A multi-paradigm modeling framework to simulate dynamic reciprocity in a bioreactor.
ABSTRACT: Despite numerous technology advances, bioreactors are still mostly utilized as functional black-boxes where trial and error eventually leads to the desirable cellular outcome. Investigators have applied various computational approaches to understand the impact the internal dynamics of such devices has on overall cell growth, but such models cannot provide a comprehensive perspective regarding the system dynamics, due to limitations inherent to the underlying approaches. In this study, a novel multi-paradigm modeling platform capable of simulating the dynamic bidirectional relationship between cells and their microenvironment is presented. Designing the modeling platform entailed combining and coupling fully an agent-based modeling platform with a transport phenomena computational modeling framework. To demonstrate capability, the platform was used to study the impact of bioreactor parameters on the overall cell population behavior and vice versa. In order to achieve this, virtual bioreactors were constructed and seeded. The virtual cells, guided by a set of rules involving the simulated mass transport inside the bioreactor, as well as cell-related probabilistic parameters, were capable of displaying an array of behaviors such as proliferation, migration, chemotaxis and apoptosis. In this way the platform was shown to capture not only the impact of bioreactor transport processes on cellular behavior but also the influence that cellular activity wields on that very same local mass transport, thereby influencing overall cell growth. The platform was validated by simulating cellular chemotaxis in a virtual direct visualization chamber and comparing the simulation with its experimental analogue. The results presented in this paper are in agreement with published models of similar flavor. The modeling platform can be used as a concept selection tool to optimize bioreactor design specifications.
Project description:Human brain organoids, 3D self-assembled neural tissues derived from pluripotent stem cells, are important tools for studying human brain development and related disorders. Suspension cultures maintained by spinning bioreactors allow for the growth of large organoids despite the lack of vasculature, but commercially available spinning bioreactors are bulky in size and have low throughput. Here, we describe the procedures for building the miniaturized multiwell spinning bioreactor Spin? from 3D-printed parts and commercially available hardware. We also describe how to use Spin? to generate forebrain, midbrain and hypothalamus organoids from human induced pluripotent stem cells (hiPSCs). These organoids recapitulate key dynamic features of the developing human brain at the molecular, cellular and structural levels. The reduction in culture volume, increase in throughput and reproducibility achieved using our bioreactor and region-specific differentiation protocols enable quantitative modeling of brain disorders and compound testing. This protocol takes 14-84 d to complete (depending on the type of brain region-specific organoids and desired developmental stages), and organoids can be further maintained over 200 d. Competence with hiPSC culture is required for optimal results.
Project description:There is a significant clinical need for engineered bone graft substitutes that can quickly, effectively, and safely repair large segmental bone defects. One emerging field of interest involves the growth of engineered bone tissue in vitro within bioreactors, the most promising of which are perfusion bioreactors. Using bioreactor systems, tissue engineered bone constructs can be fabricated in vitro. However, these engineered constructs lack inherent vasculature and once implanted, quickly develop a necrotic core, where no nutrient exchange occurs. Here, we utilized COMSOL modeling to predict oxygen diffusion gradients throughout aggregated alginate constructs, which allowed for the computer-aided design of printable vascular networks, compatible with any large tissue engineered construct cultured in a perfusion bioreactor. We investigated the effect of 3D printed macroscale vascular networks with various porosities on the viability of human mesenchymal stem cells in vitro, using both gas-permeable, and non-gas permeable bioreactor growth chamber walls. Through the use of 3D printed vascular structures in conjunction with a tubular perfusion system bioreactor, cell viability was found to increase by as much as 50% in the core of these constructs, with in silico modeling predicting construct viability at steady state.
Project description:There is an increasing interest in developing microfluidic bioreactors and organs-on-a-chip platforms combined with sensing capabilities for continual monitoring of cell-secreted biomarkers. Conventional approaches such as ELISA and mass spectroscopy cannot satisfy the needs of continual monitoring as they are labor-intensive and not easily integrable with low-volume bioreactors. This paper reports on the development of an automated microfluidic bead-based electrochemical immunosensor for in-line measurement of cell-secreted biomarkers. For the operation of the multi-use immunosensor, disposable magnetic microbeads were used to immobilize biomarker-recognition molecules. Microvalves were further integrated in the microfluidic immunosensor chip to achieve programmable operations of the immunoassay including bead loading and unloading, binding, washing, and electrochemical sensing. The platform allowed convenient integration of the immunosensor with liver-on-chips to carry out continual quantification of biomarkers secreted from hepatocytes. Transferrin and albumin productions were monitored during a 5-day hepatotoxicity assessment in which human primary hepatocytes cultured in the bioreactor were treated with acetaminophen. Taken together, our unique microfluidic immunosensor provides a new platform for in-line detection of biomarkers in low volumes and long-term in vitro assessments of cellular functions in microfluidic bioreactors and organs-on-chips.
Project description:Since 1997, the highly pathogenic influenza H5N1 virus has spread from Hong Kong. According to the WHO bulletin report, the H5N1 virus is a zoonotic disease threat that has infected more than 850 humans, causing over 450 deaths. In addition, an outbreak of another new and highly pathogenic influenza virus (H7N9) occurred in 2013 in China. These highly pathogenic influenza viruses could potentially cause a worldwide pandemic. it is crucial to develop a rapid production platform to meet this surge demand against any possible influenza pandemic. A potential solution for this problem is the use of cell-based bioreactors for rapid vaccine production. These novel bioreactors, used for cell-based vaccine production, possess various advantages. For example, they enable a short production time, allow for the handling highly pathogenic influenza in closed environments, and can be easily scaled up. In this study, two novel disposable cell-based bioreactors, BelloCell and TideCell, were used to produce H5N1 clade II and H7N9 candidate vaccine viruses (CVVs). Madin-Darby canine kidney (MDCK) cells were used for the production of these influenza CVVs. A novel bench-scale bioreactor named BelloCell bioreactor was used in the study. All culturing conditions were tested and scaled to 10 L industrial-scale bioreactor known as TideCell002. The performances of between BelloCell and TideCell were similar in cell growth, the average MDCK cell doubling time was slightly decreased to 25 hours. The systems yielded approximately 39.2 and 18.0 ?g/ml of HA protein with the 10-liter TideCell002 from the H5N1 clade II and H7N9 CVVs, respectively. The results of this study not only highlight the overall effectiveness of these bioreactors but also illustrate the potential of maintaining the same outcome when scaled up to industrial production, which has many implications for faster vaccine production. Although additional studies are required for process optimization, the results of this study are promising and show that oscillating bioreactors may be a suitable platform for pandemic influenza virus production.
Project description:Biophysical cues can potently direct a cell's or tissue's behavior. Cells interpret their biophysical surroundings, such as matrix stiffness or dynamic mechanical stimulation, through mechanotransduction. However, our understanding of the various aspects of mechanotransduction has been limited by the lack of proper analysis platforms capable of screening three-dimensional (3D) cellular behaviors in response to biophysical cues. Here, we developed a dynamic compression bioreactor to study the combinational effects of biomaterial composition and dynamic mechanical compression on cellular behavior in 3D hydrogels. The bioreactor contained multiple actuating posts that could apply cyclic compressive strains ranging from 0 to 42% to arrays of cell-encapsulated hydrogels. The bioreactor could be interconnected with other compressive bioreactors, which enabled the combinatorial screenings of 3D cellular behaviors simultaneously. As an application of the screening platform, cell spreading, and osteogenic differentiation of human mesenchymal stem cells (hMSCs) were characterized in 3D gelatin methacryloyl (GelMA) hydrogels. Increasing hydrogel concentration from 5 to 10% restricted the cell spreading, however, dynamic compressive strain increased cell spreading. Osteogenic differentiation of hMSCs was also affected by dynamic compressive strains. hMSCs in 5% GelMA hydrogel were more sensitive to strains, and the 42% strain group showed a significant increase in osteogenic differentiation compared to other groups. The interconnectable dynamic compression bioreactor provides an efficient way to study the interactions of cells and their physical microenvironments in three dimensions.
Project description:There is a growing interest to develop microfluidic bioreactors and organ-on-chip platforms with integrated sensors to monitor their physicochemical properties and to maintain a well-controlled microenvironment for cultured organoids. Conventional sensing devices cannot be easily integrated with microfluidic organ-on-chip systems with low-volume bioreactors for continual monitoring. This paper reports on the development of a multi-analyte optical sensing module for dynamic measurements of pH and dissolved oxygen levels in the culture medium. The sensing system was constructed using low-cost electro-optics including light-emitting diodes and silicon photodiodes. The sensing module includes an optically transparent window for measuring light intensity, and the module could be connected directly to a perfusion bioreactor without any specific modifications to the microfluidic device design. A compact, user-friendly, and low-cost electronic interface was developed to control the optical transducer and signal acquisition from photodiodes. The platform enabled convenient integration of the optical sensing module with a microfluidic bioreactor. Human dermal fibroblasts were cultivated in the bioreactor, and the values of pH and dissolved oxygen levels in the flowing culture medium were measured continuously for up to 3 days. Our integrated microfluidic system provides a new analytical platform with ease of fabrication and operation, which can be adapted for applications in various microfluidic cell culture and organ-on-chip devices.
Project description:Recombinant protein and virus-like particle (VLP) production based on the baculovirus expression vector system is fast, flexible, and offers high yields. Independent from the product, a multitude of parameters are screened during process development/optimisation. Early development acceleration is a key requirement for economic efficiency, and µ-scale bioreactor systems represent an attractive solution for high-throughput (HTP) experimentation. However, limited practical knowledge is available on the relevance and transferability of screening data to pilot scales and manufacturing. The main goal of the present study was to evaluate a HTP µ-bioreactor platform with respect to its aptitude as a screening platform mainly based on transferability of results to benchtop bioreactors representing the conventional production regime. Second question was to investigate to what extent the online sensors of the µ-bioreactor contribute to process understanding and development. We demonstrated that transferability of infection screening results from the HTP µ-bioreactor scale to the benchtop bioreactor was equal or better than that from shaker cultivation. However, both experimental setups turned out to be sub-optimal solutions that only allowed for a first and rough ranking with low relevance in the case of absolute numbers. Bioreactor yields were up to one order of magnitude higher than the results of screening experiments.
Project description:Analysis of perfusion-based bioreactors for organ engineering and a detailed evaluation of physical and biochemical parameters that measure dynamic changes within maturing cell-laden scaffolds are critical components of ex vivo tissue development that remain understudied topics in the tissue and organ engineering literature. Intricately designed bioreactors that house developing tissue are critical to properly recapitulate the in vivo environment, deliver nutrients within perfused media, and monitor physiological parameters of tissue development. Herein, we provide an in-depth description and analysis of two dual-purpose perfusion bioreactors that improve upon current bioreactor designs and enable comparative analyses of ex vivo scaffold recellularization strategies and cell growth performance during long-term maintenance culture of engineered kidney or liver tissues. Both bioreactors are effective at maximizing cell seeding of small-animal organ scaffolds and maintaining cell survival in extended culture. We further demonstrate noninvasive monitoring capabilities for tracking dynamic changes within scaffolds as the native cellular component is removed during decellularization and model human cells are introduced into the scaffold during recellularization and proliferate in maintenance culture. We found that hydrodynamic pressure drop (?P) across the retained scaffold vasculature is a noninvasive measurement of scaffold integrity. We further show that ?P, and thus resistance to fluid flow through the scaffold, decreases with cell loss during decellularization and correspondingly increases to near normal values for whole organs following recellularization of the kidney or liver scaffolds. Perfused media may be further sampled in real time to measure soluble biomarkers (e.g., resazurin, albumin, or kidney injury molecule-1) that indicate degree of cellular metabolic activity, synthetic function, or engraftment into the scaffold. Cell growth within bioreactors is validated for primary and immortalized cells, and the design of each bioreactor is scalable to accommodate any three-dimensional scaffold (e.g., synthetic or naturally derived matrix) that contains conduits for nutrient perfusion to deliver media to growing cells and monitor noninvasive parameters during scaffold repopulation, broadening the applicability of these bioreactor systems.
Project description:In vitro models that capture the complexity of in vivo tissue and organ behaviors in a scalable and easy-to-use format are desirable for drug discovery. To address this, we have developed a bioreactor that fosters maintenance of 3D tissue cultures under constant perfusion and we have integrated multiple bioreactors into an array in a multiwell plate format. All bioreactors are fluidically isolated from each other. Each bioreactor in the array contains a scaffold that supports formation of hundreds of 3D microscale tissue units. The tissue units are perfused with cell culture medium circulated within the bioreactor by integrated pneumatic diaphragm micropumps. Electronic controls for the pumps are kept outside the incubator and connected to the perfused multiwell by pneumatic lines. The docking design and open-well bioreactor layout make handling perfused multiwell plates similar to using standard multiwell tissue culture plates. A model of oxygen consumption and transport in the circulating culture medium was used to predict appropriate operating parameters for primary liver cultures. Oxygen concentrations at key locations in the system were then measured as a function of flow rate and time after initiation of culture to determine oxygen consumption rates. After seven days of culture, tissue formed from cells seeded in the perfused multiwell reactor remained functionally viable as assessed by immunostaining for hepatocyte and liver sinusoidal endothelial cell (LSEC) phenotypic markers.
Project description:Bioreactors of various forms have been widely used in environmental protection, healthcare, industrial biotechnology, and space exploration. Robust demand in the field stimulated the development of novel designs of bioreactor geometries and process control strategies and the evolution of the physical structure of the control system. After the introduction of digital computers to bioreactor process control, a hierarchical structure control system (HSCS) for bioreactors has become the dominant physical structure, having high efficiency and robustness. However, inherent drawbacks of the HSCS for bioreactors have produced a need for a more consolidated solution of the control system. With the fast progress in sensors, machinery, and information technology, the development of a flat organizational control system (FOCS) for bioreactors based on parallel distributed smart sensors and actuators may provide a more concise solution for process control in bioreactors. Here, we review the evolution of the physical structure of bioreactor control systems and discuss the properties of the novel FOCS for bioreactors and related smart sensors and actuators and their application circumstances, with the hope of further improving the efficiency, robustness, and economics of bioprocess control.