, cytokines, chemokines, and other bioactive molecules. In addition, such studies also could include the use of several relevant cell types simultaneously in a “co-culture” model, including established cell lines and primary cells from particular tissues, as part of a broader effort to understand particular host-pathogen interactions. The RWV bioreactor technology represents a versatile new approach for investigating infectious diseases— one that is just beginning to be tapped. Because cells generated in the RWV so closely approximate cells in native tissues, 3-D cultures offer a powerful approach for understanding host-pathogen interactions at a detailed level and will surely provide insights helpful for developing novel products and procedures for diagnosing, preventing, and treating infectious diseases. ACKNOWLEDGMENTS We thank Jim Wilson and Kerstin Höner zu Bentrup for critical reading of this manuscript; Kerstin Höner zu Bentrup for generating fluorescent images and figure panels; and Rajee Ramamurthy, Carly LeBlanc, Duane Pierson, Neal Pellis, Cindy Morris, Heather LaMarca, Steve Alexander, Kamal Emami, Tom Goodwin, Mayra Nelman-Gonzalez, Michael Schurr, Alex Carterson, Allen Honeyman, Virginia Miller, and Scott Hultgren for helpful discussions and/or sharing aspects of their work for this manuscript. We thank Synthecon Inc. for their permission to use the confocal image of the 3-D Int-407 cells that Kerstin Höner zu Bentrup generated with the assistance of Luis Marrero and Chassidy Johnson. Work in Cheryl Nickerson’s lab is supported, in part, by a grant from the National Aeronautics and Space Administration (NASA-Ames grant NAG 2–1378), and by a generous grant from the W. M. William Keck Foundation of Los Angeles, Calif. SUGGESTED READING Abbott, A. 2003. Biology’s new dimension. Nature 424:870 – 872. Carterson, A. J., C. M. Ott, M. S. Clarke, C. R. Vanderburg, C. A. Nickerson, M. J. Schurr. 2003. A549 lung epithelial cells grown as 3-D aggregates: alternative tissue culture model for P. aeruginosa pathogenesis. American Society for Microbiology, General Meeting, Washington, D.C. 2003. B-131. Hammond, T. G., and J. M. Hammond. 2001. Optimized suspension culture: the rotating-wall vessel. Am. J. Physiol. Renal Physiol. 281(1):F12–25. LaMarca, H. L., C. M. Ott, K. Höner zu Bentrup, C. L. LeBlanc, D. L. Pierson, C. A. Nickerson, A. B. Nelson, and C. A. Morris. 2003. A novel three-dimensional model of human placental cytomegalovirus infection. South Central Branch American Society for Microbiology Annual Meeting, New Orleans, La. VP#13. Long, J. P., S. Pierson, and J. H. Hughes. 1999. Suppression of Epstein-Barr virus reactivation in lymphoblastoid cells cultured in simulated microgravity. In Vitro Cell. Dev. Biol. 35(1):49 –54. Nickerson, C. A., C. M. Ott, J. W. Wilson, R. Ramamurthy, C. L. LeBlanc, K. Höner zu Bentrup, T. Hammond, and D. L. Pierson. 2003. Low shear modeled microgravity: a global environmental regulatory signal affecting bacterial gene expression, physiology, and pathogenesis. J. Microbiol. Methods 54:1–11. Nickerson, C. A., T. J. Goodwin, J. Terlonge, C. M. Ott, K. L. Buchanan, W. B. Uicker, K. Emami, C. L. Cedor, R. Ramamurthy, T. Hammond, and D. L. Pierson. 2001. Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serovar Typhimurium pathogenesis. Infect. Immun. 69:7106 –7120. O’Brien, L. E., M. P. Zegers, and K. E. Mostov. 2002. Building epithelial architecture: insights from three-dimensional culture models. Nature Rev. Mol. Cell. Biol. 3:531–537. Synthecon website. http://www.synthecon.com/ Unsworth, B. R., and P. I. Lelkes. 1998. Growing tissues in microgravity. Nature Med. 4:901–907. Volume 70, Number 4, 2004 / ASM News Y 175