High Aspect Ratio Vessel Research Articles

Alterations in amino acids metabolism in human immunocytes subjected to microgravity analog

Space flight is complex stress-inducing environment and may alter immune responsiveness. The major objective of this work was to investigate the metabolism of amino acids concurrent with altered immune responsiveness in vitro using a microgravity analog system. High Aspect Ratio Vessel (HARV) produces a time-averaged gravity vector of 10(−2) g and was used as a microgravity analog. Immunocytes from healthy donors were cultured in the HARV and in the static (STAT) environment for 48h in 5%CO2, 95% humidified atmosphere at 37oC in the presence of lipopolysachharide and phytohemagglutinin. Secretion of IL-2, IFN-γ, IL-4, IL-10, and concentrations of 41 amino-metabolites were assessed in the cell culture supernatants, in order to study effects of modeled microgravity on nutrient balance and its possible correlation with immune parameters. Secretion of IFN-γ, IL-4, and IL-10, but not IL-2 were significantly (P<0.05) decreased in HARV environment. Also, significantly lower concentrations of argenine and cysteine were registered in HARV. In the opposite, significantly higher concentrations of histidine, ornithine, serine, tryptophan, ethanolamine, and urea were registered in HARV culture compare to STAT conditions. Stress-factors of microgravity paradigm are able significantly alter metabolism of amino acids in vitro. Such alterations in the utilization or turn-over of amino acids may lead to alterations in cytokine secretion. In part this work was funded by NIH R15 GM62795-03 and NASA NSBRI through Cooperative Agreement NCC 9 58 with NASA.

Inhibition of NF‐[kappa]B activation in osteoclasts by eicosapentaenoic acid

NF-κB is a transcriptional activator of many genes. It is activated after receptor binding of a mediator such as the receptor activator of NF-κB ligand (RANKL) or TNF-α. RANKL activates distinct signaling pathways (such as NF-κB) during osteoclastogenesis and bone resorption. Eicosapentaenoic acid (EPA) is a fatty acid known to inhibit NF-κB activation in certain cells types, but the effects have not been investigated in osteoclasts. We investigated the effects of EPA on RANKL-induced differentiation of RAW264.7 monocyte/macrophage cells, and on NF-κB activation in differentiated RAW264.7 cells exposed to TNF-α or modeled microgravity using a High-Aspect Ratio Vessel (HARV). RAW264.7 cells were incubated with or without 50 μM EPA for 24 h and then differentiated into osteoclast-like cells in the presence of 50 ng/mL RANKL. In a separate experiment, differentiated RAW264.7 cells were treated with 50 μM EPA for 24 h before they were exposed to 0.2 μg/mL TNF-α or modeled microgravity in a HARV. EPA dose-dependently inhibited the RANKL-induced differentiation of RAW264.7 cells. A 24-h incubation with EPA also decreased activation of NF-κB induced by TNF-α or modeled microgravity. These data provide in vitro evidence for the potential of EPA to provide a countermeasure to mitigate bone loss associated with space flight. This study was supported by NASA.

THREE-DIMENSIONAL ADIPOSE TISSUE MODEL USING LOW SHEAR BIOREACTORS

Presented here are techniques developed to culture and analyze three-dimensional (3-D) adipose-like tissues as a means to bridge the gap between current limitations in culturing preadipocytes (PAs) and that of providing clinically relevant volumes of adipose tissue useful for soft tissue engineering strategies in reconstructive surgery. Pilot studies were performed to determine techniques to visualize and analyze 3-D PA-like tissues as well as to develop successful strategies to culture 3T3-L1 cells in a high aspect ratio vessel rotating-wall bioreactor both with and without microcarriers. Next, a series of cultures were accessed to verify these techniques as well as to compare the culture of the cells with and without microcarriers. Finally, a perfused rotating-wall bioreactor was used to further investigate the nature of the aggregates or tissues being generated. The aggregates that formed in the perfused system were analyzed via histology and in vivo animal studies. PA-like tissues as large as 4-5 mm in diameter without microcarriers that were capable of lipid-loading and composed of viable cells were achieved. We have successfully demonstrated that large tissue aggregates can be grown in bioreactor culture systems.

Functional recovery of peripheral blood mononuclear cells in modeled microgravity

Immune function is suppressed in space flight, demonstrated by reduced mitogen-stimulated proliferation of postflight astronaut peripheral blood mononuclear cells (PBMCs). While flight studies are limited, the development of rotating wall vessel (RWV) bioreactors, such as the high aspect ratio vessel (HARV), has facilitated ground-based studies of the effects of modeled microgravity (MMG) on cell-mediated immunity. Astronauts regain immune function 3 days postflight, but this recovery has not yet been demonstrated following MMG. MMG eliminated phytohemagglutinin (PHA)-stimulated proliferation of PBMCs. Upon removal from HARV, full recovery was gradually achieved over a 72 h period, in agreement with postflight studies of astronauts. Recovery from MMG delayed, but did not reduce, the maximal proliferative response compared with PHA-activated stationary cultures. Likewise, peak expression of T cell surface activation markers CD69 and CD25 was delayed upon stimulation following exposure to MMG. MMG and recovery from MMG differentially affected the detection of IL-2 and IFN in supernatants. Further development of this model of immune recovery is important for investigating the mechanisms of immune suppression and recovery in space flight, as well as possible countermeasures to prevent immunosuppression or enhance recovery. Given the analogous immune suppression observed in microgravity, MMG, and aging, further investigation may also lead to advances in anti-aging medicine.

The role of connexins in the differentiation of NT2 cells in Sertoli-NT2 cell tissue constructs grown in the rotating wall bioreactor

Neural transplantation is developing as a successful treatment for neurodegenerative diseases such as Parkinson's disease. The human Ntera-2/D1 (NT2) cell line is an attractive alternative to the use of human fetal neurons as a cell source for transplantation. We have explored combining NT2 cells, as a neuronal source, and Sertoli cells, which may act as a graft facilitator to enhance neuronal survival and differentiation, and ameliorate the host immune response, into a tissue construct for use in cell replacement therapy for neurodegenerative disease. This Sertoli-NT2-aggregated cell (SNAC) tissue construct is formed in the high aspect ratio vessel (HARV) bioreactor. NT2 cells differentiate to dopaminergic NT2N neurons within the SNAC tissue construct without retinoic acid. We report here that the gap junction protein connexin 43 is decreased among differentiated NT2N neurons. Inhibition of connexin 43 with 18beta glycyrrhetinic acid and carbenoxolone, a glycyrrhetinic acid derivative, during formation of the SNAC tissue constructs disrupts the differentiation of NT2 cells. Therefore, connexin 43 is important in the differentiation of NT2 cells in the SNAC tissue construct.

Bone Cell Survival in Microgravity: Evidence that Modeled Microgravity Increases Osteoblast Sensitivity to Apoptogens

Studies were performed to evaluate the effects of modeled microgravity on the induction of osteoblast apoptosis. MC3T3-E1 osteoblast-like cells were cultured in alginate carriers in the NASA-approved high aspect ratio vessel (HARV). This system subjects the cells to a time-averaged gravitational field (vector-averaged gravity) to simulate low gravity conditions. Cells were cultured in the HARV for five days, and then examined for apoptosis. In simulated microgravity, the cells remained vital, although analysis of expressed genes indicated that there was loss of the mature osteoblast phenotype. Additionally, we noted that there was a loss of the mitochondrial membrane potential, a low level of the antiapoptotic protein Bcl-2, as well as Akt protein, and the redox status of the cells was disturbed. All of these parameters indicated that vector-averaged gravity disrupts mitochondrial function, thereby sensitizing osteoblasts to apoptosis. We then used a challenge assay to evaluate the apoptotic sensitivity of the cells subjected to vector-averaged gravity. When challenged with staurosporine, cells subjected to vector-averaged gravity evidenced elevated levels of cell death relative to control cell populations. Another objective of the study was to improve upon conventional carriers by using alginate encapsulation to support cells in the HARV. We have demonstrated that the alginate carrier system affords a more robust system than surface-seeded carriers. This new system has the advantage of shielding cells from mechanical damage and fluid shear stresses on cells in the HARV, permitting carefully controlled studies of the effects of vector-averaged gravity.

Rapid differentiation of NT2 cells in Sertoli–NT2 cell tissue constructs grown in the rotating wall bioreactor

Cell replacement therapy is of great interest as a long-term treatment of neurodegenerative diseases such as Parkinson's disease (PD). We have previously shown that Sertoli cells (SC) provide neurotrophic support to transplants of dopaminergic fetal neurons and NT2N neurons, derived from the human clonal precursors cell line NTera2/D1 (NT2), which differentiate into dopaminergic NT2N neurons when exposed to retinoic acid. We have created SC-NT2 cell tissue constructs cultured in the high aspect ratio vessel (HARV) rotating wall bioreactor. Sertoli cells, NT2, and SC plus NT2 cells combined in starting ratios of 1:1, 1:2, 1:4 and 1:8 were cultured in the HARV in DMEM with 10% fetal bovine serum and 1% growth factor reduced Matrigel for 3 days, without retinoic acid. Conventional, non-HARV, cultures grown in the same culture medium were used as controls. The presence of tyrosine hydroxylase (TH) was assessed in all culture conditions. Sertoli-neuron-aggregated-cell (SNAC) tissue constructs grown at starting ratios of 1:1 to 1:4 contained a significant amount of TH after 3 days of culture in the HARV. No TH was detected in SC HARV cultures, or SC, NT2 or SC-NT2 conventional co-cultures. Quantitative stereology of immunolabled 1:4 SNAC revealed that approximately 9% of NT2 cells differentiate into TH-positive (TH+) NT2N neurons after 3 days of culture in the HARV, without retinoic acid. SNAC tissue constructs also released dopamine (DA) when stimulated with KCl, suggesting that TH-positive NT2N neurons in the SNAC adopted a functional dopaminergic phenotype. SNAC tissue constructs may be an important source of dopaminergic neurons for neuronal transplantation.

Quantitative analysis of three-dimensional fluid flow in rotating bioreactors for tissue engineering.

Tissue engineering has emerged as a viable alternative to the problem of organ and tissue shortage. Our laboratory has developed matrices for bone tissue engineering based on sintered spherical particles and, using bioreactor technology, has demonstrated the ability to produce highly mineralized matrices in vitro. In this study, porous microcapsule scaffolds were developed for bone tissue engineering in the high aspect ratio vessel rotating bioreactor. The motion of individual microcapsules as well as scaffolds in the bioreactor were studied by numerical simulation and in situ imaging analysis. Results show that spherical microcapsules with density less than the surrounding fluid exhibited two motions: (1) a periodic circular orbit with tangential speed equal to the free fall speed of the particle, and (2) an inward radial migration of the circular orbit toward the center of the bioreactor vessel. Lighter-than-water scaffolds were fabricated by sintering poly(lactic-co-glycolic acid) hollow microcarriers with diameter from 500 to 860 microm into a fixed three-dimensional geometry with approximately 30% pore volume and 180 to 190 microm median pore size. Scaffolds were fabricated with aggregate densities ranging from 0.65 g/mL and 0.99 g/mL by appropriate combinations of hollow and solid microcarriers within the scaffold. Scaffold velocity in the bioreactor for the above range of densities was accurately predicted by numerical simulation and ranged from 100 mm/s to 3 mm/s. Maximum shear stress estimation due to media flow over the exterior of the scaffold ranged from 0.3 N/m(2) to 0.006 N/m(2). Internal perfusion velocity through scaffolds also was calculated and ranged from 13 mm/s to 0.2 mm/s. Estimates of maximum interior shear stress ranged from 0.03 to 0.0007 N/m(2). These analytical methods provide an excellent vehicle for the study of bone tissue synthesis in three-dimensional culture with fluid flow.

Growth and membrane polarization in Pseudomonas aeruginosa UG2 grown in randomized microgravity in a high aspect ratio vessel.

Growth and membrane polarization of Pseudomonas aeruginosa UG2 cells grown under randomized microgravity (RMG) and 1xg were measured in a high aspect ratio vessel (HARV) and also in batch cultures mixed at 12 and 150 rpm in Erlenmeyer shake flasks. Membrane polarization was measured using the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). No differences were observed in the growth curves or membrane polarization values (about 0.300) under all three culture conditions. However, the net effect of RMG at the single cell level may be still unknown. It may be possible that RMG effects are species-dependent or bacterial cells with a small mass and volume may be near the threshold where RMG exerts a minimal effect.

Evaluating prostate cancer cell culturing methods: A comparison of cell morphologies and metabolic activity

LNCaP prostate cancer cells were grown under four unique cultivation conditions. Two types of bioreactor systems were used to observe the influence of low-shear culture conditions allowing for three-dimensional growth: a) a perfusion rotating wall vessel (RWV) bioreactor; and b) a high aspect ratio vessel (HARV) RWV bioreactor, with periodic medium exchanges (fed-batch). In addition, two growth methods utilized tissue culture flasks (TCFs): a) unaltered or conventional TCFs; and b) poly(2-hydroxyethyl methacrylate) [poly(HEMA)] coated TCFs, to inhibit cell attachment. Comparisons were drawn based on qualitative observation of cell morphology and quantitative metabolic data. Similar cellular metabolism was demonstrated for cells grown under each condition. The degree of aggregation, however, varies considerably. Spherical shaped aggregates with diameters of 1 to 3 mm were produced when cells were grown within the perfusion-RWV bioreactor. All other growth conditions produced irregular shaped aggregates of various sizes. Quantitative results demonstrated the expected glucose utilization concomitant with lactate accumulation. Immunohistochemical evaluations were unremarkable for all four cultivation conditions. Results demonstrate that use of the perfusion-RWV bioreactor is advantageous in obtaining spherical aggregates, while grown in a controlled environment.

Maintenance of liver functions in rat hepatocytes cultured as spheroids in a rotating wall vessel.

Rat hepatocytes were cultured initially as spheroids on culture plates and then transferred into a rotating wall vessel (high-aspect ratio vessel [HARV]) for further culturing. Morphological evaluation based on electron microscopy showed that hepatocyte spheroids cultured for 30 d in the HARV had a compact structure with tight cell-cell junctions, numerous smooth and rough endoplasmic reticulum, intact mitochondria, and bile canaliculi lined with microvilli. The viability and differentiated properties of the hepatocytes cultured in the HARV were further substantiated by the presence of both phase I oxidation and phase II conjugation drug-metabolizing enzyme activities, as well as albumin synthesis. Homogenates prepared from freshly isolated hepatocytes and hepatocytes cultured in the HARV showed similar cytochrome P450 2B activities measured as pentoxyresorufin-O-dealkylase and testosterone 16beta-hydroxylase. Further, intact hepatocytes cultured in the HARV were found to metabolize chlorzoxazone to 6-hydroxychlorzoxazone; dextromethorphan to dextrorphan, 3-methoxymorphinan, and 3-hydroxymorphinan; midazolam to 1-hydroxymidazolam and 4-hydroxymidazolam; and 7-hydroxycoumarin to its glucuronide and sulfate conjugates. In conclusion, we found that hepatocyte spheroids could be cultured in a HARV to retain cellular and physiological properties of the intact liver, including drug-metabolizing enzyme activities, plasma protein production, and long-term (1 mo) maintenance of viability and cellular function.

A PERFUSED ROTATING WALL VESSEL BIOARTIFICIAL LIVER

A novel bioartificial liver is being developed based on the rotating wall vessel (RWV) technology of NASA. We discovered that preaggregated primary rat hepatocytes retained high viability and drug metabolizing enzyme activities in the High Aspect Ratio Vessel (HARV), suggesting that a bioartificial liver could be developed using hepatocytes cultured in a RWV. The Perfused Rotating Wall Vessel (PKWV) was selected as it allowed the continuous perfusion of plasma from a patient. Porcine hepatocytes were used. The original RWV design allowed only short-term culturing of the hepatocytes, with viability dropping to less than 50% in 1 hr. Modificiation of the bioreactor via the addition of a more efficient oxygenator increased the viability to over 50% for a duration of 5 hrs. A hollow fiber column was used as a filter to prevent the entry of hepatocytes from the SLAD to the patient's systemic circulation. The efficacy of this novel bioartificial liver (Stelsys Liver Assist Device, SLAD) was demonstrated by its ability to efficiently detoxify ammonia (using 1–5 billion porcine hepatocytes, the ammonia level of a phantom patient was decreased from 130 μM to 40 μM after a 5 hr treatment period). A comparison was made between the SLAD and the conventional hollow fiber bioartificial liver. We discovered that a significantly higher mass transfer could be achieved via cross-flow than the conventional parallel flow. Ammonia detoxification as well as acetaminophen metabolism were demonstrated with the cross-flow hollow fiber bioartificial liver (SLAD-CHF).

Production of a sialylated N-linked glycoprotein in insect cells.

Under High Aspect Ratio Vessel (HARV) bioreactor culture conditions designed to simulate the microgravity of orbital space flight, insect tissue culture cells infected with a baculovirus expression vector produced a human glycoprotein with tri- and tetra-antennary complex N-linked oligosaccharides containing terminal sialic acid residues. The oligosaccharide structures were similar to those produced in human placental cells. Insect cells cultured in T-flasks only performed incomplete oligosaccharide processing. The mechanism of HARV-mediated changes in the eukaryotic N-linked glycosylation pathway was investigated and could be mimicked under T-flask growth conditions with the addition of N-acetylmannosamine to the culture medium. The significance of these investigations is discussed with respect to the production of recombinant therapeutic glycoproteins, insect physiology, and orbital space flight.

Surface transformation of bioactive glass in bioreactors simulating microgravity conditions. Part I: experimental study.

Surface modified bioactive glass with surface properties akin to those of the bone mineral phase is an attractive candidate for use as a microcarrier material for 3-D growth of bone-like tissue in rotating wall vessel bioreactors (RWVs). The critical surface properties of this material are the result of reaction in solution. Because an RWV environment is completely different from conditions previously employed for bioactive glass testing, a detailed study of the surface reactions is warranted. Under properly chosen conditions, RWVs can also provide a simulated microgravity environment for the bioactive glass (BG) particles. In this sense, this study is also a report on the behavior of a bioactive material under microgravity conditions simulated on earth. A high aspect ratio vessel (HARV) and carefully selected experimental conditions enabled the simulation of microgravity in our laboratory. A complimentary numerical study was simultaneously conducted to ascertain the appropriateness of the experimental parameters (particle size, particle density, medium density, medium viscosity, and rotational speed) that ensure simulated microgravity conditions for the glass particles in the HARV. Physiological solutions (pH 7.4) with and without electrolytes, and also with serum proteins, were used to study the change in surface character resulting from simulated microgravity. Control tests at normal gravity, both static and dynamic, were also conducted. Solution and surface analyses revealed major effects of simulated microgravity. The rates of leaching of constituent ions (Si-, Ca-, and P-ions) were greatly increased in all solutions tested. The enhanced dissolution was followed by the enhanced formation of bone-like minerals at the BG surface. This enhancement is expected to affect adsorption of serum proteins and attachment molecules, which, in turn, may favorably affect bone cell adhesion and function. The findings of the study are important for the use of bioactive materials as microcarriers to generate and analyze 3-D bone-like tissue structures in bioreactors under microgravity conditions or otherwise.

Surface transformation of bioactive glass in bioreactors simulating microgravity conditions. Part II: numerical simulations.

The effects of simulated microgravity on the surface modification of bioactive glass (BG) in solution were studied using a numerical method. Models were developed for estimating the mass transfers of different chemical species from the surface of bioactive glass particles (microcarriers) suspended in the rotating liquid medium of a NASA-designed high aspect ratio vessel (HARV) bioreactor and on the bottom surface of a static vial. The concentration profiles resulting from chemical reactions and ionic transports were ascertained. Numerical results for the transport under simulated microgravity in the HARV and at normal gravity in the static vial were compared. These results were also compared with those of experiments to verify the enhancement of the reaction kinetics under simulated microgravity conditions. The experimental and numerical studies confirm that simulated microgravity conditions lead to the quick achievement of bioactive glass surface modification.

The simulated microgravity environment maintains key metabolic functions and promotes aggregation of primary porcine hepatocytes

The high aspect ratio vessel allows the culture of primary porcine hepatocytes in an environment of low shear stress and simulated microgravity. Primary porcine hepatocytes have been difficult to maintain in culture long term while preserving their metabolic functions. This study was carried out in order to characterise key metabolic functions of cell aggregates formed by primary porcine hepatocytes cultured in a high aspect ratio vessel for a predetermined period of 21 days. 108 porcine hepatocytes were loaded into the high aspect ratio vessel and continuously rotated during the experiments. 0.7 ml of the culture medium was sampled on days 1, 2, 4, 7, 10, 14 and 21. 1H nuclear magnetic resonance spectroscopy of the culture medium, using the presaturation technique, assessed the following: glucose metabolism, glutamine synthesis and ketogenesis. There was glucose breakdown anaerobically during the first 10 days as manifested by lactate production and pyruvate and threonine consumption. After day 10 there was significantly smaller lactate production (day 1 vs day 10 P<0.01), and significantly smaller pyruvate (day 1 vs day 14 P<0.03) and threonine consumption (day 1 vs day 10 P<0.002), indicative of an aerobic metabolic pattern. Significantly more glutamate was produced after day 10 (day 1 vs day 10 P<0.031), and more glutamine was consumed after day 14. There was a steadily diminishing production of acetate which reached a minimum on day 14 (day 2 vs day 14 P<0.00014). After an initial 10 day period of acclimatisation cell aggregates formed in the high aspect ratio vessel switched from the anaerobic pattern of metabolism to the more efficient aerobic pattern, which was exhibited until the experiments were terminated. The high aspect ratio vessel is suitable for long-term culture of porcine hepatocytes and it is worthwhile carrying out scale-up feasibility studies.

Altered TNF-α, glucose, insulin, and amino acids in islets of Langerhans cultured in a microgravity model system

The present studies were designed to determine effects of a microgravity model system upon lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha) activity and indexes of insulin and fuel homeostasis of pancreatic islets of Langerhans. Islets (1,726 +/- 117, 150 islet equivalent units) from Wistar-Furth rats were treated as 1) high aspect ratio vessel (HARV) cell culture, 2) HARV plus LPS, 3) static culture, and 4) static culture plus LPS. TNF-alpha (L929 cytotoxicity assay) was significantly increased in LPS-induced HARV and static cultures; yet the increase was more pronounced in the static culture group (P < 0.05). A decrease in insulin concentration was demonstrated in the LPS-stimulated HARV culture (P < 0.05). We observed a greater glucose concentration and increased disappearance of arginine in islets cultured in HARVs. Although nitrogenous compound analysis indicated a ubiquitous reliance on glutamine in all experimental groups, arginine was converted to ornithine at a twofold greater rate in the islets cultured in the HARV microgravity model system (P < 0.05). These studies demonstrate alterations in LPS-induced TNF-alpha production of pancreatic islets of Langerhans, favoring a lesser TNF activity in the HARV. These alterations in fuel homeostasis may be promulgated by gravity-averaged cell culture methods or by three-dimensional cell assembly.

NEURAL PRECURSOR CELLS FORM RUDIMENTARY TISSUE-LIKE STRUCTURES IN A ROTATING-WALL VESSEL BIOREACTOR

We have analyzed the biology of embryonic, epidermal growth factor-responsive murine neural precursor cells cultured in the high-aspect ratio vessel (HARV). Within 2-3 d of rotary-cell culture, such cells formed multiple, macroscopic, three-dimensional structures that were orders of magnitude larger than the cellular clusters ("neurospheres") formed by these cells in conventional stationary-flask cultures. Each HARV structure was composed of a multilayered cellular shell surrounding one or more central cavities that were bordered by pyknotic cell nuclei. Although the cells in the HARV structures were more pleomorphic than those in neurospheres, the structures did not appear to represent primitive neural tumors: the formation of HARV structures by precursor cells was not an irreversible phenotypic change, and the structures did not originate from the clonal expansion of single-progenitor cells; the growth rate and invasiveness of the cells in HARVs were less than those in flasks; and HARV-cultured cells did not form tumors after subcutaneous inoculation into the flanks of NOD-scid/scid mice. Immunohistochemical analysis suggested that HARV structures might be novel "prototissues" characterized by a crude, but organized, architecture, with a surface layer of immature proliferating cells (nestin- and proliferating cell nuclear antigen-positive) that enclosed strata of more differentiated cells (beta-tubulin III- and glial fibrillary acidic protein-positive) within. Rotary-cell culture may have significant implications for the eventual utility of neural precursors for clinical neurotransplantation.

Effect of culture in a rotating wall bioreactor on the physiology of differentiated neuron-like PC12 and SH-SY5Y cells.

A variety of evidence suggests that nervous system function is altered during microgravity, however, assessing changes in neuronal physiology during space flight is a non-trivial task. We have used a rotating wall bioreactor with a high aspect ratio vessel (HARV), which simulates the microgravity environment, to investigate the how the viability, neurite extension, and signaling of differentiated neuron-like cells changes in different culture environments. We show that culture of differentiated PC12 and SH-SY5Y cells in the simulated microgravity HARV bioreactor resulted in high cell viability, moderate neurite extension, and cell aggregation accompanied by NO production. Neurite extension was less than that seen in static cultures, suggesting that less than optimal differentiation occurs in simulated microgravity relative to normal gravity. Cells grown in a mixed vessel under normal gravity (a spinner flask) had low viability, low neurite extension, and high glutamate release. This work demonstrates the feasibility of using a rotating wall bioreactor to explore the effects of simulated microgravity on differentiation and physiology of neuron-like cells.

Neonatal rat heart cells cultured in simulated microgravity.

In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA-designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organization of cardiac cells in vitro.