High Aspect Ratio Vessel Research Articles

Modelled microgravity impacts Vibrio fischeri population structure in a mutualistic association with an animal host.

Perturbations to host-microbe interactions, such as environmental stress, can alter and disrupt homeostasis. In this study, we examined the effects of a stressor, simulated microgravity, on beneficial bacteria behaviours when colonising their host. We studied the bacterium Vibrio fischeri, which establishes a mutualistic association in a symbiosis-specific organ within the bobtail squid, Euprymna scolopes. To elucidate how animal-microbe interactions are affected by the stress of microgravity, squid were inoculated with different bacterial strains exhibiting either a dominant- or sharing-colonisation behaviour in High Aspect Ratio Vessels, which simulate the low-shear environment of microgravity. The colonisation behaviours of the sharing and dominant strains under modelled microgravity conditions were determined by counting light-organ homogenate of squids as well as confocal microscopy to assess the partitioning of different strains within the light organ. The results indicated that although the colonisation behaviours of the strains did not change, the population levels of the sharing strains were at lower relative abundance in single-colonised animals exposed to modelled microgravity compared to unit gravity; in addition, there were shifts in the relative abundance of strains in co-colonised squids. Together these results suggest that the initiation of beneficial interactions between microbes and animals can be altered by environmental stress, such as simulated microgravity.

Staphylococcus aureus Sensitivity to Membrane Disrupting Antibacterials Is Increased under Microgravity.

In a survey of the International Space Station (ISS), the most common pathogenic bacterium identified in samples from the air, water and surfaces was Staphylococcus aureus. While growth under microgravity is known to cause physiological changes in microbial pathogens, including shifts in antibacterial sensitivity, its impact on S. aureus is not well understood. Using high-aspect ratio vessels (HARVs) to generate simulated microgravity (SMG) conditions in the lab, we found S. aureus lipid profiles are altered significantly, with a higher presence of branch-chained fatty acids (BCFAs) (14.8% to 35.4%) with a concomitant reduction (41.3% to 31.4%) in straight-chain fatty acids (SCFAs) under SMG. This shift significantly increased the sensitivity of this pathogen to daptomycin, a membrane-acting antibiotic, leading to 12.1-fold better killing under SMG. Comparative assays with two additional compounds, i.e., SDS and violacein, confirmed S. aureus is more susceptible to membrane-disrupting agents, with 0.04% SDS and 0.6 mg/L violacein resulting in 22.9- and 12.8-fold better killing in SMG than normal gravity, respectively. As humankind seeks to establish permanent colonies in space, these results demonstrate the increased potency of membrane-active antibacterials to control the presence and spread of S. aureus, and potentially other pathogens.

Co-adaptation of Streptococcus mutans to simulated microgravity and silver nitrate

To sustain life on extended space missions, it is essential to maintain clean potable water. NASA currently uses iodine as the primary biocide in the potable water dispenser on the International Space Station and has recently proposed a potential switch to silver-based antimicrobials. Streptococcus mutans is the primary etiological agent of dental caries, part of the normal oral flora, and would endure direct exposure to water from the potable water dispenser. In our previous work, we examined the 100-day adaptive response of Streptococcus mutans to simulated microgravity (sMG). Here, we examined the evolutionary co-adaptation of S. mutans under sMG and silver nitrate (AgNO3) to evaluate the consequences of using silver as a primary biocide in space and the impact on the evolution of microbes from the oral microbiome. To do this, we adapted four populations of S. mutans under sMG and co-adapted four populations in simulated microgravity and silver nitrate using high-aspect ratio vessels for 100 days. Genomic analysis at multiple time points showed that S. mutans in sMG evolved variants consistent with our previous findings (SMU_1307c and SMU_399) while also acquiring novel mutations in the glutathione reductase gorA. The co-adapted populations showed mutations specific for the environment in ciaH/R, PBP1a, trkA, and trkB. We also assessed virulence phenotypes, and while simulated microgravity increased antibiotic susceptibility, sucrose-dependent adhesion, and, in some populations, acid tolerance, co-adaptation to silver nitrate reversed these effects. Overall, these data show that the use of silver as a biocide in simulated microgravity can evolve strains with novel genotypic and phenotypic traits that could alter virulence.

Characterization and Analysis of HEK293/Adenovirus Type 5 Cell Cultures under Simulated Microgravity Using Differential Neural Network Modeling

HEK293 cells are the common system to produce adenoviral vectors for vectorized vaccines and gene therapy products. Therefore, the improvement of this type of cell culture is highly relevant nowadays. Furthermore, the high aspect ratio vessel (HARV) bioreactor provides a simulated microgravity condition (SMGC) which allows the establishment of in vitro models of mammalian cells. The present work is aimed at showing the characterization of HEK293 cell cultures in serum‐free medium under SMGC and to identify a set of adaptable time differential equations from experimental data through a nonparametric mathematical modeling using differential neural networks (DNN). Results showed that the HARV bioreactor enables to obtain a gravity percentage equal to 〖%g〗_maximum = −0.053% (where minus sign implies against the gravity direction). In addition, the characterization of HEK293 culture under SMGC revealed that such condition favored the production of infective viral particles as well as the uptake of basic nutrients such as glucose and glutamine, but the cell concentration was not significantly affected. As shown, these results contribute to improve the adenoviral vector production, and the DNN analysis is useful to model the kinetic changes and to build the foundations for control strategies and optimization of adenoviral production in further studies.

Adaptation to simulated microgravity in Streptococcus mutans

Long-term space missions have shown an increased incidence of oral disease in astronauts’ and as a result, are one of the top conditions predicted to impact future missions. Here we set out to evaluate the adaptive response of Streptococcus mutans (etiological agent of dental caries) to simulated microgravity. This organism has been well studied on earth and treatment strategies are more predictable. Despite this, we are unsure how the bacterium will respond to the environmental stressors in space. We used experimental evolution for 100-days in high aspect ratio vessels followed by whole genome resequencing to evaluate this adaptive response. Our data shows that planktonic S. mutans did evolve variants in three genes (pknB, SMU_399 and SMU_1307c) that can be uniquely attributed to simulated microgravity populations. In addition, collection of data at multiple time points showed mutations in three additional genes (SMU_399, ptsH and rex) that were detected earlier in simulated microgravity populations than in the normal gravity controls, many of which are consistent with other studies. Comparison of virulence-related phenotypes between biological replicates from simulated microgravity and control orientation cultures generally showed few changes in antibiotic susceptibility, while acid tolerance and adhesion varied significantly between biological replicates and decreased as compared to the ancestral populations. Most importantly, our data shows the importance of a parallel normal gravity control, sequencing at multiple time points and the use of biological replicates for appropriate analysis of adaptation in simulated microgravity.

Effects of Simulated Microgravity on the Proteome and Secretome of the Polyextremotolerant Black Fungus Knufia chersonesos.

Black fungi are a group of melanotic microfungi characterized by remarkable polyextremotolerance. Due to a broad ecological plasticity and adaptations at the cellular level, it is predicted that they may survive in a variety of extreme environments, including harsh niches on Earth and Mars, and in outer space. However, the molecular mechanisms aiding survival, especially in space, are yet to be fully elucidated. Based on these premises, the rock-inhabiting black fungus Knufia chersonesos (Wt) and its non-melanized mutant (Mut) were exposed to simulated microgravity—one of the prevalent features characterizing space conditions—by growing the cultures in high-aspect-ratio vessels (HARVs). Qualitative and quantitative proteomic analyses were performed on the mycelia and supernatant of culture medium (secretome) to assess alterations in cell physiology in response to low-shear simulated microgravity (LSSMG) and to ultimately evaluate the role of cell-wall melanization in stress survival. Differential expression was observed for proteins involved in carbohydrate and lipid metabolic processes, transport, and ribosome biogenesis and translation via ribosomal translational machinery. However, no evidence of significant activation of stress components or starvation response was detected, except for the scytalone dehydratase, enzyme involved in the synthesis of dihydroxynaphthalene (DNH) melanin, which was found to be upregulated in the secretome of the wild type and downregulated in the mutant. Differences in protein modulation were observed between K. chersonesos Wt and Mut, with several proteins being downregulated under LSSMG in the Mut when compared to the Wt. Lastly, no major morphological alterations were observed following exposure to LSSMG. Similarly, the strains’ survivability was not negatively affected. This study is the first to characterize the response to simulated microgravity in black fungi, which might have implications on future astrobiological missions.

Modeled microgravity alters lipopolysaccharide and outer membrane vesicle production of the beneficial symbiont Vibrio fischeri

Reduced gravity, or microgravity, can have a pronounced impact on the physiology of animals, but the effects on their associated microbiomes are not well understood. Here, the impact of modeled microgravity on the shedding of Gram-negative lipopolysaccharides (LPS) by the symbiotic bacterium Vibrio fischeri was examined using high-aspect ratio vessels. LPS from V. fischeri is known to induce developmental apoptosis within its symbiotic tissues, which is accelerated under modeled microgravity conditions. In this study, we provide evidence that exposure to modeled microgravity increases the amount of LPS released by the bacterial symbiont in vitro. The higher rates of shedding under modeled microgravity conditions are associated with increased production of outer-membrane vesicles (OMV), which has been previously correlated to flagellar motility. Mutants of V. fischeri defective in the production and rotation of their flagella show significant decreases in LPS shedding in all treatments, but levels of LPS are higher under modeled microgravity despite loss of motility. Modeled microgravity also appears to affect the outer-membrane integrity of V. fischeri, as cells incubated under modeled microgravity conditions are more susceptible to cell-membrane-disrupting agents. These results suggest that, like their animal hosts, the physiology of symbiotic microbes can be altered under microgravity-like conditions, which may have important implications for host health during spaceflight.

Growth of Staphylococcus aureus Using a Rotary Cell Culture System.

The Rotary Cell Culture System (RCCS) is an apparatus that was originally designed by NASA engineers to simulate microgravity conditions for growth of both eukaryotic and bacterial cell cultures. The RCCS growth environment is also characterized by low fluid shear stress, thereby also providing an in vitro growth condition relevant to certain in vivo environments encountered during bacterial infection. This chapter describes a method for growing Staphylococcus aureus under simulated microgravity conditions using the RCCS and disposable High Aspect Ratio Vessels (HARVs). Small samples can be removed and replaced with fresh media during the experiment (continuous sampling method) or the whole culture can be removed at the end of the experiment (end-point sampling method) for larger sample volumes required for follow-up studies such as RNAseq or proteomics.

Comparisons of Transcriptome Profiles from Bacillus subtilis Cells Grown in Space versus High Aspect Ratio Vessel (HARV) Clinostats Reveal a Low Degree of Concordance.

Although clinostats have long been used in space microbiology studies as ground-based analogs of spaceflight, few studies to date have systematically compared -omics data from clinostats versus spaceflight. This study compared the transcriptomic response of the Gram-positive bacterium Bacillus subtilis flown in space with corresponding transcriptomes derived from 2-D clinostat (High Aspect Ratio Vessel: HARV) experiments performed under the same conditions of bacterial strain, growth medium, temperature, and incubation time. High-quality total RNA (RNA Integrity Number >9.6) was isolated from multiple biological replicates from each treatment, transcripts were quantified by RNA-seq, and raw data was processed through a previously described standardized bioinformatics pipeline. Transcriptome data sets from spaceflight-grown and corresponding clinostat-grown cells were compared by using three different methods: (i) principal component analysis, (ii) analysis of differentially expressed genes, and (iii) gene set enrichment analysis of KEGG pathways. All three analyses found a low degree of concordance between the spaceflight and corresponding clinostat transcriptome data sets, ranging from 0.9% to 5.3% concordance. These results are in agreement with prior studies that also revealed low concordances between spaceflight and clinostat transcriptomes of the Gram-negative bacteria Rhodospirillum rubrum and Pseudomonas aeruginosa. The results are discussed from the perspective of several potential confounding factors, and suggestions are offered with the aim of achieving increased concordance between clinostat and spaceflight data.

Insight into Pseudomonas aeruginosa pyocyanin production under low-shear modeled microgravity.

Long-term space flight impairs the immune system of astronauts, rendering them vulnerable to opportunistic infections. Pseudomonas aeruginosa causes opportunistic infections, particularly in individuals with a compromised immune system; it can be a major health hazard for astronauts during space flight missions. Hence, the production of the most abundant redox active virulence factor, pyocyanin by P. aeruginosa, was assessed under low-shear modeled microgravity (LSMMG) conditions, simulated using a high aspect ratio vessel. Moreover, we evaluated changes in the expression of genes involved in pyocyanin biosynthesis and genes involved in the MexGHI-OpmD operon quorum sensing. Extracellular DNA and H2O2 production were measured, and their correlation with pyocyanin production was examined. Interestingly, the pyocyanin quantity was 2.58-fold lower in the LSMMG conditions compared to the normal gravity. LSMMG caused downregulation of the genes associated with pyocyanin biosynthesis. Interestingly, extracellular DNA and H2O2 release were significantly high in the normal gravity environment. Scanning electron microscopy revealed aggregation and elongated cells under LSMMG. Taken together, these findings suggest that LSMMG did not induce pyocyanin secretion in P. aeruginosa.

Micro–Nano‐Texturing Inner Surfaces of Small‐Caliber High Aspect Ratio and Superhydrophobic Artificial Vessels using Femtosecond Laser Filamenting Pulses

AbstractCardiovascular diseases, the leading cause of death worldwide in the last two decades, are mainly due to the pathological changes inside the heart or blood vessels. Current treatment prescription is to replace obstructed blood vessels by synthetic alternatives, but it can only cure patients effectually when the vessel diameter is larger than a certain value because the cell attachment capacity on a small‐caliber artificial vessel is usually unacceptable for long‐term patency. Here a femtosecond filamenting laser‐based fabrication approach that can produce in situ microstructures on the inner surface of small‐caliber tubules is reported. It is shown that the inner‐surface fabrication with an aspect ratio as high as 10:1 can be achieved and the processed samples exhibit significant changes in physical properties including topography, roughness, hydrophobicity, as well as in biological property with improved ability for Hela cells to adhere and grow. The results provide a possibility toward fabricating small‐caliber artificial vessels that might be suitable for long‐term patency use.

Investigation of simulated microgravity effects on Streptococcus mutans physiology and global gene expression

Astronauts have been previously shown to exhibit decreased salivary lysozyme and increased dental calculus and gingival inflammation in response to space flight, host factors that could contribute to oral diseases such as caries and periodontitis. However, the specific physiological response of caries-causing bacteria such as Streptococcus mutans to space flight and/or ground-based simulated microgravity has not been extensively investigated. In this study, high aspect ratio vessel S. mutans simulated microgravity and normal gravity cultures were assessed for changes in metabolite and transcriptome profiles, H2O2 resistance, and competence in sucrose-containing biofilm media. Stationary phase S. mutans simulated microgravity cultures displayed increased killing by H2O2 compared to normal gravity control cultures, but competence was not affected. RNA-seq analysis revealed that expression of 153 genes was up-regulated ≥2-fold and 94 genes down-regulated ≥2-fold during simulated microgravity high aspect ratio vessel growth. These included a number of genes located on extrachromosomal elements, as well as genes involved in carbohydrate metabolism, translation, and stress responses. Collectively, these results suggest that growth under microgravity analog conditions promotes changes in S. mutans gene expression and physiology that may translate to an altered cariogenic potential of this organism during space flight missions.

Exposure of Mycobacterium marinum to low-shear modeled microgravity: effect on growth, the transcriptome and survival under stress.

Waterborne pathogenic mycobacteria can form biofilms, and certain species can cause hard-to-treat human lung infections. Astronaut health could therefore be compromised if the spacecraft environment or water becomes contaminated with pathogenic mycobacteria. This work uses Mycobacterium marinum to determine the physiological changes in a pathogenic mycobacteria grown under low-shear modeled microgravity (LSMMG). M. marinum were grown in high aspect ratio vessels (HARVs) using a rotary cell culture system subjected to LSMMG or the control orientation (normal gravity, NG) and the cultures used to determine bacterial growth, bacterium size, transcriptome changes, and resistance to stress. Two exposure times to LSMMG and NG were examined: bacteria were grown for ~40 h (short), or 4 days followed by re-dilution and growth for ~35 h (long). M. marinum exposed to LSMMG transitioned from exponential phase earlier than the NG culture. They were more sensitive to hydrogen peroxide but showed no change in resistance to gamma radiation or pH 3.5. RNA-Seq detected significantly altered transcript levels for 562 and 328 genes under LSMMG after short and long exposure times, respectively. Results suggest that LSMMG induced a reduction in translation, a downregulation of metabolism, an increase in lipid degradation, and increased chaperone and mycobactin expression. Sigma factor H (sigH) was the only sigma factor transcript induced by LSMMG after both short and long exposure times. In summary, transcriptome studies suggest that LSMMG may simulate a nutrient-deprived environment similar to that found within macrophage during infection. SigH is also implicated in the M. marinum LSMMG transcriptome response.

Engineering three dimensional micro nerve tissue using postnatal stem cells from human dental apical papilla.

The in vitro generation of cell-based three dimensional (3D) nerve tissue is an attractive subject to improve graft survival and integration into host tissue for neural tissue regeneration or to model biological events in stem cell differentiation. Although 3D organotypic culture strategies are well established for 3D nerve tissue formation of pluripotent stem cells to study underlying biology in nerve development, cell-based nerve tissues have not been developed using human postnatal stem cells with therapeutic potential. Here, we established a culture strategy for the generation of in vitro cell-based 3D nerve tissue from postnatal stem cells from apical papilla (SCAPs) of teeth, which originate from neural crest-derived ectomesenchyme cells. A stem cell population capable of differentiating into neural cell lineages was generated during the ex vivo expansion of SCAPs in the presence of EGF and bFGF, and SCAPs differentiated into neural cells, showing neural cell lineage-related molecular and gene expression profiles, morphological changes and electrophysical property under neural-inductive culture conditions. Moreover, we showed the first evidence that 3D cell-based nerve-like tissue with axons and myelin structures could be generated from SCAPs via 3D organotypic culture using an integrated bioprocess composed of polyethylene glycol (PEG) microwell-mediated cell spheroid formation and subsequent dynamic culture in a high aspect ratio vessel (HARV) bioreactor. In conclusion, the culture strategy in our study provides a novel approach to develop in vitro engineered nerve tissue using SCAPs and a foundation to study biological events in the neural differentiation of postnatal stem cells. Biotechnol. Bioeng. 2017;114: 903-914. © 2016 Wiley Periodicals, Inc.

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA's Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Production of Highly Aligned Collagen Scaffolds by Freeze-drying of Self-assembled, Fibrillar Collagen Gels.

Matrix and cellular alignment are critical factors in the native function of many tissues, including muscle, nerve, and ligaments. Collagen is frequently a component of these aligned tissues, and collagen biomaterials are widely used in tissue engineering applications. However, the generation of aligned collagen scaffolds that maintain the native architecture of collagen fibrils has not been straightforward, with many methods requiring specialized equipment or technical procedures, extensive incubation times, or denaturing of the collagen. Herein, we present a simple, rapid method for fabrication of highly aligned collagen scaffolds. Collagen was assembled to form a fibrillar hydrogel in a cylindrical conduit with high aspect ratio and then frozen and lyophilized. The resulting collagen scaffolds demonstrated highly aligned topographical features along the scaffold surface. This presence of an initial fibrillar network and the high-aspect ratio vessel were both required to generate alignment. The diameter of fabricated scaffolds was found to vary significantly with both the collagen concentration of the hydrogel suspension and the diameter of conduits used for fabrication. Additionally, the size of individual aligned topographical features was significantly dependent on the conduit diameter and the freezing temperature. When cultured on aligned collagen scaffolds, both rat dermal fibroblasts and axons emerging from chick dorsal root ganglia explants demonstrated elongated, aligned morphology and growth on the aligned topographical features. Overall, this method presents a simple means for generating aligned collagen scaffolds that can be applied to a wide variety of tissue types, particularly those where such alignment is critical to native function.

Numerical simulation of coupled cell motion and nutrient transport in NASA’s rotating bioreactor

Abstract Rotating bioreactor, such as the NASA bioreactor, which was designed by the National Aeronautics and Space Administration (NASA), USA, can be used to mimic micro-gravity conditions and simulate the effects of microgravity on cells growth. The cell growth in the bioreactor depends on the nutrient availability which in turn depends on the cell density and distribution within the bioreactor. In this work, we use a numerical model of suspended particle motion to simulate the cell motion and distribution in a specific variant of the NASA bioreactor, namely, the high aspect ratio vessel (HARV) bioreactor. The nutrient distribution in the bioreactor is simulated based on a convection–diffusion-reaction supplemented by laboratory experiments aimed at obtaining the required data. We present the modelling framework in this paper and discuss the most salient simulated results. For example, the simulation results show that the distributions of the cells in the bioreactor appear as concentric circles and that the cells density is higher in the middle of the HARV bioreactor. These cell distributions imply that they may accumulate in the middle of the bioreactor at sufficiently high cell density. The results also demonstrate that the concentration of nutrient is fairly uniform in the bioreactor but decreases slightly from the outer radius of the HARV bioreactor to the inner radius. This is possibly caused by the higher consumption of glucose due to the higher cell density in the middle of the radius. We expect that the modelling framework in this paper would help optimise the culture conditions for the cells in HARV bioreactors.

Microwell-mediated micro cartilage-like tissue formation of adipose-derived stem cell

In cartilage tissue engineering, various technical approaches using postnatal stem cells, three-dimensional (3D) scaffolds composed of synthetic or natural polymers, and culture systems have been applied to develop 3D cartilage-like tissue. In this study, scaffold-free 3D micro-cartilage-like tissue was developed via microwell-mediated cell spheroid formation and 3D dynamic chondrogenic culture in a bioreactor. First, homogenous micro-cell spheroids were generated by the self-condensation of adipose tissue-derived stem cells (ADSCs) in microfabricated poly(ethylene glycol) (PEG) hydrogel microwells. Next, chondrogenic differentiation of the micro-ADSC spheroids was induced in the presence of transforming growth factor-beta 3 under dynamic 3D culture conditions using a high aspect ratio vessel bioreactor. Several hundred viable ADSC spheroids could be generated at a time from ADSC culture in PEG microwells. The 3D dynamic chondrogenic culture of ADSCs in the bioreactor facilitated the chondrogenic mRNA expression of proteins such as sox-9, runx2, osterix, type II collagen, and aggrecan, and the well deposition of glycosaminoglycan and type II collagen, which finally generated micro-cartilage-like tissue. Therefore, the hydrogel microwell arrays could be useful for efficiently deriving initial cell condensation-mediated chondrogenic differentiation, and for developing 3D cell-based micro-cartilage-like tissue with stem cells in a controlled manner.

In vitro micro-mineralized tissue formation by the combinatory condition of adipose-derived stem cells, macroporous PLGA microspheres and a bioreactor

For the final purpose of accelerating the restoration of defected bone tissue, researches for developing in vitro three dimensional (3D) mineralized tissue using various stem cells, scaffolds and culture systems have been extensively done. In this research, an integrated bioprocess to generate stem cell-based 3D mineralized construct was developed using adipose-derived stem cell (ADSC), highly porous biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres and a high-aspect ratio vessel (HARV) bioreactor system. First, ADSCs adhered uniformly on poly-L-ornithine-coated macroporous PLGA microspheres, and the ADSC/microsphere composites were cultured in the presence of osteogenic supplements in a HARV bioreactor. Alkaline phosphatase (ALPase) activity assay, immunocytochemical staining and quantitative real-time polymerase chain reaction (qPCR) analysis showed temporal increase of ALPase activity, molecular expressions of type I collagen, osteocalcin and runx2 and upregulation of runx2, Sp7, type I collagen and bone sialoprotein mRNA during 3D osteogenic culture of ADSC/microsphere composites. Finally, 3D dynamic osteogenic culture generated highly mineralized micro tissues as validated by alizarin red-S staining and SEM-EDS. The results demonstrated that cell-based 3D micro-mineralized tissue could be generated by integrated bioprocess, and potentially utilized for bone tissue regeneration. The integrated bioprocess in this study may provide an efficient and scalable culture system for application in bone tissue engineering. Open image in new window

Mixing time in high aspect ratio vessels stirred with multiple impellers

Mixing time is a key parameter for evaluating the blending effectiveness of stirred tanks but, in contrast with the case of single impeller configurations, a general purpose correlation for multiple impeller geometries is not available to date. In this work, a lumped parameter equation for mixing time in the turbulent regime in high aspect ratio tanks stirred with multiple identical impellers is presented. The experimental data are collected by conductivity probes on a variety of stirred tank configurations, dimensions and impeller types with the purpose of identifying the influence of two main geometrical parameters: (i) the impeller number at fixed vessel aspect ratio, and (ii) the vessel aspect ratio at constant impeller distance. The results show that a single equation adding the impeller efficiency, the impeller number and the liquid height to the well-known correlation due to Grenville and Nienow (Grenville, R.K., Nienow, A.W., 2004. Handbook of Industrial Mixing: Science and Practice. In: Paul, E.L., Atiemo-Obeng, V.A., Kresta, S.M. (Eds.), Wiley-Interscience, Hoboken, NJ, USA) properly represents the whole experimental data relevant to axial and mixed-flow impellers collected in this work and in some of previous literature studies. For multiple radial turbines giving rise to compartmentalization of the vessel volume, a modified model based on a simplified fluid dynamic schematization of the vessel as a cascade of ideal stages with backmixing is proposed.