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. Copyright John Wiley & Sons, Inc.     

Simulated conditions of microgravity suppress progesterone production by luteal cells of the pregnant rat

The purpose of this study was to assess whether simulated conditions of microgravity induce changes in the production of progesterone by luteal cells of the pregnant rat ovary using an in vitro model system. The microgravity environment was simulated using either a high aspect ratio vessel (HARV) bioreactor with free fall or a clinostat without free fall of cells. A mixed population of luteal cells isolated from the corpora lutea of day 8 pregnant rats was attached to cytodex microcarrier beads (cytodex 3). These anchorage dependent cells were placed in equal numbers in the HARV or a spinner flask control vessel in culture conditions. It was found that HARV significantly reduced the daily production of progesterone from day 1 through day 8 compared to controls. Scanning electron microscopy showed that cells attached to the microcarrier beads throughout the duration of the experiment in both types of culture vessels. Cells cultured in chamber slide flasks and placed in a clinostat yielded similar results when compared to those in the HARV. Also, when they were stained by Oil Red-O for lipid droplets, the clinostat flasks showed a larger number of stained cells compared to control flasks at 48 h. Further, the relative amount of Oil Red-O staining per milligram of protein was found to be higher in the clinostat than in the control cells at 48 h. It is speculated that the increase in the level of lipid content in cells subjected to simulated conditions of microgravity may be due to a disruption in cholesterol transport and/or lesions in the steroidogenic pathway leading to a fall in the synthesis of progesterone. Additionally, the fall in progesterone in simulated conditions of microgravity could be due to apoptosis of luteal cells.     

Gramicidin S Production by Bacillus brevis in Simulated Microgravity

In a continuing study of microbial secondary metabolism in simulated microgravity, we have examined gramicidin S (GS) production by Bacillus brevis strain Nagano in NASA High Aspect Rotating Vessels (HARVs), which are designed to simulate some aspects of microgravity. Growth and GS production were found to occur under simulated microgravity. When performance under simulated microgravity was compared with that under normal gravity conditions in the bioreactors, GS production was found to be unaffected by simulated microgravity. The repressive effect of glycerol in flask fermentations was not observed in the HARV. Thus the negative effect of glycerol on specific GS formation is dependent on shear and/or vessel geometry, not gravity. Received: 7 August 1996 / Accepted: 17 September 1996     

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.     

Altered TNF-alpha, 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.     

Effects of simulated microgravity on DU 145 human prostate carcinoma cells

The high aspect rotating-wall vessel (HARV) was recently designed by NASA to cultivate animal cells in an environment that simulates microgravity. This work examines the effects of HARV cultivation on DU 145 human prostate carcinoma cells. In the HARV, these prostate cells grew in suspension on Cytodex-3 microcarrier beads to form bead aggregates with extensive three-dimensional growth between beads and on the aggregate surface. HARV and spinner-flask control cultures of DU 145 cells had similar doubling times, but the former was characterized by a higher percentage of G(1)-phase cells, larger bead aggregates, enhanced development of filopodia and microvilli-like structures on the aggregate surface, and stronger staining for select cytoskeletal proteins (cytokeratins 8 and 18, actin, and vimentin). When compared with static controls grown in a T-flask and Transwell insert, HARV cultures grew more slowly and differences in the cell cycle and immunostaining became more pronounced. These results suggest that HARV cultivation produced a culture that was less aggressive from the perspective of proliferation, more differentiated and less pliant than any of the three control cultures examined in this work. Possible factors effecting this change are discussed including turbulence and three-dimensional growth. (c) 1996 John Wiley & Sons, Inc.     

Epstein-barr virus latently infected cells are selectively deleted in simulated-microgravity cultures

Summary Rotating-wall vessels (RWVs) allow for the cultivation of cells in simulated microgravity. Previously, we showed that the cultivation of lymphoblastoid cells in simulated microgravity results in the suppression of Epstein—Barr virus (EBV) reactivation. To determine if the suppression generated by simulated microgravity could be reversed by changing to static culture conditions, cells were cultured in an RWV for 5 d, and then switched to static conditions. Following the switch to static conditions, viral reactivation remained suppressed (significantly lower) relative to static control cultures over a 4-d period. Additionally, experiments were conducted to determine if chemical treatment could induce viral reactivation in cells from simulated-microgravity cultures. Cells were cultured in static flask cultures and in simulated microgravity in RWVs for 4–7 d. The cells were then transferred to 50-cm³ tubes, and treated with 3 mM n-butyrate for 48 h, or 18 ng/ml of phorbol ester, viz., 12-0-tetradecanoylphorbol-13 acetate (TPA) for either 2 or 48 h, under static conditions. Although EBV was inducible, the cells from simulated-microgravity cultures treated withn-butyrate displayed significantly lower levels of viral-antigen expression compared with the treated cells from static cultures. Also, incubation with TPA for 2–3 h, but not for 48 h, reactivated EBV in cells from RWV cultures. In contrast, EBV was inducible in cells from static cultures treated for either 2–3 or 48 h with TPA. TPA reactivation of EBV following a 2–3-h period of treatment indicates that the protein kinase C signal-transduction pathway is not impaired in lymphoblastoid cells cultured in simulated microgravity. However, the exposure of B-lymphoblastoid cells from simulated-microgravity cultures to TPA for more than 3–4 h triggered a lytic event (apoptosis or necrosis), which prevented replication of the virus. Thus, EBV-infected cells in simulated microgravity were negatively selected in the absence of any cytotoxic cells.     

模拟失重对白假丝酵母菌增殖和毒性的影响

【背景】近年来研究发现,失重条件可对一些致病微生物的增殖和毒性产生影响,白假丝酵母菌(Candida albicans)是典型的条件性致病真菌,在太空环境和人体中普遍存在,研究失重条件下白假丝酵母菌的增殖和毒性意义重大。【目的】利用旋转细胞培养系统(Rotary cell culture system,RCCS)模拟失重环境对白假丝酵母菌进行连续传代培养,检测模拟失重环境对白假丝酵母菌增殖情况、毒性以及基因表达的变化。【方法】将白假丝酵母菌接种在旋转生物反应器(High aspect rotating vessel,HARV)中,利用旋转细胞培养系统连续传代培养14 d,然后对菌株进行增殖速率测定、不同pH条件下增殖能力测定、生物膜相对形成能力测定和细胞毒性和动物毒力测定;利用转录组测序技术找出差异表达基因,结合性状分析模拟失重可能对白假丝酵母菌增殖和毒力的影响。【结果】与对照组相比,模拟失重组白假丝酵母菌对数期提前,增殖速率加快,在适宜pH条件下的增殖能力普遍提高,但其生物膜形成能力相对减弱,对LoVo细胞和小鼠的毒性减弱;转录组测序发现,模拟失重组共有280个基因表达差异达1.5倍以上(P0.05),其中248个上调、32个下调。差异基因经基因功能注释(Gene ontology,GO)和京都基因及基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析发现,相关胞膜形成及细胞分裂基因表达上调,生物膜形成、细胞黏附及共生粘连宿主基因表达下调。【结论】模拟失重环境可引起白假丝酵母菌增殖和毒性水平发生变化,相关改变可为研究失重环境对微生物的影响提供参考。     

Influence of simulated microgravity on the longevity of insect-cell culture

Simulated microgravity within the NASA High Aspect Rotating-Wall Vessel (HARV) provides a quiescent environment to culture fragile insect cells. In this vessel, the duration of stationary and death phase for cultures of Spodoptera frugiperda cells was greatly extended over that achieved in shaker-flask controls. For both HARV and control cultures, S. frugiperda cells grew to concentrations in excess of 1 x 10(7) viable cells ml-1 with viabilities greater than 90%. In the HARV, stationary phase was maintained 9-15 days in contrast to 4-5 days in the shaker flask. Furthermore, the rate of cell death was reduced in the HARV by a factor of 20-90 relative to the control culture and was characterized with a death rate constant of 0.01-0.02 day-1. Beginning in the stationary phase and continuing in the death phase, there was a significant decrease in population size in the HARV versus an increase in the shaker flask. This phenomenon could represent cell adaptation to simulated microgravity and/or a change in the ratio of apoptotic to necrotic cells. Differences observed in this research between the HARV and its control were attributed to a reduction in hydrodynamic forces in the microgravity vessel.     

Neonatal rat heart cells cultured in simulated microgravity

Summary 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 organizations of cardiac cells in vitro.     

Effect of simulated microgravity on the production of IL-12 by PBMCs

During space flight immunity is altered. This phenomenon is partly due to the microgravity condition itself. Our earlier space experiments (INTERFERON) indicated that microgravity has a significant effect at the cellular level. In our subsequent terrestrial studies we applied the Rotating Cell Culture System (RCCS) developed by NASA to mimick microgravity on ground. Previously we reported that human peripheral blood mononuclear cells (PBMCS) respond to simulated microgravity conditions with elevated tumor necrosis factor-alpha (TNF-alpha) production. We extended our investigations to the production of interleukin (IL)-12 under modelled microgravity conditions by separated PBMCs. In simulated microgravity we found significantly elevated level of secreted IL-12 compared to static, standard tissue culture conditions. Following a maximum of TNF-alpha production at 24 hours, the peak of IL-12 production was observed at 48 hours after the start of the experiment. Our results suggest that simulated microgravity favors the establishment of a Th1 type cytokine response.     

Simulated microgravity culture system for a 3-D carcinoma tissue model

An in vitro organotypic culture model is needed to understand the complexities of carcinoma tissue consisting of carcinoma cells, stromal cells, and extracellular matrices. We developed a new in vitro model of carcinoma tissue using a rotary cell culture system with four disposable vessels (RCCS-4D) that provides a simulated microgravity condition. Solid collagen gels containing human pancreatic carcinoma NOR-P1 cells and fibroblasts or minced human pancreatic carcinoma tissue were cultured under a simulated microgravity condition or a static Ig condition for seven days. NOR-P1 cultures subjected to the simulated microgravity condition showed greater numbers of mitotic, cycling (Ki-67-positive), nuclear factor-kappa B-activating cells, and a lower number of apoptotic cells than were shown by cultures subjected to the static Ig condition. In addition, human pancreatic carcinoma specimens cultured under the simulated microgravity condition maintained the heterogeneous composition and cellular activity (determined by the cycling cell ratio and mitotic index) of the original carcinoma tissue better than static culture conditions. This new 3-D rotary cell culture system with four disposal vessels may be useful for in vitro studies of complex pancreatic carcinoma tissue.     

Reduced shear stress: A major component in the ability of mammalian tissues to form three-dimensional assemblies in simulated microgravity

BHK-21 cells were cultured under various shear stress conditions in an Integrated Rotating-Wall Vessel (IRWV). Shear ranged from 0.5 dyn/cm² (simulated microgravity) to 0.92 dyn/cm². Under simulated microgravity conditions, BHK-21 cells complexed into three-dimensional cellular aggregates attaining 6 × 10⁶ cells/ml as compared to growth under 0.92 dyn cm² conditions. Glucose utilization in simulated microgravity was reduced significantly, and cellular damage at the microcarrier surface was kept to a minimum. Thus, the integrated rotating wall vessel provides a quiescent environment for the culture of mammalian cells. © 1993 Wiley-Liss, Inc.     

Growth and photosynthesis of Japanese flowering cherry under simulated microgravity conditions.

The photosynthetic rate, the leaf characteristics related to photosynthesis, such as the chlorophyll content, chlorophyll a/b ratio and density of the stomata, the leaf area and the dry weight in seedlings of Japanese flowering cherry grown under normal gravity and simulated microgravity conditions were examined. No significant differences were found in the photosynthetic rates between the two conditions. Moreover, leaf characteristics such as the chlorophyll content, chlorophyll a/b ratio and density of the stomata in the seedlings grown under the simulated microgravity condition were not affected. However, the photosynthetic product of the whole seedling under the simulated microgravity condition increased compared with the control due to its leaf area increase. The results suggest that dynamic gravitational stimulus controls the partitioning of the products of photosynthesis.     

Growth and development, and auxin polar transport of transgenic Arabidopsis under simulated microgravity conditions on a three-dimensional clinostat.

Growth and development, and auxin polar transport in Arabidopsis thaliana transformed with iaaH gene were studied under simulated microgravity conditions on a three-dimensional (3-D) clinostat. Simulated microgravity conditions on a 3-D clinostat did not affect the number of rosette leaves but promoted the growth and development (fresh weight of plant and the elongation of flower stalk) of transformants. Final growth of transformants under simulated microgravity conditions on a 3-D clinostat was almost equivalent to that grown on 1 g conditions in the presence of 1 micromoles IAM (indole-3-acetamide). The activities of auxin polar transport in the segments of flower stalk (inflorescence axis) of transformants grown on 1 g conditions were significantly promoted by the addition of IAM. Interestingly, simulated microgravity conditions on a 3-D clinostat also promoted the activities of auxin polar transport of transformants grown on the medium with or without IAM. Based on the results in this study, transgenic plants may not have an efficient homeostatic mechanism for the control of growth and development, and auxin polar transport activity in microgravity conditions in space.     

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.     

Immunohistochemical evidence that culture in the high aspect rotating vessel can up-regulate hormone expression in growth dedifferentiated PHHI-derived islet cells

Islet cells derived from patients with persistent hyperinsulinemic hypoglycemia of infancy (PHHI) have the ability to grow readily in simple culture media. However, as with primary islets and cell lines, they lose hormone expression upon growth. In this study, we have investigated the role of three-dimensional cell-to-cell contact in the reinitiation of hormone expression in growth dedifferentiated PHHI-derived cells. Two main methods of cell aggregation were studied; the promotion of pseudoislets through petri dish culture and the creation of cell aggregates in the microgravity environment of the high aspect ratio vessel (HARV). Immunohistochemical analysis and ELISA assay showed that petri dish culture did not re-establish endocrine expression in any of the five cultures tested. However, through HARV technology, we have demonstrated that it is possible to reactivate insulin, glucagon, somatostatin, and GAD expression in PHHI-derived cells that had previously stopped expressing these markers. These results indicate that the unique environment of the HARV can be conducive to the upregulation of endocrine expression of islet-derived cells and optimization of culture conditions may prove useful in the sphere of β cell proliferation.     

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.     

3D Bone tissue engineered with bioactive microspheres in simulated microgravity

Three-dimensional (3D) osteoblast cell cultures were obtained in rotating-wall vessels (RWV), simulating microgravity. Three types of bioactive microcarriers, specifically modified bioactive glass particles, bioceramic hollow microspheres, and biodegradable bioactive glass-polymer composite microspheres, were developed and used with osteoblasts. The surfaces of composite microspheres fully transformed into bone apatite after 2-wk immersion in simulated physiological fluid, which demonstrated their bone-bonding ability. The motion of microcarriers in RWVs was photographically recorded and numerically analyzed. The trajectories of hollow microspheres showed that they migrated and eventually stayed around at the central region of the RWV. At their surfaces, shear stresses were low. In contrast, solid glass or polymer particles moved toward and finally bounced off the outer wall of the RWVs. Cell culture studies in the RWV using bone marrow stromal cells showed that the cells attached to and formed 3D aggregates with the hollow microspheres. Extracellular matrix and mineralization were observed in the aggregates. Cell culture studies also confirmed the ability of the composite microspheres to support 3D bone-like tissue formation. These data suggest that the new hollow bioceramic microspheres and degradable composite microspheres can be used as microcarriers for 3D bone tissue engineering in microgravity. They also have potential applications as drug delivery systems.