Cell-based high content screening using an integrated microfluidic device

High content screening (HCS) has quickly established itself as a core technique in the early stage of drug discovery for secondary compound screening. It allows several independent cellular parameters to be measured in a single cell or populations of cells in a single assay. In this work, we describe high content screening for the multiparametric measurement of cellular responses in human liver carcinoma (HepG2) cells using an integrated microfluidic device. This device consists of multiple drug gradient generators and parallel cell culture chambers, in which the processes of liquid dilution and diffusion, micro-scale cell culture, cell stimulation and cell labeling can be integrated into a single device. The simple assay provides multiparametric measurements of plasma membrane permeability, nuclear size, mitochondrial transmembrane potential and intracellular redox states in anti-cancer drug-induced apoptosis of HepG2 cells. The established platform is able to rapidly extract the maximum of information from tumor cells in response to several drugs varying in concentration, with minimal sample and less time, which is very useful for basic biomedical research and cancer treatment.     

Apoptotic cell death dynamics of HL60 cells studied using a microfluidic cell trap device

This paper presents the design, fabrication and first results of a microfluidic cell trap device for analysis of apoptosis. The microfluidic silicon-glass chip enables the immobilization of cells and real-time monitoring of the apoptotic process. Induction of apoptosis, either electric field mediated or chemically induced with tumour necrosis factor (TNF-alpha), in combination with cycloheximide (CHX), was addressed. Exposure of cells to the appropriate fluorescent dyes, FLICA and PI, allows one to discriminate between viable, apoptotic and necrotic cells. The results showed that the onset of apoptosis and the transitions during the course of the cell death cascade were followed in chemically induced apoptotic HL60 cells. For the case of electric field mediated cell death, the distinction between apoptotic and necrotic stage was not clear. This paper presents the first results to analyse programmed cell death dynamics using this apoptosis chip and a first step towards an integrated apoptosis chip for high-throughput drug screening on a single cellular level.     

A radial microfluidic concentration gradient generator with high-density channels for cell apoptosis assay

An integrated microfluidic concentration gradient chip was developed for generating stepwise concentrations in high-density channels and applied to high-throughput apoptosis analysis of human uterine cervix cancer (HeLa) cells. The concentration gradient was generated by repeated splitting-and-mixing of the source solutions in a radial channel network which consists of multiple concentric circular channels and an increasing number of branch channels. The gradients were formed over hundreds of branches with predictable concentrations in each branch channel. This configuration brings about some distinctive advantages, e.g., more compact and versatile design, high-density of channels and wide concentration ranges. This concentration gradient generator was used in perfusion culture of HeLa cells and a drug-induced apoptosis assay, demonstrated by investigating the single and combined effects of two model anticancer drugs, 5-fluorouracil and Cyclophosphamide, which were divided into 65 concentrations of the two drugs respectively and 65 of their combinatorial concentrations. The gradient generation, the cell culture/stimulation and staining were performed in a single chip. The present device offers a unique platform to characterize various cellular responses in a high-throughput fashion.     

Sox-4 is a positive regulator of Hep3B and HepG2 cells' apoptosis induced by prostaglandin (PG)A(2) and delta(12)-PGJ(2)

We reported earlier that expression of Sox-4 was found to be elevated during prostaglandin (PG) A(2) and delta(12)-PGJ(2) induced apoptosis in human hepatocarcinoma Hep3B cells. In this study, the role of Sox-4 was examined using human Hep3B and HepG2 cell lines. Sox-4 induction by several apoptotic inducer such as A23187 (Ca(2+) ionophore) and etoposide (topoisomerase II inhibitor) and Sox-4 transfection into the cells were able to induce apoptosis as observed by the cellular DNA fragmentation. Antisense oligonucleotide of Sox-4 inhibited the induction of Sox-4 expression and blocked the formation of DNA fragmentation by PGA(2) and delta(12)-PGJ(2) in Hep3B and HepG2 cells. Sox-4-induced apoptosis was accompanied with caspase-1 activation indicating that caspase cascade was involved in this apoptotic pathway. These results indicate that Sox-4 is involved in Hep3B and HepG2 cells apoptosis as an important apoptotic mediator.     

Rapid spatial and temporal controlled signal delivery over large cell culture areas

Controlled chemical delivery in microfluidic cell culture devices often relies on slowly evolving diffusive gradients, as the spatial and temporal control provided by fluid flow results in significant cell-perturbation. In this paper we introduce a microfluidic device architecture that allows for rapid spatial and temporal soluble signal delivery over large cell culture areas without fluid flow over the cells. In these devices the cell culture well is divided from a microfluidic channel located directly underneath the chamber by a nanoporous membrane. This configuration requires chemical signals in the microchannel to only diffuse through the thin membrane into large cell culture area, rather than diffuse in from the sides. The spatial chemical pattern within the microfluidic channel was rapidly transferred to the cell culture area with good fidelity through diffusion. The cellular temporal response to a step-function signal showed that dye reached the cell culture surface within 45 s, and achieved a static concentration in under 6 min. Chemical pulses of less than one minute were possible by temporally alternating the signal within the microfluidic channel, enabling rapid flow-free chemical microenvironment control for large cell culture areas.     

Cytotoxicity of quantum dots assay on a microfluidic 3D-culture device based on modeling diffusion process between blood vessels and tissues

In this work, a novel quantum dot (QD) cytotoxicity assay platform on a microfluidic three-dimensional (3D) culture device via imitating the diffusion process between blood vessels and tissues was developed. The device is composed of a main channel and two sets of cell culture chambers. The cell culture chambers were located at different distances from the main channel and were divided into "close chambers" and "far chambers". HepG2 cells were cultured in an agarose matrix under 3D conditions and kept at high viability for at least three days. Fluorescein sodium and fluorescein isothiocyanate conjugated to bovine serum albumin (FITC-BSA) were used as models to demonstrate the diffusion process between main channel and cell culture chambers. QD cytotoxicity was evaluated by determining cell apoptosis, intracellular reactive oxygen species (ROS) and glutathione (GSH) with specific fluorescence probes. Cell autophagy inhibitor 3-methyladenine (3-MA) could reduce cell apoptosis at low concentrations of QDs, which proves that cell autophagy plays a key role in QD cytotoxicity. The effect of a series of 3-MA solutions on cell apoptosis at QD concentration of 40 μg mL(-1) was investigated, which showed that the percentage of cell apoptosis decreased ～15% from 0 to 12 mM 3-MA. The device shows potential as a high-throughput, low-cost and time-saving platform and constructs a more vivid biomimetic microenvironment for the QD cytotoxicity study.     

Perfusion-based microfluidic device for three-dimensional dynamic primary human hepatocyte cell culture in the absence of biological or synthetic matrices or coagulants

We describe a perfusion-based microfluidic device for three-dimensional (3D) dynamic primary human hepatocyte cell culture. The microfluidic device was used to promote and maintain 3D tissue-like cellular morphology and cell-specific functionality of primary human hepatocytes by restoring membrane polarity and hepatocyte transport function in vitro without the addition of biological or synthetic matrices or coagulants. A unique feature of our dynamic cell culture device is the creation of a microenvironment, without the addition of biological or synthetic matrices or coagulants, that promotes the 3D organization of hepatocytes into cord-like structures that exhibit functional membrane polarity as evidenced by the expression of gap junctions and the formation of an extended, functionally active, bile canalicular network.     

Supercritical carbon dioxide extraction of ethyl p-methoxycinnamate from Kaempferia galanga L. rhizome and its apoptotic induction in human HepG2 cells

In this study, supercritical carbon dioxide extraction of ethyl p-methoxycinnamate from Kaempferia galanga L. rhizome and its apoptotic induction in human HepG2 cells are reported for the first time. By using supercritical carbon dioxide extraction, the yield of ethyl p-methoxycinnamate identified by gas chromatography mass spectrometry (GC-MS) was as high as 2.5% with respect to the raw materials. In the anticancer assay, it was found that ethyl p-methoxycinnamate could inhibit the proliferation of the human hepatocellular liver carcinoma HepG2 cell line in a dose-dependent manner and induce the significant increase of the subG0 cell population. After treatment with ethyl p-methoxycinnamate, phosphatidylserine of HepG2 cells could significantly translocate to the surface of the membrane. The increase of an early apoptotic population was observed by both annexin-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining. It was concluded that ethyl p-methoxycinnamate not only induced cells to enter into apoptosis, but also affected the progress of the cell cycle.     

Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel

Deciphering the signaling pathways that govern stimulation of na?ve CD4+ T helper cells by antigen-presenting cells via formation of the immunological synapse is key to a fundamental understanding of the progression of successful adaptive immune response. The study of T cell-APC interactions in vitro is challenging, however, due to the difficulty of tracking individual, non-adherent cell pairs over time. Studying single cell dynamics over time reveals rare, but critical, signaling events that might be averaged out in bulk experiments, but these less common events are undoubtedly important for an integrated understanding of a cellular response to its microenvironment. We describe a novel application of microfluidic technology that overcomes many limitations of conventional cell culture and enables the study of hundreds of passively sequestered hematopoietic cells for extended periods of time. This microfluidic cell trap device consists of 440 18 micromx18 micromx10 microm PDMS, bucket-like structures opposing the direction of flow which serve as corrals for cells as they pass through the cell trap region. Cell viability analysis revealed that more than 70% of na?ve CD4+ T cells (TN), held in place using only hydrodynamic forces, subsequently remain viable for 24 hours. Cytosolic calcium transients were successfully induced in TN cells following introduction of chemical, antibody, or cellular forms of stimulation. Statistical analysis of TN cells from a single stimulation experiment reveals the power of this platform to distinguish different calcium response patterns, an ability that might be utilized to characterize T cell signaling states in a given population. Finally, we investigate in real time contact- and non-contact-based interactions between primary T cells and dendritic cells, two main participants in the formation of the immunological synapse. Utilizing the microfluidic traps in a daisy-chain configuration allowed us to observe calcium transients in TN cells exposed only to media conditioned by secretions of lipopolysaccharide-matured dendritic cells, an event which is easily missed in conventional cell culture where large media-to-cell ratios dilute cellular products. Further investigation into this intercellular signaling event indicated that LPS-matured dendritic cells, in the absence of antigenic stimulation, secrete chemical signals that induce calcium transients in T(N) cells. While the stimulating factor(s) produced by the mature dendritic cells remains to be identified, this report illustrates the utility of these microfluidic cell traps for analyzing arrays of individual suspension cells over time and probing both contact-based and intercellular signaling events between one or more cell populations.     

Imitation of drug metabolism in human liver and cytotoxicity assay using a microfluidic device coupled to mass spectrometric detection

In this work, we developed a microfluidic device for the imitation of drug metabolism in human liver and its cytotoxicity on cells. The integrated microfluidic device consists of three sections: (1) bioreactors containing poly(ethylene) glycol (PEG) hydrogel encapsulated human liver microsomes (HLMs); (2) cell culture chambers for cytotoxicity assay; and (3) integrated micro solid-phase extraction (SPE) columns to desalt and concentrate the products of enzymatic reaction. To verify the feasibility of the integrated microchip, we studied uridine 5'-diphosphate-glucuronosyltransferase (UGT) metabolism of acetaminophen (AP) and the cytotoxicity of products on HepG2 cells. The products of the reaction in one region of the device were injected into the cell culture chamber for cytotoxicity assay, while those in another region were directly detected online with an electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-Q-TOF MS) after micro-SPE pre-treatment. Semiquantitative analysis achieved in the experiments could be related to the drug-induced HepG2 cell cytotoxicity. Total analysis time for one product was about 30 min and only less than 4 μg HLM protein was required for one reaction region. The results demonstrated that the established platform could be used to imitate drug metabolism occurring in the human liver, thereby replacing animal experiments in the near future. In addition, the integrated microchip will be a useful tool for drug metabolism studies and cytotoxicity assays, which are pivotal in drug development.     

Low energy proton beam induces tumor cell apoptosis through reactive oxygen species and activation of caspases

Proton beam is useful to target tumor tissue sparing normal cells by allowing precise dose only into tumor cells. However, the cellular and molecular mechanisms by which proton beam induces tumor cell death are still undefined. We irradiated three different tumor cells (LLC, HepG2, and Molt-4) with low energy proton beam (35 MeV) with spread out Bragg peak (SOBP) in vitro, and investigated cell death by MTT or CCK-8 assay at 24 h after irradiation. LLC and HepG2 cells were sensitive to proton beam at over 10 Gy to induce apoptosis whereas Molt-4 showed rather low sensitivity. Relative biological effectiveness (RBE) values for the death rate relative to gamma-ray were ranged from 1.1 to 2.3 in LLC and HepG2 but from 0.3 to 0.7 in Molt-4 at 11 d after irradiation by colony formation assay. The typical apoptotic nuclear DNA morphological pattern was observed by staining with 4'-6-diamidino-2-phenylindole (DAPI). Tiny fragmented DNA was observed in HepG2 but not in Molt-4 by the treatment of proton in apoptotic DNA fragment assay. By FACS analysis after stained with FITC-Annexin-V, early as well as median apoptotic fractions were clearly increased by proton treatment. Proton beam-irradiated tumor cells induced a cleavage of poly (ADP-ribose) polymerase-1 (PARP-1) and procaspases-3 and -9. Activity of caspases was highly enhanced after proton beam irradiation. Reactive oxygen species (ROS) were significantly increased and N-acetyl cysteine pretreatment restored the apoptotic cell death induced by proton beam. Furthermore, p38 and JNK but not ERK were activated by proton and dominant negative mutants of p38 and JNK revived proton-induced apoptosis, suggesting that p38 and JNK pathway may be activated through ROS to activate apoptosis. In conclusion, our data clearly showed that single treatment of low energy proton beam with SOBP increased ROS and induced cell death of solid tumor cells (LLC and HepG2) in an apoptotic cell death program by the induction of caspases activities.     

An integrated microfluidic culture device to regulate endothelial cell differentiation from embryonic stem cells

We developed an integrated microfluidic culture device to regulate embryonic stem (ES) cell fate. The integrated microfluidic culture device consists of an air control channel and a fluidic channel with 4×4 micropillar arrays. We hypothesized that the microscale posts within the micropillar arrays would enable the control of uniform cell docking and shear stress profiles. We demonstrated that ES cells cultured for 6 days in the integrated microfluidic culture device differentiated into endothelial cells. Therefore, our integrated microfluidic culture device is a potentially powerful tool for directing ES cell fate.     

Multi-step microfluidic device for studying cancer metastasis

This paper describes a multi-step microfluidic device for studying the deformation and extravasation of primary tumor cells. Prior to extravasation, primary tumor cells undergo sequential steps of deformation through the capillaries, before adhering and transmigrating through the endothelial lining and basement membrane. To study this cascade of events, we fabricated a multi-step microfluidic device whose microgaps were coated with Matrigel to mimic the basement membrane. The microchannel was lined with human microvascular endothelial cells (HMECs) to replicate the endothelial lining. Analysis of deformation, biological and migratory capabilities of various tumor cell lines viz. HepG2, HeLa, and MDA-MB 435S were quantified using the fabricated device. After deformation, the cells' viabilities were significantly reduced and their doubling times were simultaneously increased, indicating changes in their biological capability. However, cell deformation did not significantly reduce their cell motility. Cell motility was co-assessed using the cell's migration rate and the overall population's percentage migration under various conditions (no barrier, Matrigel and Matrigel-HMEC). The device was also used to quantify the effects of Matrigel and the endothelial lining on cell migration. Our results suggest that both played an independent role in inhibiting cell extravasation, with the Matrigel significantly slowing down cell movement and the endothelial lining reducing the total number of transmigrated cells.     

Generation of oxygen gradients in microfluidic devices for cell culture using spatially confined chemical reactions

This paper reports a microfluidic device capable of generating oxygen gradients for cell culture using spatially confined chemical reactions with minimal chemical consumption. The microfluidic cell culture device is constructed by single-layer polydimethylsiloxane (PDMS) microfluidic channels, in which the cells can be easily observed by microscopes. The device can control the oxygen gradients without the utilization of bulky pressurized gas cylinders, direct addition of oxygen scavenging agents, or tedious gas interconnections and sophisticated flow control. In addition, due to the efficient transportation of oxygen within the device using the spatially confined chemical reactions, the microfluidic cell culture device can be directly used in conventional cell incubators without altering their gaseous compositions. The oxygen gradients generated in the device are numerically simulated and experimentally characterized using an oxygen-sensitive fluorescence dye. In this paper, carcinomic human alveolar basal epithelial (A549) cells have been cultured in the microfluidic device with a growth medium and an anti-cancer drug (Tirapazamine, TPZ) under various oxygen gradients. The cell experiment results successfully demonstrate the hyperoxia-induced cell death and hypoxia-induced cytotoxicity of TPZ. In addition, the results confirm the great cell compatibility and stable oxygen gradient generation of the developed device. Consequently, the microfluidic cell culture device developed in this paper is promising to be exploited in biological labs with minimal instrumentation to study cellular responses under various oxygen gradients.     

A novel 3D mammalian cell perfusion-culture system in microfluidic channels

Mammalian cells cultured on 2D surfaces in microfluidic channels are increasingly used in drug development and biological research applications. These systems would have more biological or clinical relevance if the cells exhibit 3D phenotypes similar to the cells in vivo. We have developed a microfluidic channel based system that allows cells to be perfusion-cultured in 3D by supporting them with adequate 3D cell-cell and cell-matrix interactions. The maximal cell-cell interaction was achieved by perfusion-seeding cells through an array of micropillars; and 3D cell-matrix interactions were achieved by a polyelectrolyte complex coacervation process to form a thin layer of matrix conforming to the 3D cell shapes. Carcinoma cell lines (HepG2, MCF7), primary differentiated (hepatocytes) and primary progenitor cells (bone marrow mesenchymal stem cells) were perfusion-cultured for 72 hours to 1 week in the microfluidic channel, which preserved their 3D cyto-architecture and cell-specific functions or differentiation competence. This transparent 3D microfluidic channel-based cell culture system also allows direct optical monitoring of cellular events for a wide range of applications.     

Ischemia/reperfusion injury of primary porcine cardiomyocytes in a low-shear microfluidic culture and analysis device

Ischemia/reperfusion (I/R) injury was induced in primary porcine cardiomyocytes in a low-shear microfluidic culture chip. The chip was capable of sustaining the cardiomyocyte culture and inducing I/R injury by subjecting the cells to periods of hypoxia lasting 3-4 hours followed by normoxia. Mitochondrial membrane potential was assayed using MitoTracker Red to follow mitochondrial depolarization, the earliest stage of apoptosis. Cell adhesion and morphology were also determined simultaneously with fluorescence measurements. Changes in membrane potential were observed earlier than previously reported, with mitochondria becoming depolarized as early as 2 hours into the ischemia period. The cells with depolarized mitochondria were deemed apoptotic. Out of 38-61 cells per time frame, the fraction of apoptotic cells was found to be similar to control samples (3%) at two hours of ischemia, which increased up to 22% at the end of the ischemia period as compared to 0% in the control samples. Morphological analysis of cells showed that 4 hours of ischemia followed by reperfusion produced blebbing cells within 2 hours of restoring oxygen to the chip. This approach is a versatile method for cardiomyocyte stress, and in future work additional analytical probes can be incorporated for a multi-analyte assay of cardiomyocyte apoptosis.     

A microfluidic magnetic bead impact generator for physical stimulation of osteoblast cell

We developed a novel microfluidic cell culture device in which magnetic beads repetitively collide with osteoblast cells, MC3T3‐E1, owing to attractive forces generated by pulsed electromagnetic fields and consequently the cells were physically stimulated by bead impacts. Our device consists of an on‐chip microelectromagnet and a microfluidic channel which were fabricated by a microelectromechanical system technique. The impact forces and stresses acting on a cell were numerically analyzed and experimentally generated with different sizes of bead (4.5, 7.6 and 8.4 μm) and at various pulse frequencies (60 Hz, 1 kHz and 1 MHz). Cells were synchronized at each specific phase of the cell cycle before stimulation in order to determine the most susceptible phase against bead impacts. The cells were stimulated with different sizes of bead at various pulse frequencies for 1 min at G1, S and G2 phases, respectively, and then counted immediately after one doubling time. The growth rate of cells was highly accelerated when they were stimulated with 4.5 μm beads at G1 phase and a pulse frequency of 1 MHz. Almost all of the cells were viable after stimulation, indicating that our cell stimulator did not cause any cellular damage and is suitable for use in new physical stimulus modalities.     

Characterization of a microfluidic dispensing system for localised stimulation of cellular networks

We present a 3-D microfluidic device designed for localized drug delivery to cellular networks. The device features a flow cell comprising a main channel for nutrient delivery as well as multiple channels for drug delivery. This device is one key component of a larger, fully integrated system now under development, based upon a microelectrode array (MEA) with on-chip CMOS circuitry for recording and stimulation of electrogenic cells (e.g. neurons, cardiomyocytes). As a critical system unit, the microfluidics must be carefully designed and characterized to ensure that candidate drugs are delivered to specific regions of the culture at known concentrations. Furthermore, microfluidic design and functionality is dictated by the size, geometry, and material/electrical characteristics of the CMOS MEA. Therefore, this paper reports on the design considerations and fabrication of the flow cell, including theoretical and experimental analysis of the mass transfer properties of the nutrient and drug flows, which are in good agreement with one another. To demonstrate proof of concept, the flow cell was mounted on a dummy CMOS chip, which had been plated with HL-1 cardiomyocytes. A test chemical compound was delivered to the cell culture in a spatially resolved manner. Envisioned applications of this stand-alone system include simultaneous toxicological testing of multiple compounds and chemical stimulation of natural neural networks for neuroscience investigations.     

Human neural stem cell growth and differentiation in a gradient-generating microfluidic device

This paper describes a gradient-generating microfluidic platform for optimizing proliferation and differentiation of neural stem cells (NSCs) in culture. Microfluidic technology has great potential to improve stem cell (SC) cultures, whose promise in cell-based therapies is limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms should provide much greater control over cell microenvironment and rapid optimization of media composition using relatively small numbers of cells. Our platform exposes cells to a concentration gradient of growth factors under continuous flow, thus minimizing autocrine and paracrine signaling. Human NSCs (hNSCs) from the developing cerebral cortex were cultured for more than 1 week in the microfluidic device while constantly exposed to a continuous gradient of a growth factor (GF) mixture containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). Proliferation and differentiation of NSCs into astrocytes were monitored by time-lapse microscopy and immunocytochemistry. The NSCs remained healthy throughout the entire culture period, and importantly, proliferated and differentiated in a graded and proportional fashion that varied directly with GF concentration. These concentration-dependent cellular responses were quantitatively similar to those measured in control chambers built into the device and in parallel cultures using traditional 6-well plates. This gradient-generating microfluidic platform should be useful for a wide range of basic and applied studies on cultured cells, including SCs.     

An on-chip intestine-liver model for multiple drugs absorption and metabolism behavior simulation

A double-layer microfluidic chip integrated with a hollow fiber(HF)was developed to reconstitute the intestine-liver functionality for studying the absorption and metabolism of combination drugs.Caco-2 cells were inoculated in the HF cavity at the top of the serpentine channel to simulate the intestinal tissue for drug absorption and transport studied,and Hep G2 cells,seeded in the bottom chamber,were used to mimic the liver for metabolism-related studies.Genistein and dacarbazine were selected for combination drug therapy and its effects on cell viability,hepatotoxicity,and cell cycle arrest under drug-conditioned culture were investigated.The results suggested that the combined concentration below-100μg/m L had no significant inhibitory effect on Hep G2 cell viability,and therefore Hep G2 cells maintained their drug metabolism ability.When the drug concentration was increased above 250μg/m L,Hep G2 cells underwent apoptosis.Detection of metabolites by mass spectrometry proved the effective metabolism in the microchip model.This dynamic,co-culture microchip successfully provided a podium for long-term observation of absorption,transport,and metabolism of combination drugs,and could be an effective in vitro simulation model for further clinical research.