Microfluidic Cell Culture System Research Articles

Hollow fibres integrated in a microfluidic cell culture system

Article Hollow fibres integrated in a microfluidic cell culture system was published on August 31, 2012 in the journal Biomedical Engineering / Biomedizinische Technik (volume 57, issue SI-1-Track-G).

The application of an optically switched dielectrophoretic (ODEP) force for the manipulation and assembly of cell-encapsulating alginate microbeads in a microfluidic perfusion cell culture system for bottom-up tissue engineering

This study reports the utilisation of an optically switched dielectrophoretic (ODEP) force for the manipulation and assembly of cell-encapsulating alginate microbeads in a microfluidic perfusion cell culture system for bottom-up tissue engineering. One of the key features of this system is the ODEP force-based mechanism, which allows a commercial projector to be coupled with a computer to manipulate and assemble cell-encapsulating microbeads in an efficient, manageable, and user-friendly manner. Another distinctive feature is the design of the microfluidic cell culture chip, which allows the patterned cell-encapsulating microbeads to be cultivated on site under culture medium perfusion conditions. For demonstrating its application in bottom-up cartilage tissue engineering, chondrocyte-encapsulating alginate microbeads varying in encapsulated cell densities were generated. The manipulation forces associated with operating the alginate microbeads were experimentally evaluated. The results revealed that the measured manipulation forces increased with increases in both the applied electric voltage and the number of cells in the alginate microbeads. Nevertheless, the observed manipulation force was found to be independent of the size of the cell-free alginate microbeads. It can be speculated that the friction force may influence the estimation of the ODEP force within the experimental conditions investigated. In this study, chondrocyte-encapsulating alginate microbeads with three different cell densities were manipulated and assembled in the proposed microfluidic system to form a compact sheet-like cell culture construct that imitates the cell distribution in the cross-section of native articular cartilage. Moreover, the demonstration case also showed that the cell viability of the cultured cells in the microfluidic system remained as high as 96 ± 2%. In this study, four sheet-like cell culture constructs were stacked to create a larger assembled cell culture construct. The cell distribution inside the cell culture construct was further confirmed by a confocal microscopy observation, which showed that the distribution was similar to that in native articular cartilage. As a whole, the proposed system holds great promise as a platform for engineering tissue constructs with easily tunable inner cell distributions.

Fluid flow stress induced contraction and re-spread of mesenchymal stem cells: a microfluidic study

Mesenchymal stem cells (MSCs), the multipotent progenitor cells, are sensitive to fluid shear stress (FSS). MSCs can migrate through the blood stream by intravasation into the circulatory system to transfer to distant positions through the blood stream. During the transferring process, MSCs may differentiate into cells of corresponding tissues for repair, or remain undifferentiated and initiate ectopic tissue formation, lipid accumulation, or calcification, which are closely related to the pathology of atherosclerosis. However, how the MSCs sense and respond to vascular FSS stimulation and lead to subsequent biological effects remains elusive. In this study, by using an in situ time-lapse microfluidic cell culture and observation system, we found that rat mesenchymal stem cells (rMSCs) presented a contraction and re-spread (CRS) process when they were initially subjected to a physiological FSS (1.3 Pa). Our subsequent studies demonstrated that integrin and cilia played key roles in sensing FSS. Calcium, F-actin, and Rho-kinase were key molecules in the mechanotransduction of the CRS of the rMSCs. Our study revealed the immediate response of the rMSCs to FSS. It will be helpful for the understanding of MSC-related tissue repair and the role of MSCs in the initiation of atherosclerosis.

A self-contained, programmable microfluidic cell culture system with real-time microscopy access

Utilizing microfluidics is a promising way for increasing the throughput and automation of cell biology research. We present a complete self-contained system for automated cell culture and experiments with real-time optical read-out. The system offers a high degree of user-friendliness, stability due to simple construction principles and compactness for integration with standard instruments. Furthermore, the self-contained system is highly portable enabling transfer between work stations such as laminar flow benches, incubators and microscopes. Accommodation of 24 individual inlet channels enables the system to perform parallel, programmable and multiconditional assays on a single chip. A modular approach provides system versatility and allows many different chips to be used dependent upon application. We validate the system's performance by demonstrating on-chip passive switching and mixing by peristaltically driven flows. Applicability for biological assays is demonstrated by on-chip cell culture including on-chip transfection and temporally programmable gene expression.

A disposable and multifunctional capsule for easy operation of microfluidic elastomer systems

The global lab-on-chip and microfluidic markets for cell-based assays have been predicted to grow considerably, as novel microfluidic systems enable cell biologists to perform in vitro experiments at an unprecedented level of experimental control. Nevertheless, microfluidic assays must, in order to compete with conventional assays, be made available at easily affordable costs, and in addition be made simple to operate for users having no previous experience with microfluidics. We have to this end developed a multifunctional microfluidic capsule that can be mass-produced at low cost in thermoplastic material. The capsule enables straightforward operation of elastomer inserts of optional design, here exemplified with insert designs for molecular gradient formation in microfluidic cell culture systems. The integrated macro–micro interface of the capsule ensures reliable connection of the elastomer fluidic structures to an external perfusion system. A separate compartment in the capsule filled with superabsorbent material is used for internal waste absorption. The capsule assembly process is made easy by integrated snap-fits, and samples within the closed capsule can be analyzed using both inverted and upright microscopes. Taken together, the capsule concept presented here could help accelerate the use of microfluidic-based biological assays in the life science sector.

Development of an Integrated Microfluidic Perfusion Cell Culture System for Real-Time Microscopic Observation of Biological Cells

This study reports an integrated microfluidic perfusion cell culture system consisting of a microfluidic cell culture chip, and an indium tin oxide (ITO) glass-based microheater chip for micro-scale perfusion cell culture, and its real-time microscopic observation. The system features in maintaining both uniform, and stable chemical or thermal environments, and providing a backflow-free medium pumping, and a precise thermal control functions. In this work, the performance of the medium pumping scheme, and the ITO glass microheater were experimentally evaluated. Results show that the medium delivery mechanism was able to provide pumping rates ranging from 15.4 to 120.0 μL·min−1. In addition, numerical simulation and experimental evaluation were conducted to verify that the ITO glass microheater was capable of providing a spatially uniform thermal environment, and precise temperature control with a mild variation of ±0.3 °C. Furthermore, a perfusion cell culture was successfully demonstrated, showing the cultured cells were kept at high cell viability of 95 ± 2%. In the process, the cultured chondrocytes can be clearly visualized microscopically. As a whole, the proposed cell culture system has paved an alternative route to carry out real-time microscopic observation of biological cells in a simple, user-friendly, and low cost manner.

Microengineering methods for cell-based microarrays and high-throughput drug-screening applications

Screening for effective therapeutic agents from millions of drug candidates is costly, time consuming, and often faces concerns due to the extensive use of animals. To improve cost effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems has facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell-based drug-screening models which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell-based drug-screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds great potential to provide repeatable 3D cell-based constructs with high temporal, spatial control and versatility.

Wnt5a-mediating neurogenesis of human adipose tissue-derived stem cells in a 3D microfluidic cell culture system

In stem cell biology, cell plasticity refers to the ability of stem cells to differentiate into a variety of cell lineages. Recently, cell plasticity has been used to refer to the ability of a given cell type to reversibly de-differentiate, re-differentiate, or transdifferentiate in response to specific stimuli. These processes are regulated by multiple intracellular and extracellular growth and differentiation factors, including low oxygen. Our recent study showed that 3D microfluidic cell culture induces activation of the Wnt5A/β-catenin signaling pathway in hATSCs (human Adipose Tissue-derived Stem Cells). This resulted in self renewal and transdifferentiation of hATSCs into neurons. To improve neurogenic potency of hATSCs in response to low oxygen and other unknown physical factors, we developed a gel-free 3D microfluidic cell culture system (3D-μFCCS). The functional structure was developed for the immobilization of 3D multi-cellular aggregates in a microfluidic channel without the use of a matrix on the chip. Growth of hATSCs neurosphere grown on a chip was higher than the growth of control cells grown in a culture dish. Induction of differentiation in the Chip system resulted in a significant increase in the induction of neuronal-like cell structures and the presentation of TuJ or NF160 positive long neuritis compared to control cells after active migration from the center of the microfluidic channel layer to the outside of the microfluidic channel layer. We also observed that the chip neurogenesis system induced a significantly higher level of GABA secreting neurons and, in addition, almost 60% of cells were GABA + cells. Finally, we observed that 1 month of after the transplantation of each cell type in a mouse SCI lesion, chip cultured and neuronal differentiated hATSCs exhibited the ability to effectively transdifferentiate into NF160 + motor neurons at a high ratio. Interestingly, our CHIP/PCR analysis revealed that HIF1α-induced hATSCs neurogenesis on the chip. This induction was a result of the direct binding of HIF1α to the regulatory regions of the Oct4 and β-catenin genes in nucleus. In the Chip culture of hATSCs that we developed, a low oxygen microenvironment was induced. The low oxygen level induced HIF1α expression, which resulted in increased expression of Wnt5A/β-catenin and Oct4 via the direct binding of HIF1α to the regulatory regions of β-catenin and Oct4.

Interstitial flow influences direction of tumor cell migration through competing mechanisms

Interstitial flow is the convective transport of fluid through tissue extracellular matrix. This creeping fluid flow has been shown to affect the morphology and migration of cells such as fibroblasts, cancer cells, endothelial cells, and mesenchymal stem cells. A microfluidic cell culture system was designed to apply stable pressure gradients and fluid flow and allow direct visualization of transient responses of cells seeded in a 3D collagen type I scaffold. We used this system to examine the effects of interstitial flow on cancer cell morphology and migration and to extend previous studies showing that interstitial flow increases the metastatic potential of MDA-MB-435S melanoma cells [Shields J, et al. (2007) Cancer Cell 11:526-538]. Using a breast carcinoma line (MDA-MB-231) we also observed cell migration along streamlines in the presence of flow; however, we further demonstrated that the strength of the flow as well as the cell density determined directional bias of migration along the streamline. In particular, we found that cells either at high seeding density or with the CCR-7 receptor inhibited migration against, rather than with the flow. We provide further evidence that CCR7-dependent autologous chemotaxis is the mechanism that leads to migration with the flow, but also demonstrate a competing CCR7-independent mechanism that causes migration against the flow. Data from experiments investigating the effects of cell concentration, interstitial flow rate, receptor activity, and focal adhesion kinase phosphorylation support our hypothesis that the competing stimulus is integrin mediated. This mechanism may play an important role in development of metastatic disease.

Transfection of Molecular Beacons in Microchannels for Single-Cell Gene-Expression Analysis

Efficient transfection of molecular beacons has to be performed in the microscale in order to fully utilize the potential of molecular beacons and microfluidics for studying the real-time gene-expression dynamics in living cells. Nevertheless, there has been relatively little study on transfection of molecular beacons in microfluidic channels. In this work, the differences between transfection in conventional cell culture systems and in microfluidic cell culture systems were investigated systematically with a combination of computational and experimental methods. Comparison between a no-flow microchannel and a 96-well plate revealed that the scale-dependence of reaction-diffusion kinetics contributes to the reduced transfection efficiency in the no-flow microchannel. Study on transfection in the microfluidic system under flow conditions suggested that the fluid flow enhances mass transfer, while the fluid shear stress can reduce the transfection efficiency. The results of this study will provide useful guidelines in optimizing molecular beacon transfection efficiency in microfluidic systems for studying gene-expression dynamics in living cells.

Reproducible fashion of the HSP70B' promoter‐induced cytotoxic response on a live cell‐based biosensor by cell cycle synchronization

Live cell-based sensors potentially provide functional information about the cytotoxic effect of reagents on various signaling cascades. Cells transfected with a reporter vector derived from a cytotoxic response promoter can be used as intelligent cytotoxicity sensors (i.e., sensor cells). We have combined sensor cells and a microfluidic cell culture system that can achieve several laminar flows, resulting in a reliable high-throughput cytotoxicity detection system. These sensor cells can also be applied to single cell arrays. However, it is difficult to detect a cellular response in a single cell array, due to the heterogeneous response of sensor cells. The objective of this study was cell homogenization with cell cycle synchronization to enhance the response of cell-based biosensors. Our previously established stable sensor cells were brought into cell cycle synchronization under serum-starved conditions and we then investigated the cadmium chloride-induced cytotoxic response at the single cell level. The GFP positive rate of synchronized cells was approximately twice as high as that of the control cells, suggesting that cell homogenization is an important step when using cell-based biosensors with microdevices, such as a single cell array.

Microfluidic cell culture system studies and computational fluid dynamics

Microfluidic platforms are microfabricated tools that are gaining popularity for studying of cellular biology. These platforms can allow precise control of the environment surrounding individual cells and have achieved amazing progress in application to cell culture in recent several years. After a brief description of the background and the major characteristics of the microfluidic chip and the corresponding advantages for cell culture, the development of cell patterning techniques is discussed. The microfluidic cell culture platforms are classified into: (i) single cell-type culture; and, (ii) multiple cell-type culture (i.e., cell coculture). Several microfluidic cell culture systems integrated with other functionalities using microfabricated technology are reviewed. This review also aims at discussing the rationale of using computational fluid dynamics (CFD) in microfluidic cell culture system studies, as fluid flow processes and phenomena have direct implications on cellular response such as migration and/or proliferation.

A hemispherical microfluidic channel for the trapping and passive dissipation of microbubbles

In this paper, we present that trapping and dissipating of bubbles in a microfluidic cell culture system can be simultaneously achieved by utilizing curved geometry principles. For this end, a simple and cost-effective method to fabricate a curved hemispherical microfluidic channel is presented. On the basis of an analytical model, the mechanism that the hemispherical well can trap various sizes of bubbles better than the cylindrical well is described, and we present a quantitative comparison of the trapping capabilities of the hemispherical versus conventional cylindrical wells through experiments. The surface tension is another important factor to trap bubbles, which was also verified through the analysis and experiments. In the hemispherical wells, the trapped bubbles were spontaneously dissipated under the flowing condition without using any active source, and we characterized the degassing process by measuring the area of bubbles occupied in the well over time. For an application to a biomedical system, a cell culture chamber was combined with the bubble trapping system, and the performance of the system was verified by culturing HeLa cells with the flowing bubbled culture media. Conclusively, the suggested method demonstrated excellent performance in trapping of microbubbles and dissipation without using any peripheral device, and will be broadly applied in biomedical microfluidic research.

Concentration gradients in microfluidic 3D matrix cell culture systems

Microfluidic technology enables the creation of well-defined cell culture environments, which integrate the control of multiple biophysical and biochemical cues for designing novel in vitro assays. Growth-factor concentration gradients play a critical role in a wide range of biological processes ranging from development to cancer, guiding cell migration and influencing cell signaling. We present a microfluidic device capable of generating stable concentration gradients in a 3D matrix, while allowing for direct imaging of cellular behavior. The design consists of polydimethylsiloxane microchannels interconnected through 3D matrices. Optical access of the 3D matrix permits direct observation of invasive properties of cells seeded inside the channels or embedded in the matrix. An important characteristic of the microfluidic platform is the capability to generate reproducible, stable and quantifiable concentration gradients that are essential for systematic studies of soluble factor signaling in chemotaxis as...

Microfluidic cell culture systems for drug research

In pharmaceutical research, an adequate cell-based assay scheme to efficiently screen and to validate potential drug candidates in the initial stage of drug discovery is crucial. In order to better predict the clinical response to drug compounds, a cell culture model that is faithful to in vivo behavior is required. With the recent advances in microfluidic technology, the utilization of a microfluidic-based cell culture has several advantages, making it a promising alternative to the conventional cell culture methods. This review starts with a comprehensive discussion on the general process for drug discovery and development, the role of cell culture in drug research, and the characteristics of the cell culture formats commonly used in current microfluidic-based, cell-culture practices. Due to the significant differences in several physical phenomena between microscale and macroscale devices, microfluidic technology provides unique functionality, which is not previously possible by using traditional techniques. In a subsequent section, the niches for using microfluidic-based cell culture systems for drug research are discussed. Moreover, some critical issues such as cell immobilization, medium pumping or gradient generation in microfluidic-based, cell-culture systems are also reviewed. Finally, some practical applications of microfluidic-based, cell-culture systems in drug research particularly those pertaining to drug toxicity testing and those with a high-throughput capability are highlighted.

PDMS/glass microfluidic cell culture system for cytotoxicity tests and cells passage

In this paper, hybrid (PDMS/glass) microfluidic cell culture system (MCCS) integrated with the concentration gradient generator (CGG) is presented. PDMS gas permeability enabled cells’ respiration in the fabricated microdevices and excellent glass hydrophilicity allowed successful cells’ seeding. The human lung carcinoma cells (A549) were cultured in the microdevice for several days. The growth and proliferation of cells was monitored using an inverted fluorescence microscope. After the cells’ confluence was achieved in the microchambers, the novel method of cells’ passaging in the designed microdevice was developed and successfully tested. The MCCS microdevice is fully reusable, i.e. it can be used several times for various cell culture and cytotoxic experiments. The suitability of designed MCCS for cell-based cytotoxicity assay application was verified using 1,4-dioxane as a model toxic agent. The series of cytotoxicity tests in the microdevice as well as in classic way using 96-well cell culture plates were performed to compare results obtained in micro- and macroscale. Fluorescein dibutyrate (FDB) and iodide propidine (PI) were used as viable and dead cells’ markers, respectively. Fabricated MCCS microdevices were reproducible and apart from cell culture for long period of time, including cell passaging, it allowed cell-based cytotoxicity assays performance. The MCCS can be applied in high-throughput cell-based assays providing important informations on potential drug targets, substances’ bioactivity, defining the lowest toxic level of tested substances etc.

A self-contained microfluidic cell culture system

Conventional in vitro cell culture that utilizes culture dishes or microtiter plates is labor-intensive and time-consuming, and requires technical expertise and specific facilities to handle cell harvesting, media exchange and cell subculturing procedures. A microfluidic array platform with eight microsieves in each cell culture chamber is presented for continuous cell culture. With the help of the microsieves, uniform cell loading and distribution can be obtained. Within the arrays, cells grown to the point of confluency can be trypsinized and recovered from the device. Cells trapped in the microsieves after trypsinization function to seed the chambers for subsequent on-chip culturing, creating a sustainable platform for multiple cycles. The capability of the microfluidic array platform was demonstrated with a BALB/3T3 (murine embryonic fibroblast) cell line. The present microfluidic cell culture platform has potential to develop into a fully automated cell culture system integrated with temperature control, fluidic control, and micropumps, maximizing cell culture health with minimal intervention.

Quantitative inference of cellular parameters from microfluidic cell culture systems

Microfluidic cell culture systems offer a convenient way to measure cell biophysical parameters in conditions close to the physiological environment. We demonstrate the application of a mathematical model describing the spatial distribution of nutrient and growth factor concentrations in inferring cellular oxygen uptake rates from experimental measurements. We use experimental measurements of oxygen concentrations in a poly(dimethylsiloxane) (PDMS) microreactor culturing human hepatocellular liver carcinoma cells (HepG2) to infer quantitative information on cellular oxygen uptake rates. We use a novel microchannel design to avoid the parameter correlation problem associated with simultaneous cellular uptake and diffusion of oxygen through the PDMS surface. We find that the cellular uptake of oxygen is dependent on the cell density and can be modeled using a logistic term in the Michaelis-Menten equation. Our results are significant not only for the development of novel assays to quantitatively infer cell response to stimuli, but also for the development, design, and optimization of novel in vitro systems for drug discovery and tissue engineering.

Prevention of air bubble formation in a microfluidic perfusion cell culture system using a microscale bubble trap

Formation of air bubbles is a serious obstacle to a successful operation of a long-term microfluidic systems using cell culture. We developed a microscale bubble trap that can be integrated with a microfluidic device to prevent air bubbles from entering the device. It consists of two PDMS (polydimethyldisiloxane) layers, a top layer providing barriers for blocking bubbles and a bottom layer providing alternative fluidic paths. Rather than relying solely on the buoyancy of air bubbles, bubbles are physically trapped and prevented from entering a microfluidic device. Two different modes of a bubble trap were fabricated, an independent module that is connected to the main microfluidic system by tubes, and a bubble trap integrated with a main system. The bubble trap was tested for the efficiency of bubble capture, and for potential effects a bubble trap may have on fluid flow pattern. The bubble trap was able to efficiently trap air bubbles of up to 10 mul volume, and the presence of captured air bubbles did not cause alterations in the flow pattern. The performance of the bubble trap in a long-term cell culture with medium recirculation was examined by culturing a hepatoma cell line in a microfluidic cell culture device. This bubble trap can be useful for enhancing the consistency of microfluidic perfusion cell culture operation.

Towards a human-on-chip: Culturing multiple cell types on a chip with compartmentalized microenvironments

We have developed a multi-channel 3D microfluidic cell culture system (multi-channel 3D-microFCCS) with compartmentalized microenvironments for potential application in human drug screening. To this end, the multi-channel 3D-microFCCS was designed for culturing different 3D cellular aggregates simultaneously to mimic multiple organs in the body. Four human cell types (C3A, A549, HK-2 and HPA) were chosen to represent the liver, lung, kidney and the adipose tissue, respectively. Cellular functions were optimized by supplementing the common medium with growth factors. However, TGF-beta1 was found to enhance A549 functions but inhibit C3A functions. Therefore, TGF-beta1 was specifically controlled-released inside the A549 compartment by means of gelatin microspheres mixed with cells, thus creating a cell-specific microenvironment. The function of A549 cells was enhanced while the functions of C3A, HK-2 and HPA cells were uncompromised, demonstrating the limited cross-talk between cell culture compartments similar to the in vivo situation. Such a multi-channel 3D-microFCCS could be potentially used to supplement or even replace animal models in drug screening.