Cultivation of cell-polymer tissue constructs in simulated microgravity

Tissue-engineered cartilage was cultivated under conditions of simulated microgravity using rotating bioreactors. Rotation randomized the effects of gravity on inoculated cells (chondrocytes) and permitted their attachment to three-dimensional (3D) synthetic, biodegradable polymer scaffolds that were freely suspended within the vessel. After 1 week of cultivation, the cells regenerated a cartilaginous extracellular matrix (ECM) consisting of glycosaminoglycan (GAG) and collagen types I and II. Tissue constructs grown in simulated microgravity had higher GAG contents and thinner outer capsules than control constructs grown in turbulent spinner flasks. Two fluid dynamic regimes of simulated microgravity were identified, depending on the vessel rotation speed: (i) a settling regime in which the constructs were maintained in a state of continuous free-fall close to a stationary point within the vessel and (ii) an orbiting regime in which the constructs orbited around the vessel spin axis. In the settling regime, the numerically calculated relative fluid-construct velocity was comparable to the experimentally measured construct settling velocity (2-3 cm/s). A simple mathematical model was used in conjunction with measured construct physical properties to determine the hydrodynamic drag force and to estimate the hydrodynamic stress at the construct surface (1.5 dyn/cm(2)). Rotating bioreactors thus provide a powerful research tool for cultivating tissue-engineered cartilage and studying 3D tissue morphogenesis under well-defined fluid dynamic conditions. (c) 1995 John Wiley & Sons, Inc.     

Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage

Tissue engineered cartilage can be grown in vitro if the necessary physical and biochemical factors are present in the tissue culture environment. Cell metabolism and tissue composition were studied for engineered cartilage cultured for 5 weeks using bovine articular chondrocytes, polymer scaffolds (5 mm diameter x 2 mm thick fibrous discs), and rotating bioreactors. Medium pH and concentrations of oxygen, carbon dioxide, glucose, lactate, ammonia, and glycosoaminoglycan (GAG) were varied by altering the exchange rates of gas and medium in the bioreactors. Cell-polymer constructs were assessed with respect to histomorphology, biochemical composition and metabolic activity. Low oxygen tension ( approximately 40 mmHg) and low pH ( approximately 6.7) were associated with anaerobic cell metabolism (yield of lactate on glucose, YL/G, of 2.2 mol/mol) while higher oxygen tension ( approximately 80 mmHg) and higher pH ( approximately 7.0) were associated with more aerobic cell metabolism (YL/G of 1.65-1.79 mol/mol). Under conditions of infrequent medium replacement (50% once per week), cells utilized more economical pathways such that glucose consumption and lactate production both decreased, cell metabolism remained relatively aerobic (YL/G of 1.67 mol/mol) and the resulting constructs were cartilaginous. More aerobic conditions generally resulted in larger constructs containing higher amounts of cartilaginous tissue components, while anaerobic conditions suppressed chondrogenesis in 3D tissue constructs.     

Cartilaginous tissue formation from bone marrow cells using rotating wall vessel (RWV) bioreactor

This is the first successful report of the rapid regeneration of three-dimensional large and homogeneous cartilaginous tissue from rabbit bone marrow cells without a scaffold using a rotating wall vessel (RWV) bioreactor, which simulates a microgravity environment for cells. Bone marrow cells cultured for 3 weeks in DMEM were resuspended and cultured for 4 weeks in the chondrogenic medium within the vessel. Large cylindrical cartilaginous tissue with dimensions of (1.25 +/- 0.06) x (0.60 +/- 0.08) cm (height x diameter) formed. Their cartilage marker expression was confirmed by mRNA expressions of aggrecan, collagen type I and II, and glycosaminoglycan (GAG)/DNA ratio. Their cartilaginous properties were demonstrated by toluidine blue, safranin-O staining, and polarization.     

The effect of continuous culture on the growth and structure of tissue‐engineered cartilage

The use of bioreactors for cartilage tissue engineering has become increasingly important as traditional batch‐fed culture is not optimal for in vitro tissue growth. Most tissue engineering bioreactors rely on convection as the primary means to provide mass transfer; however, convective transport can also impart potentially unwanted and/or uncontrollable mechanical stimuli to the cells resident in the construct. The reliance on diffusive transport may not necessarily be ineffectual as previous studies have observed improved cartilaginous tissue growth when the constructs were cultured in elevated volumes of media. In this study, to approximate an infinite reservoir of media, we investigated the effect of continuous culture on cartilaginous tissue growth in vitro. Isolated bovine articular chondrocytes were seeded in high density, 3D culture on Millicell? filters. After two weeks of preculture, the constructs were cultivated with or without continuous media flow (5–10 μL/min) for a period of one week. Tissue engineered cartilage constructs grown under continuous media flow significantly accumulated more collagen and proteoglycans (increased by 50–70%). These changes were similar in magnitude to the reported effect of through‐thickness perfusion without the need for large volumetric flow rates (5–10μL/min as opposed to 240–800 μL/min). Additionally, tissues grown in the reactor displayed some evidence of the stratified morphology of native cartilage as well as containing stores of intracellular glycogen. Future studies will investigate the effect of long‐term continuous culture in terms of extracellular matrix accumulation and subsequent changes in mechanical function. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Tissue engineering of human cartilage in bioreactors using single and composite cell-seeded scaffolds

Chondrocytes isolated from human fetal epiphyseal cartilage were seeded under mixed conditions into 15-mm-diameter polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to generate cartilage constructs. After seeding, the cell distributions in thick (4.75 mm) and thin (2.15 mm) PGA disks were nonuniform, with higher cell densities accumulating near the top surfaces. Composite scaffolds were developed by suturing together two thin PGA disks after seeding to manipulate the initial cell distribution before bioreactor culture. The effect of medium flow direction in the bioreactors, including periodic reversal of medium flow, was also investigated. The quality of the tissue-engineered cartilage was assessed after 5 weeks of culture in terms of the tissue wet weight, glycosaminoglycan (GAG), total collagen and collagen type II contents, histological analysis of cell, GAG and collagen distributions, and immunohistochemical analysis of collagen types I and II. Significant enhancement in construct quality was achieved using composite scaffolds compared with single PGA disks. Operation of the bioreactors with periodic medium flow reversal instead of unidirectional flow yielded further improvements in tissue weight and GAG and collagen contents with the composite scaffolds. At harvest, the constructs contained GAG concentrations similar to those measured in ex vivo human adult articular cartilage; however, total collagen and collagen type II levels were substantially lower than those in adult tissue. This study demonstrates that the location of regions of high cell density in the scaffold coupled with application of dynamic bioreactor operating conditions has a significant influence on the quality of tissue-engineered cartilage.     

Modulation of the mechanical properties of tissue engineered cartilage

Cartilaginous constructs have been grown in vitro using chondrocytes, biodegradable polymer scaffolds, and tissue culture bioreactors. In the present work, we studied how the composition and mechanical properties of engineered cartilage can be modulated by the conditions and duration of in vitro cultivation, using three different environments: static flasks, mixed flasks, and rotating vessels. After 4-6 weeks, static culture yielded small and fragile constructs, while turbulent flow in mixed flasks induced the formation of an outer fibrous capsule; both environments resulted in constructs with poor mechanical properties. The constructs that were cultured freely suspended in a dynamic laminar flow field in rotating vessels had the highest fractions of glycosaminoglycans and collagen (respectively 75% and 39% of levels measured in native cartilage), and the best mechanical properties (equilibrium modulus, hydraulic permeability, dynamic stiffness, and streaming potential were all about 20% of values measured in native cartilage). Chondrocytes in cartilaginous constructs remained metabolically active and phenotypically stable over prolonged cultivation in rotating bioreactors. The wet weight fraction of glycosaminoglycans and equilibrium modulus of 7 month constructs reached or exceeded the corresponding values measured from freshly explanted native cartilage. Taken together, these findings suggest that functional equivalents of native cartilage can be engineered by optimizing the hydrodynamic conditions in tissue culture bioreactors and the duration of tissue cultivation.     

Bioreactor studies of native and tissue engineered cartilage

Functional tissue engineering of cartilage involves the use of bioreactors designed to provide a controlled in vitro environment that embodies some of the biochemical and physical signals known to regulate chondrogenesis. Hydrodynamic conditions can affect in vitro tissue formation in at least two ways: by direct effects of hydrodynamic forces on cell morphology and function, and by indirect flow-induced changes in mass transfer of nutrients and metabolites. In the present work, we discuss the effects of three different in vitro environments: static flasks (tissues fixed in place, static medium), mixed flasks (tissues fixed in place, unidirectional turbulent flow) and rotating bioreactors (tissues dynamically suspended in laminar flow) on engineered cartilage constructs and native cartilage explants. As compared to static and mixed flasks, dynamic laminar flow in rotating bioreactors resulted in the most rapid tissue growth and the highest final fractions of glycosaminoglycans and total collagen in both tissues. Mechanical properties (equilibrium modulus, dynamic stiffness, hydraulic permeability) of engineered constructs and explanted cartilage correlated with the wet weight fractions of glycosaminoglycans and collagen. Current research needs in the area of cartilage tissue engineering include the utilization of additional physiologically relevant regulatory signals, and the development of predictive mathematical models that enable optimization of the conditions and duration of tissue culture.     

Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development

A concentric cylinder bioreactor has been developed to culture tissue engineered cartilage constructs under hydrodynamic loading. This bioreactor operates in a low shear stress environment, has a large growth area for construct production, allows for dynamic seeding of constructs, and provides for a uniform loading environment. Porous poly-lactic acid constructs, seeded dynamically in the bioreactor using isolated bovine chondrocytes, were cultured for 4 weeks at three seeding densities (60, 80, 100 x 10(6) cells per bioreactor) and three different shear stresses (imposed at 19, 38, and 76 rpm) to characterize the effect of chondrocyte density and hydrodynamic loading on construct growth. Construct seeding efficiency with chondrocytes is greater than 95% within 24 h. Extensive chondrocyte proliferation and matrix deposition are achieved so that after 28 days in culture, constructs from bioreactors seeded at the highest cell densities contain up to 15 x 10(6) cells, 2 mg GAG, and 3.5 mg collagen per construct and exhibit morphology similar to that of native cartilage. Bioreactors seeded with 60 million chondrocytes do not exhibit robust proliferation or matrix deposition and do not achieve morphology similar to that of native cartilage. In cultures under different steady hydrodynamic loading, the data demonstrate that higher shear stress suppresses matrix GAG deposition and encourages collagen incorporation. In contrast, under dynamic hydrodynamic loading conditions, cartilage constructs exhibit robust matrix collagen and GAG deposition. The data demonstrate that the concentric cylinder bioreactor provides a favorable hydrodynamic environment for cartilage construct growth and differentiation. Notably, construct matrix accumulation can be manipulated by hydrodynamic loading. This bioreactor is useful for fundamental studies of construct growth and to assess the significance of cell density, nutrients, and hydrodynamic loading on cartilage development. In addition, studies of cartilage tissue engineering in the well-characterized, uniform environment of the concentric cylinder bioreactor will develop important knowledge of bioprocessing parameters critical for large-scale production of engineered tissues.     

Design and characterization of a rotating bed system bioreactor for tissue engineering applications

The main challenge in the development of bioreactors for tissue engineering is the delivery of a sufficient nutrient and oxygen supply for cell growth in a 3D environment. Thus, a new rotating bed system bioreactor for tissue engineering applications was developed. The system consists of a culture vessel as well as an integrated rotating bed of special porous ceramic discs and a process control unit connected with the reactor to ensure optimal culturing conditions. The aim of the project was the design and construction of a fully equipped rotating bed reactor, and in particular, the characterization and optimization of the system with regard to technical parameters such as mixing time and pH-control to guarantee optimal conditions for cell growth and differentiation. Furthermore, the applicability of the developed system was demonstrated by cultivation of osteoblast precursor cells. The porous structure of the ceramic discs and the external medium circulation loop provide an optimal environment for tissue generation in long-term cultivations. Mass transfer limitations were minimized by the slow rotation, which also provides the cells with sufficient nutrients and oxygen through alternate contact to air and medium. An osteoblast precursor cell line was successfully cultivated in this bioreactor for 28 days.     

Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization.

Cardiac tissue engineering has been motivated by the need to create functional tissue equivalents for scientific studies and cardiac tissue repair. We previously demonstrated that contractile cardiac cell-polymer constructs can be cultivated using isolated cells, 3-dimensional scaffolds, and bioreactors. In the present work, we examined the effects of (1) cell source (neonatal rat or embryonic chick), (2) initial cell seeding density, (3) cell seeding vessel, and (4) tissue culture vessel on the structure and composition of engineered cardiac muscle. Constructs seeded under well-mixed conditions with rat heart cells at a high initial density ((6-8) x 10(6) cells/polymer scaffold) maintained structural integrity and contained macroscopic contractile areas (approximately 20 mm(2)). Seeding in rotating vessels (laminar flow) rather than mixed flasks (turbulent flow) resulted in 23% higher seeding efficiency and 20% less cell damage as assessed by medium lactate dehydrogenase levels (p < 0.05). Advantages of culturing constructs under mixed rather than static conditions included the maintenance of metabolic parameters in physiological ranges, 2-4 times higher construct cellularity (p &le 0.0001), more aerobic cell metabolism, and a more physiological, elongated cell shape. Cultivations in rotating bioreactors, in which flow patterns are laminar and dynamic, yielded constructs with a more active, aerobic metabolism as compared to constructs cultured in mixed or static flasks. After 1-2 weeks of cultivation, tissue constructs expressed cardiac specific proteins and ultrastructural features and had approximately 2-6 times lower cellularity (p < 0.05) but similar metabolic activity per unit cell when compared to native cardiac tissue.     

Novel technique for online characterization of cartilaginous tissue properties

The goal of tissue engineering is to use substitutes to repair and restore organ function. Bioreactors are an indispensable tool for monitoring and controlling the unique environment for engineered constructs to grow. However, in order to determine the biochemical properties of engineered constructs, samples need to be destroyed. In this study, we developed a novel technique to nondestructively online-characterize the water content and fixed charge density of cartilaginous tissues. A new technique was developed to determine the tissue mechano-electrochemical properties nondestructively. Bovine knee articular cartilage and lumbar annulus fibrosus were used in this study to demonstrate that this technique could be used on different types of tissue. The results show that our newly developed method is capable of precisely predicting the water volume fraction (less than 3% disparity) and fixed charge density (less than 16.7% disparity) within cartilaginous tissues. This novel technique will help to design a new generation of bioreactors which are able to actively determine the essential properties of the engineered constructs, as well as regulate the local environment to achieve the optimal conditions for cultivating constructs.     

Effect of simulated microgravity and shear stress on microcin B17 production by Escherichia coli and on its excretion into the medium.

Production of the antibacterial polypeptide microcin B17 (MccB17) by Escherichia coli ZK650 was inhibited by simulated microgravity. The site of MccB17 accumulation was found to be different, depending on whether the organism was grown in shaking flasks or in rotating bioreactors designed to establish a simulated microgravity environment. In flasks, the accumulation was cellular, but in the reactors, virtually all the microcin was found in the medium. The change from a cellular site to an extracellular one was apparently not a function of gravity, since extracellular production occurred in these bioreactors, irrespective of whether they were operated in the simulated microgravity or normal gravity mode. More probably, excretion is due to the much lower degree of shear stress in the bioreactors. Addition of even a single glass bead to the 50-ml medium volume in the bioreactor created enough shear to change the site of MccB17 accumulation from the medium to the cells.     

Bi-zonal cartilaginous tissues engineered in a rotary cell culture system

In this study, we aimed at validating a rotary cell culture system (RCCS) bioreactor with medium recirculation and external oxygenation, for cartilage tissue engineering. Primary bovine and human culture-expanded chondrocytes were seeded into non-woven meshes of esterified hyaluronan (HYAFF-11), and the resulting constructs were cultured statically or in the RCCS, in the presence of insulin and TGFbeta3, for up to 4 weeks. Culture in the RCCS did not induce significant differences in the contents of glycosaminoglycans (GAG) and collagen deposited, but markedly affected their distribution. In contrast to statically grown tissues, engineered cartilage cultured in the RCCS had a bi-zonal structure, consisting of an outgrowing fibrous capsule deficient in GAG and rich in collagen, and an inner region more positively stained for GAG. Structurally, trends were similar using primary bovine or expanded human chondrocytes, although the human cells deposited inferior amounts of matrix. The use of the presented RCCS, in conjunction with the described medium composition, has the potential to generate bi-zonal tissues with features qualitatively resembling the native meniscus.     

Temporal assessment of ribose treatment on self-assembled articular cartilage constructs

Articular cartilage cannot repair itself in response to degradation from injury or osteoarthritis. As such, there is a substantial clinical need for replacements of damaged cartilage. Tissue engineering aims to fulfill this need by developing replacement tissues in vitro. A major goal of cartilage tissue engineering is to produce tissues with robust biochemical and biomechanical properties. One technique that has been proposed to improve these properties in engineered tissue is the use of non-enzymatic glycation to induce collagen crosslinking, an attractive solution that may avoid the risks of cytotoxicity posed by conventional crosslinking agents such as glutaraldehyde. The objectives of this study were (1) to determine whether continuous application of ribose would enhance biochemical and biomechanical properties of self-assembled articular cartilage constructs, and (2) to identify an optimal time window for continuous ribose treatment. Self-assembled constructs were grown for 4 weeks using a previously established method and were subjected to continuous 7-day treatment with 30 mM ribose during culture weeks 1, 2, 3, or 4, or for the entire 4-week culture. Control constructs were grown in parallel, and all groups were evaluated for gross morphology, histology, cellularity, collagen and sulfated glycosaminoglycan (GAG) content, and compressive and tensile mechanical properties. Compared to control constructs, it was found that treatment with ribose during week 2 and for the entire duration of culture resulted in significant 62% and 40% increases in compressive stiffness, respectively; significant 66% and 44% increases in tensile stiffness; and significant 50% and 126% increases in tensile strength. Similar statistically significant trends were observed for collagen and GAG. In contrast, constructs treated with ribose during week 1 had poorer biochemical and biomechanical properties, although they were significantly larger and more cellular than all other groups. We conclude that non-enzymatic glycation with ribose is an effective method for improving tissue engineered cartilage and that specific temporal intervention windows exist to achieve optimal functional properties.     

Tissue engineering strategies for cartilage generation--micromass and three dimensional cultures using human chondrocytes and a continuous cell line

Utilizing ATDC5 murine chondrogenic cells and human articular chondrocytes, this study sought to develop facile, reproducible three-dimensional models of cartilage generation with the application of tissue engineering strategies, involving biodegradable poly(glycolic acid) scaffolds and rotating wall bioreactors, and micromass pellet cultures. Chondrogenic differentiation, assessed by histology, immunohistochemistry, and gene expression analysis, in ATDC5 and articular chondrocyte pellets was evident by the presence of distinct chondrocytes, expressing Sox-9, aggrecan, and type II collagen, in lacunae embedded in a cartilaginous matrix of type II collagen and proteoglycans. Tissue engineered explants of ATDC5 cells were reminiscent of cartilaginous structures composed of numerous chondrocytes, staining for typical chondrocytic proteins, in lacunae embedded in a matrix of type II collagen and proteoglycans. In comparison, articular chondrocyte explants exhibited areas of Sox-9, aggrecan, and type II collagen-expressing cells growing on fleece, and discrete islands of chondrocytic cells embedded in a cartilaginous matrix.     

The role of bioreactors in cartilage tissue engineering

Cartilage tissue engineering is concerned with developing in vitro cartilage implants that closely match the properties of native cartilage, for eventual implantation to replace damaged cartilage. The three components to cartilage tissue engineering are cell source, such as in vitro expanded autologous chondrocytes or mesenchymal progenitor cells, a scaffold onto which the cells are seeded and a bioreactor which attempts to recreate the in vivo physicochemical conditions in which cartilage develops. Although much progress has been made towards the goal of developing clinically useful cartilage constructs, current constructs have inferior physicochemical properties than native cartilage. One of the reasons for this is the neglect of mechanical forces in cartilage culture. Bioreactors have been defined as devices in which biological or biochemical processes can be re-enacted under controlled conditions e.g. pH, temperature, nutrient supply, O2 tension and waste removal. The purpose of this review is to detail the role of bioreactors in the engineering of cartilage, including a discussion of bioreactor designs, current state of the art and future perspectives.     

Growth Factor Stimulation Improves the Structure and Properties of Scaffold-Free Engineered Auricular Cartilage Constructs

The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.     

A modular approach to creating large engineered cartilage surfaces

Native articular cartilage has limited capacity to repair itself from focal defects or osteoarthritis. Tissue engineering has provided a promising biological treatment strategy that is currently being evaluated in clinical trials. However, current approaches in translating these techniques to developing large engineered tissues remains a significant challenge. In this study, we present a method for developing large-scale engineered cartilage surfaces through modular fabrication. Modular Engineered Tissue Surfaces (METS) uses the well-known, but largely under-utilized self-adhesion properties of de novo tissue to create large scaffolds with nutrient channels. Compressive mechanical properties were evaluated throughout METS specimens, and the tensile mechanical strength of the bonds between attached constructs was evaluated over time. Raman spectroscopy, biochemical assays, and histology were performed to investigate matrix distribution. Results showed that by Day 14, stable connections had formed between the constructs in the METS samples. By Day 21, bonds were robust enough to form a rigid sheet and continued to increase in size and strength over time. Compressive mechanical properties and glycosaminoglycan (GAG) content of METS and individual constructs increased significantly over time. The METS technique builds on established tissue engineering accomplishments of developing constructs with GAG composition and compressive properties approaching native cartilage. This study demonstrated that modular fabrication is a viable technique for creating large-scale engineered cartilage, which can be broadly applied to many tissue engineering applications and construct geometries.     

Cartilage Tissue Engineering: Controversy in the Effect of Oxygen

Articular cartilage lacks the ability to repair itself and consequently defects in this tissue do not heal. Tissue engineering approaches, employing a scaffold material and cartilage producing cells (chondrocytes), hold promise for the treatment of such defects. In these strategies the limitation of nutrients, such as oxygen, during in vitro culture are of major concern and will have implications for proper bioreactor design. We recently demonstrated that oxygen gradients are indeed present within tissue engineered cartilaginous constructs. Interestingly, oxygen, besides being an essential nutrient, is also a controlling agent of developmental processes including cartilage formation. However, the specific role of oxygen in these processes is still obscure despite the recent advances in the field. In particular, the outcome of published investigations is inconsistent regarding the effect of oxygen tension on chondrocytes. Therefore, this article describes the possible roles of oxygen gradients during embryonic cartilage development and reviews the data reported on the effect of oxygen tension on in vitro chondrocyte proliferation and differentiation from a tissue engineering perspective. Furthermore, possible causes for the variance in the data are discussed. Finally, recommendations are included that may reduce the variation, resulting in more reliable and comparable data.     

Erythroid cell growth and differentiation in vitro in the simulated microgravity environment of the nasa rotating wall vessel bioreactor

Prolonged exposure of humans and experimental animals to the altered gravitational conditions of space flight has adverse effects on the lymphoid and erythroid hematopoietic systems. Although some information is available regarding the cellular and molecular changes in lymphocytes exposed to microgravity, little is known about the erythroid cellular changes that may underlie the reduction in erythropoiesis and resultant anemia. We now report a reduction in erythroid growth and a profound inhibition of erythropoietin (Epo)-induced differentiation in a ground-based simulated microgravity model system. Rauscher murine erythroleukemia cells were grown either in tissue culture vessels at 1 x g or in the simulated microgravity environment of the NASA-designed rotating wall vessel (RWV) bioreactor. Logarithmic growth was observed under both conditions; however, the doubling time in simulated microgravity was only one-half of that seen at 1 x g. No difference in apoptosis was detected. Induction with Epo at the initiation of the culture resulted in differentiation of approximately 25% of the cells at 1 x g, consistent with our previous observations. In contrast, induction with Epo at the initiation of simulated microgravity resulted in only one-half of this degree of differentiation. Significantly, the growth of cells in simulated microgravity for 24 h prior to Epo induction inhibited the differentiation almost completely. The results suggest that the NASA RWV bioreactor may serve as a suitable ground-based microgravity simulator to model the cellular and molecular changes in erythroid cells observed in true microgravity.