Microfluidic Bioreactor Research Articles

Analysis of oxygen transport in microfluidic bioreactors for cell culture and organ‐on‐chip applications

In the recent decade, development of microfluidic bioreactors and organ‐on‐chip platforms for drug screening and disease modeling has been rising significantly. Prediction of oxygen level within the microfluidic bioreactors to create physiologically relevant oxygen tension for realistic cellular behavior is of critical importance. This article presented an analytical method to calculate oxygen tension in microchannel parallel plate bioreactors. Two‐dimensional convection‐diffusion equation was solved analytically with considering diffusion in two directions. Cellular oxygen consumption was assumed to follow Michaelis‐Menten kinetics. Effects of oxygenator design, gas permeability of the microfluidic channels, as well as flow rate and cell density on the oxygen distribution within the bioreactor were examined. In addition, a mathematical model was developed to predict oxygen tension in a fluidic circuit containing two interconnected bioreactors with and without recirculation of the media for the culture of hepatocytes and cardiomyocytes. The oxygenators were used to maintain normoxia in the bioreactors independently. The model allowed for the prediction and independent modulation of oxygen tension in the two bioreactors through changing the length of the oxygenators. In overall, the obtained results provide critical insights required for the design and operation of microfluidic bioreactors with desired levels of oxygen tension.

Characterisation of osteogenic and vascular responses of hMSCs to Ti-Co doped phosphate glass microspheres using a microfluidic perfusion platform

Using microspherical scaffolds as building blocks to repair bone defects of specific size and shape has been proposed as a tissue engineering strategy. Here, phosphate glass (PG) microcarriers doped with 5 mol % TiO2 and either 0 mol % CoO (CoO 0%) or 2 mol % CoO (CoO 2%) were investigated for their ability to support osteogenic and vascular responses of human mesenchymal stem cells (hMSCs). Together with standard culture techniques, cell-material interactions were studied using a novel perfusion microfluidic bioreactor that enabled cell culture on microspheres, along with automated processing and screening of culture variables. While titanium doping was found to support hMSCs expansion and differentiation, as well as endothelial cell-derived vessel formation, additional doping with cobalt did not improve the functionality of the microspheres. Furthermore, the microfluidic bioreactor enabled screening of culture parameters for cell culture on microspheres that could be potentially translated to a scaled-up system for tissue-engineered bone manufacturing.

A Drug Repurposing and Protein-Protein Interaction Network Study of Ribosomopathies Using Yeast as a Model System.

Ribosomopathies result in various cancers, neurodegenerative and viral diseases, and other pathologies such as Diamond-Blackfan anemia and Shwachman-Diamond syndrome. Their pathophysiology at a proteome and functional level remains to be determined. Protein networks and highly connected hub proteins for ribosome biogenesis in Saccharomyces cerevisiae offer a potential as a model system to inform future therapeutic innovation in ribosomopathies. In this context, we report a ribosome biogenesis protein-protein interaction network in S. cerevisiae, created with 1772 proteins and 22,185 physical interactions connecting them. Moreover, by network decomposition analysis, we determined the linear pathways between the transcription factors and target proteins with a view to drug repurposing. While considering only the paths containing the three C/D box proteins (Nop56, Nop58, and Nop1), the most frequently encountered proteins were Aft1, Htz1, Ssa1, Ssb1, Ssb2, Gcn5, Cka1, Tef1, Nop1, Cdc28, Act1, Krr1, Rpl8B, and Tor1, which were then identified as potential drug targets. For drug repurposing, these candidate proteins were further searched in the DrugBank to find other diseases associated with them, as well as the drugs used to treat these diseases. To support the computational results, an experimental study was conducted using in-house manufactured microfluidic bioreactor platform, while the effect of the drug temsirolimus, Tor1 inhibitor, on yeast cells was investigated by following Nop56 protein expression. In conclusion, these results inform the ways in which ribosomopathies and associated common complex human diseases materialize and how drug repurposing might accelerate therapeutic innovation through bioinformatic studies of yeast.

Microfluidic Sensor Based on Composite Left-Right Handed Transmission Line

In this paper, we propose a novel metamaterial-based microfluidic sensor that permits the monitoring of properties of the fluid flowing in the microfluidic reservoir embedded between the composite left–right handed (CLRH) microstrip line and the ground plane. The sensor’s working principle is based on the phase shift measurement of the two signals, the referent one that is guided through conventional microstrip line and measurement signal guided through the CLRH line. At the operating frequency of 1.275 GHz, the CLRH line supports electromagnetic waves with group and phase velocities that are antiparallel, and therefore the phase “advance” occurs in the case of CLRH line, while phase delay arises in the right-handed (RH) frequency band. The change of the fluid’s properties that flow in the microfluidic reservoir causes the change of effective permittivity of the microstrip substrate, and subsequently the phase velocity changes, as well as the phase shift. This effect was used in the design of the microfluidic sensor for the measurement of characteristics of the fluid that flows in the microfluidic reservoir placed under the CLRH line. The complete measurement system was developed including the Wilkinson power divider that splits the signal between conventional RH and CLRH section, transmission lines with the microfluidic reservoirs, and a detection circuit for phase shift measurement. Measurement results for different fluids confirm that the proposed sensor is characterized by relatively high sensitivity and good linearity (R2 = 0.94). In this study, the practical application of the proposed sensor is demonstrated for the biomass estimation inside the microfluidic bioreactors, which are used for the cultivation of MRC-5 fibroblasts.

Nano and Microtechnologies for the Study of Magnetotactic Bacteria

AbstractMagnetotactic bacteria (MTB) naturally synthesize magnetic nanoparticles that are wrapped in lipid membranes. These membrane‐bound particles, which are known as magnetosomes, are characterized by their narrow size distribution, high colloidal stability, and homogenous magnetic properties. These characteristics of magnetosomes confer them with significant value as materials for biomedical and industrial applications. MTB are also a model system to study key biological questions relating to formation of bacterial organelles, metal homeostasis, biomineralization, and magnetoaerotaxis. The similar size scale of nano and microfluidic systems to MTB and ease of coupling to local magnetic fields make them especially useful to study and analyze MTB. In this Review, a summary of nano‐ and microtechnologies that are developed for purposes such as MTB sorting, genetic engineering, and motility assays is provided. The use of existing platforms that can be adapted for large‐scale MTB processing including microfluidic bioreactors is also described. As this is a relatively new field, future synergistic research directions coupling MTB, and nano‐ and microfluidics are also suggested. It is hoped that this Review could start to bridge scientific communities and jump‐start new ideas in MTB research that can be made possible with nano‐ and microfluidic technologies.

Dynamic Culture and Selective Extraction of Target Microbial Cells in Self-Assembled Particle Membrane-Integrated Microfluidic Bioreactor Array.

Various microfluidic devices have overcome many disadvantages common to conventional bioreactor systems by enabling active manipulation of cell-culture conditions, monitoring of cellular responses in high-throughput mode, and extraction of target cells in a relatively rapid and low-cost manner. However, existing microfluidic devices still have limitations, including the complexity of their operation and a lack of availability of dynamic control of the chemical environment. Here, we present a novel microfluidic bioreactor array device capable of not only the stable and dynamic programing of cell-culture environments but also the selective extraction of target cells. This device comprises 64 microchambers in a 16 × 4 array format, and each microchamber is integrated with a robust and nanoporous membrane on one side and an H-shaped entrance on the other. The membrane made of self-assembled particles allowed continuous and sequential delivery of various nutrients containing gene inducers to compartmentalized microbial cells, thereby enabling dynamic cell culturing. Additionally, the H-shaped entrance was used for local and selective blocking of the microchamber by employing UV-curable material, thereby enabling the retrieval of target cells from the device while sequestering nontarget cells in the microchambers. Our results demonstrated that the targeted rare cells could be isolated and separated from a mixture of cells by repeating the extraction procedure. Therefore, we anticipate that this microfluidic bioreactor array device will be widely used for not only screening/extraction but also off-chip postanalyses of various microorganisms.

Online Measurement of Glucose Consumption from HepG2 Cells Using an Integrated Bioreactor and Enzymatic Assay.

Hepatocytes help to maintain glucose homeostasis in response to a variety of signals, including pancreatic hormones such as insulin. Insulin is released from the pancreas with variable dynamics, yet the role that these play in regulating glucose metabolism in the liver is still unclear. In this study, a modular microfluidic system was developed to quantitatively measure the effect of insulin dynamics on glucose consumption by a human hepatocarcinoma cell line, HepG2. A microfluidic bioreactor that contained 106 HepG2 cells was cultured for up to 10 days in an incubator. For glucose consumption experiments, the bioreactor was removed from the incubator and connected with reagents for an enzymatic glucose assay. The mixed components were then delivered into a droplet-based microfluidic system where the intensity of the fluorescent product of the enzyme assay was used to quantify the glucose concentration. By optimizing the mixing time of the reagents, the dynamic range of the enzymatic assay was adjusted to 0–12 mM glucose and had a time resolution of 96 ± 12 s. The system was used to observe rapid changes in insulin-induced glucose consumption from HepG2 cells. This assay format is versatile and can be expanded to measure a variety of hepatic metabolites, such as lactate, pyruvate, or ketone bodies, which will enable the correlation of pancreatic hormone dynamics to liver metabolism.

Influence of the static magnetic field on cell response in a miniaturized optically accessible bioreactor for 3D cell culture

Hydraulic sealing is a crucial condition for the maintenance of sterility during long term operation of microfluidic bioreactors. We developed a miniaturized optically accessible bioreactor (MOAB) allowing perfused culture of 3D cellularised constructs. In the MOAB, the culture chambers are sealed by magnets that generate a weak static magnetic field (SMF). Here, we predicted computationally the exact level of SMF to which cells are subjected during culture in the MOAB and we assessed its influence on the viability, metabolic activity and gene expression of neuroblastoma-derived cells cultured up to seven days. The predicted SMF ranged from 0.32 to 0.57 T using an axial-symmetric model of a single chamber, whereas it ranged from 0.35 to 0.62 T using a 3D model of the complete device. Cell function was evaluated in SH-SY5Y neuroblastoma cells at 2 and 7 days of culture in the MOAB, compared to 2D monolayer, 3D non-perfused constructs, and 3D perfused constructs cultured in a modified MOAB with magnet-free sealing. We measured the cell metabolic activity normalized by the DNA content and the expression levels of heat-shock protein 70 (Hsp-70), Bcl-2 and Bax. We found that the level of SMF applied to cells in the MOAB did not influence their metabolic activity and exerted a stressful effect in 2D monolayer, not confirmed in 3D conditions, neither static not perfused. Instead, the magnets provided a significantly greater hydraulic sealing in long-term culture, thus the MOAB might be potentially exploitable for the development of reliable in vitro models of neurodegeneration.

Fish-gut-on-chip: development of a microfluidic bioreactor to study the role of the fish intestine in vitro.

In this study we present the first fish-gut-on-chip model. This model is based on the reconstruction of the intestinal barrier by culturing two intestinal cell lines from rainbow trout, namely epithelial RTgutGC and fibroblastic RTgutF, in an artificial microenvironment. For a realistic mimicry of the interface between the intestinal lumen and the interior of the organism we i) developed ultrathin and highly porous silicon nitride membranes that serve as basement membrane analogues and provide a culture interface for the fish cells; ii) constructed a unique micro-well plate-based microfluidic bioreactor that enables parallelization of experiments and creates realistic fluid flow exposure scenarios for the cells; iii) integrated electrodes in the reactor for non-invasive impedance sensing of cellular well-being. In a first approach, we used this reactor to investigate the response of epithelial fish cells to in vivo-like shear stress rates of 0.002-0.06 dyne per cm2, resulting from fluid flow within the intestinal lumen. Moreover, we investigated the interplay of epithelial and fibroblast cells under optimal flow conditions to carefully evaluate the benefits and drawbacks of the more complex reconstruction of the intestinal architecture. With our fish-gut-on-chip model we open up new strategies for a better understanding of basic fish physiology, for the refinement of fish feed in aquaculture and for predicting chemical uptake and bioaccumulation in fish for environmental risk assessment. The basic principles of our reactor prototype, including the use of ultrathin membranes, an open microfluidic circuit for perfusion and the micro-well plate-based format for simplified handling and avoidance of air-bubbles, will as well be of great value for other barrier-on-chip models.

A Compartmented Flow Microreactor System for Automated Optimization of Bioprocesses Applying Immobilized Enzymes.

In the course of their development, industrial biocatalysis processes have to be optimized in small-scale, e. g., within microfluidic bioreactors. Recently, we introduced a novel microfluidic reactor device, which can handle defined reaction compartments of up to 250 μL in combination with magnetic micro carriers. By transferring the magnetic carriers between subsequent compartments of differing compositions, small scale synthesis, and bioanalytical assays can be conducted. In the current work, this device is modified and extended to broaden its application range to the screening and optimization of bioprocesses applying immobilized enzymes. Besides scaling the maximum compartment volume up to 3 mL, a temperature control module, as well as a focused infrared spot were integrated. By adjusting the pump rate, compartment volumes can be accurately dosed with an error <5% and adjusted to the requested temperature within less than a minute. For demonstration of bioprocess parameter optimization within such compartments, the influence of pH, temperature, substrate concentration, and enzyme carrier loading was automatically screened for the case of transferring UDP-Gal onto N-acetylglucosamine linked to a tert-butyloxycarbonyl protected amino group using immobilized β1,4-galactosyltransferase-1. In addition, multiple recycling of the enzyme carriers and the use of increased compartment volumes also allows the simple production of preparative amounts of reaction products.

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography.

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

Long-Term Retinal Differentiation of Human Induced Pluripotent Stem Cells in a Continuously Perfused Microfluidic Culture Device.

Understanding how microenvironmental cues influence cellular behavior will enable development of efficient and robust pluripotent stem cell differentiation protocols. Unlike traditional cell culture dishes, microfluidic bioreactors can provide stable microenvironmental conditions by continuous medium perfusion at a controlled rate. The aim of this study is to investigate whether a microfluidic culture device could be used as a perfused platform for long-term cell culture processes such as the retinal differentiation of human induced pluripotent stem cells. The perfusion flow rate is established based on the degradation and consumption of growth factors (DKK-1, Noggin, IGF-1, and bFGF) and utilizing the Péclet number. The device's performance analyzed by qRT-PCR show improvements compared to the well-plate control as characterized by significantly higher expression of the markers Pax6, Chx10, and Crx on Day 5, Nrl on day 10, Crx, and Rhodopsin on day 21. Optimization of perfusion rate is an important operating variable in development of robust processes for differentiation cultures. Result demonstrates convective delivery of nutrients via perfusion has a significant impact upon the expression of key retinal markers. This study is the first continuously perfused long-term (21 days) retinal differentiation of hiPSCs in a microfluidic device.

Design of microfluidic bioreactors: Transport regimes

AbstractAn advection–diffusion–reaction transport model for a continuous reactor characterized by a simple geometry is simulated to provide a design guidance for the realization of an optimized experimental prototype consisting of a rectangular channel hosting a scaffold slab that provides an immobilizing support for the cells. A counter‐current carrier flow crossing the channel feeds the oxygen and growth factors and ensures the removal of metabolic products. A more complex sinusoidal configuration, which has been recently used as for hepatocyte culture, is proposed and evaluated. Silico tests shows how the designed device is able to replicate the endothelial–parenchymal interface of a liver sinusoid.

A low cost PS based microfluidic platform to investigate cell cycle towards developing a therapeutic strategy for cancer.

Inhibition of DNA damage response pathway in combination with DNA alkylating agents may enhance the selective killing of cancer cells leading to better therapeutic effects. MDM2 binding protein (MTBP) in human has a role in G1 phase (interphase of cell cycle) and its overexpression leads to breast and ovarian cancers. Sld7 is an uncharacterized protein in budding yeast and a potential functional homologue of MTBP. To investigate the role of Sld7 as a therapeutic target, the behavior of the wild-type cells and sld7∆ mutants were monitored in 0.5nL microbioreactors. The brightfield microscopy images were used to analyze the change in the cell size and to determine the durations of G1 and S/G2/M phases of wild type cells and mutants. With the administration of the alkylating agent, the cell size decreased and the duration of cell cycle increased. The replacement of the medium with the fresh one enabled the cells to repair their DNA. The application of calorie restriction together with DNA alkylating agent to mutant cells resulted in smaller cell size and longer G1 phase compared to those in control environment. For therapeutic purposes, the potential of MTBP in humans or Sld7 in yeast as a drug target deserves further exploration. The fabrication simplicity, robustness and low-cost of this microfluidic bioreactor made of polystyrene allowed us to perform yeast culturing experiments and show a potential for further cell culturing studies. The device can successfully be used for therapeutic applications including the discovery of new anti-microbial, anti-inflammatory, anti-cancer drugs.

Journey into Bone Models: A Review.

Bone is a complex tissue with a variety of functions, such as providing mechanical stability for locomotion, protection of the inner organs, mineral homeostasis and haematopoiesis. To fulfil these diverse roles in the human body, bone consists of a multitude of different cells and an extracellular matrix that is mechanically stable, yet flexible at the same time. Unlike most tissues, bone is under constant renewal facilitated by a coordinated interaction of bone-forming and bone-resorbing cells. It is thus challenging to recreate bone in its complexity in vitro and most current models rather focus on certain aspects of bone biology that are of relevance for the research question addressed. In addition, animal models are still regarded as the gold-standard in the context of bone biology and pathology, especially for the development of novel treatment strategies. However, species-specific differences impede the translation of findings from animal models to humans. The current review summarizes and discusses the latest developments in bone tissue engineering and organoid culture including suitable cell sources, extracellular matrices and microfluidic bioreactor systems. With available technology in mind, a best possible bone model will be hypothesized. Furthermore, the future need and application of such a complex model will be discussed.

Engineered microfluidic bioreactor for examining the three-dimensional breast tumor microenvironment.

The interaction of cancer cells with the stromal cells and matrix in the tumor microenvironment plays a key role in progression to metastasis. A better understanding of the mechanisms underlying these interactions would aid in developing new therapeutic approaches to inhibit this progression. Here, we describe the fabrication of a simple microfluidic bioreactor capable of recapitulating the three-dimensional breast tumor microenvironment. Cancer cell spheroids, fibroblasts, and endothelial cells co-cultured in this device create a robust microenvironment suitable for studying in real time the migration of cancer cells along matrix structures laid down by fibroblasts within the 3D tumor microenvironment. This system allows for ready evaluation of response to targeted therapy.

Development of a 3D in vitro platform for functional and structural interrogation of neuronal circuits

Development of a 3D in vitro platform for functional and structural interrogation of neuronal circuits

Numerical Simulation of the Behavior of Toroidal and Spheroidal Multicellular Aggregates in Microfluidic Devices with Microwell and U-Shaped Barrier.

A microfluidic system provides an excellent platform for cellular studies. Most importantly, a three-dimensional (3D) cell culture model reconstructs more accurately the in vivo microenvironment of tissue. Accordingly, microfluidic 3D cell culture devices could be ideal candidates for in vitro cell culture platforms. In this paper, two types of 3D cellular aggregates, i.e., toroid and spheroid, are numerically studied. The studies are carried out for microfluidic systems containing U-shaped barrier as well as microwell structure. For the first time, we obtain oxygen and glucose concentration distributions inside a toroid aggregate as well as the shear stress on its surface and compare its performance with a spheroid aggregate of the same volume. In particular, we obtain the oxygen concentration distributions in three areas, namely, oxygen-permeable layer, multicellular aggregates and culture medium. Further, glucose concentration distributions in two regions of multicellular aggregates and culture medium are investigated. The results show that the levels of oxygen and glucose in the system containing U-shaped barriers are far more than those in the system containing microwells. Therefore, to achieve high levels of oxygen and nutrients, a system with U-shaped barriers is more suited than the conventional traps, but the choice between toroid and spheroid depends on their volume and orientation. The results indicate that higher oxygen and glucose concentrations can be achieved in spheroid with a small volume as well as in horizontal toroid with a large volume. The vertical toroid has the highest levels of oxygen and glucose concentration while the surface shear stress on its surface is also maximum. These findings can be used as guidelines for designing an optimum 3D microfluidic bioreactor based on the desired levels of oxygen, glucose and shear stress distributions.

SCWISh network is essential for survival under mechanical pressure

Cells that proliferate within a confined environment build up mechanical compressive stress. For example, mechanical pressure emerges in the naturally space-limited tumor environment. However, little is known about how cells sense and respond to mechanical compression. We developed microfluidic bioreactors to enable the investigation of the effects of compressive stress on the growth of the genetically tractable model organism Saccharomyces cerevisiae We used this system to determine that compressive stress is partly sensed through a module consisting of the mucin Msb2 and the cell wall protein Sho1, which act together as a sensor module in one of the two major osmosensing pathways in budding yeast. This signal is transmitted via the MAPKKK kinase Ste11. Thus, we term this mechanosensitive pathway the "SMuSh" pathway, for Ste11 through Mucin/Sho1 pathway. The SMuSh pathway delays cells in the G1 phase of the cell cycle and improves cell survival in response to growth-induced pressure. We also found that the cell wall integrity (CWI) pathway contributes to the response to mechanical compressive stress. These latter results are confirmed in complimentary experiments in Mishra et al. [Mishra R, et al. (2017) Proc Natl Acad Sci USA, 10.1073/pnas.1709079114]. When both the SMuSh and the CWI pathways are deleted, cells fail to adapt to compressive stress, and all cells lyse at relatively low pressure when grown in confinement. Thus, we define a network that is essential for cell survival during growth under pressure. We term this mechanosensory system the SCWISh (survival through the CWI and SMuSh) network.

Protein/polysaccharide-based scaffolds mimicking native extracellular matrix for cardiac tissue engineering applications.

Tissue engineering has emerged as a viable approach to treat disease or repair damage in tissues and organs. One of the key elements for the success of tissue engineering is the use of a scaffold serving as artificial extracellular matrix (ECM). The ECM hosts the cells and improves their survival, proliferation, and differentiation, enabling the formation of new tissue. Here, we propose the development of a class of protein/polysaccharide-based porous scaffolds for use as ECM substitutes in cardiac tissue engineering. Scaffolds based on blends of a protein component, collagen or gelatin, with a polysaccharide component, alginate, were produced by freeze-drying and subsequent ionic and chemical crosslinking. Their morphological, physicochemical, and mechanical properties were determined and compared with those of natural porcine myocardium. We demonstrated that our scaffolds possessed highly porous and interconnected structures, and the chemical homogeneity of the natural ECM was well reproduced in both types of scaffolds. Furthermore, the alginate/gelatin (AG) scaffolds better mimicked the native tissue in terms of interactions between components and protein secondary structure, and in terms of swelling behavior. The AG scaffolds also showed superior mechanical properties for the desired application and supported better adhesion, growth, and differentiation of myoblasts under static conditions. The AG scaffolds were subsequently used for culturing neonatal rat cardiomyocytes, where high viability of the resulting cardiac constructs was observed under dynamic flow culture in a microfluidic bioreactor. We therefore propose our protein/polysaccharide scaffolds as a viable ECM substitute for applications in cardiac tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 769-781, 2018.