High Aspect Ratio Vessel Bioreactor Research Articles

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.

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

Fabrication, characterization, and in vitro evaluation of poly(lactic acid glycolic acid)/nano‐hydroxyapatite composite microsphere‐based scaffolds for bone tissue engineering in rotating bioreactors

Dynamic flow culture bioreactor systems have been shown to enhance in vitro bone tissue formation by facilitating mass transfer and providing mechanical stimulation. Our laboratory has developed a biodegradable poly (lactic acid glycolic acid) (PLAGA) mixed scaffold consisting of lighter-than-water (LTW) and heavier-than-water (HTW) microspheres as potential matrices for engineering tissue using a high aspect ratio vessel (HARV) rotating bioreactor system. We have demonstrated enhanced osteoblast differentiation and mineralization on PLAGA scaffolds in the HARV rotating bioreactor system when compared with static culture. The objective of the present study is to improve the mechanical properties and bioactivity of polymeric scaffolds by designing LTW polymer/ceramic composite scaffolds suitable for dynamic culture using a HARV bioreactor. We employed a microsphere sintering method to fabricate three-dimensional PLAGA/nano-hydroxyapatite (n-HA) mixed scaffolds composed of LTW and HTW composite microspheres. The mechanical properties, pore size and porosity of the composite scaffolds were controlled by varying parameters, such as sintering temperature, sintering time, and PLAGA/n-HA ratio. The PLAGA/n-HA (4:1) scaffold sintered at 90 degrees C for 3 h demonstrated the highest mechanical properties and an appropriate pore structure for bone tissue engineering applications. Furthermore, evaluation human mesenchymal stem cells (HMSCs) response to PLAGA/n-HA scaffolds was performed. HMSCs on PLAGA/n-HA scaffolds demonstrated enhanced proliferation, differentiation, and mineralization when compared with those on PLAGA scaffolds. Therefore, PLAGA/n-HA mixed scaffolds are promising candidates for HARV bioreactor-based bone tissue engineering applications.

Increased Filamentous Growth of Candida albicans in Simulated Microgravity

Knowledge of simulated microgravity (SMG)-induced changes in the pathogenicity of microorganisms is important for success of long-term spaceflight. In a previous study using the high aspect ratio vessel bioreactor, we showed that the yeast species Saccharomyces cerevisiae underwent a significant phenotypic response when grown in modeled microgravity, which was reflected in the analysis of gene expression profiles. In this study, we establish that Candida albicans responds to SMG in a similar fashion, demonstrating that there is a conserved response among yeast to this environmental stress. We also report that the growth of C. albicans in SMG results in a morphogenic switch that is consistent with enhanced pathogenicity. Specifically, we observed an increase in filamentous forms of the organism and accompanying changes in the expression of two genes associated with the yeast-hyphal transition. The morphological response may have significant implications for astronauts’ safety, as the fungal pathogen may become more virulent during spaceflight.

Gene Expression Profiling of Human Epidermal Keratinocytes in Simulated Microgravity and Recovery Cultures

Simulated microgravity (SMG) bioreactors and DNA microarray technology are powerful tools to identify “space genes” that play key roles in cellular response to microgravity. We applied these biotechnology tools to investigate SMG and post-SMG recovery effects on human epidermal keratinocytes by exposing cells to SMG for 3, 4, 9, and 10 d using the high aspect ratio vessel bioreactor followed by recovery culturing for 15, 50, and 60 d in normal gravity. As a result, we identified 162 differentially expressed genes, 32 of which were “center genes” that were most consistently affected in the time course experiments. Eleven of the center genes were from the integrated stress response pathways and were coordinately down-regulated. Another seven of the center genes, which are all metallothionein MT-I and MT-II isoforms, were coordinately up-regulated. In addition, HLA-G, a key gene in cellular immune response suppression, was found to be significantly up-regulated during the recovery phase. Overall, more than 80% of the differentially expressed genes from the shorter exposures (≤4 d) recovered in 15 d; for longer (≥9 d) exposures, more than 50 d were needed to recover to the impact level of shorter exposures. The data indicated that shorter SMG exposure duration would lead to quicker and more complete recovery from the microgravity effect.

Tissue growth in a rotating bioreactor. Part II: fluid flow and nutrient transport problems

Fluid flow and nutrient transport around a growing tissue construct within a cylindrical bioreactor of circular cross-section are considered. The bioreactor is filled with nutrient-rich culture medium, and the growing tissue construct is modelled as a cylindrical obstacle, also of circular cross-section, at a given (moving) position within the nutrient solution. The bioreactor rotates about its cylindrical axis, and its axial length is small relative to its radius (the high-aspect ratio vessel bioreactor). This small-aspect ratio means that a simple idealized model may be considered, in which (leading order) quantities are averaged across the axial direction. The leading-order fluid flow is then of Hele-Shaw type, and may be solved for explicitly. The trajectory of the tissue construct within the rotating bioreactor is determined by analysis of the various forces acting on it. Several different modes of motion are found to be possible, depending on the experimental conditions, and examples of each type of motion are presented. Additionally, we solve the problem for the nutrient transport around the tissue construct in the special case in which the construct remains fixed in the laboratory frame, and (as the cells proliferate in response to the nutrient available locally) deduce growth rates for the construct. Finally, we discuss our results in the light of possible experimental bioreactor set-ups. We note the present model's limitations, and consider how our work could be extended and improved to inform experimental protocols in future.

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.

Skeletal muscle satellite cells cultured in simulated microgravity.

Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and, therefore, provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75% satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D culture and 3-D HARV culture. Plating efficiency (cells attached divided by cells plated x 100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and nonsatellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability because glucose levels in medium from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARV were joined together by cells into 3-D aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a 3-D level of organization that could provide a more suitable model to study postnatal muscle development than is currently available with standard culture methods.