Cell Culture Chamber Research Articles

A droplet-based heterogeneous immunoassay for screening single cells secreting antigen-specific antibodies.

High throughput heterogeneous immunoassays that screen antigen-specific antibody secreting cells are essential to accelerate monoclonal antibody discovery for therapeutic applications. Here, we introduce a heterogeneous single cell immunoassay based on alginate microparticles as permeable cell culture chambers. Using a microfluidic device, we encapsulated single antibody secreting cells in 35-40 μm diameter alginate microbeads. We functionalized the alginate to capture the secreted antibodies inside the microparticles, enabling single cell analysis and preventing the cross-talk between the neighboring encapsulated cells. We demonstrated non-covalent functionalization of alginate microparticles by adding three secondary antibodies to the alginate solution to form high molecular weight complexes that become trapped in the porous nanostructure of alginate and capture the secreted antibodies. We screened anti-TNF-alpha antibody-secreting cells from a mixture of antibody-secreting cells.

Fabricating microfluidic valve master molds in SU-8 photoresist

Multilayer soft lithography has become a powerful tool in analytical chemistry, biochemistry, material and life sciences, and medical research. Complex fluidic micro-circuits require reliable components that integrate easily into microchips. We introduce two novel approaches to master mold fabrication for constructing in-line micro-valves using SU-8. Our fabrication techniques enable robust and versatile integration of many lab-on-a-chip functions including filters, mixers, pumps, stream focusing and cell-culture chambers, with in-line valves. SU-8 created more robust valve master molds than the conventional positive photoresists used in multilayer soft lithography, but maintained the advantages of biocompatibility and rapid prototyping. As an example, we used valve master molds made of SU-8 to fabricate PDMS chips capable of precisely controlling beads or cells in solution.

A novel three-dimensional microfluidic platform for on chip multicellular tumor spheroid formation and culture

The formation of three-dimensional (3D) multicellular cell spheroids such as microspheres and embryoid bodies has recently gained much attention as a useful cell culture technique, but few studies have investigated the suitability of glass for spheroids formation and culture. In this work, we present a novel three-dimensional microfluidic device made of poly(dimethylsiloxane) (PDMS) and glass for the easy and rapid synthesis and culture of tumor spheroid. The cell culture unit is composed of an array of microwells on the bottom of a glass plate, bigger microwells and elastomeric microchannels on the top of a PDMS plate. Cell suspension can be easily introduced into the cell culture unit and exchange with the external liquid environment by the microfluidic channels. A single tumor spheroid can be formed and cultured in each glass cell culture chamber, the surface of which was modified with poly(vinyl alcohol) to render it to be resistant to cell adhesion. As the cell culture medium could be replaced, spheroids of the human breast cancer (MCF-7) cells were cultured on the chip for 3 days, reaching the diameters of about 150 μm. Furthermore, the MCF-7 cells were successfully cultured on the chip in 2D and 3D culture modes. Results have shown that glass is well suitable for multicellular tumor spheroids culture. The established platform provides a convenient and rapid method for tumor spheroid culture, which is also adaptable for anticancer drug screening and fundamental biomedical research in cell biology.

A Microfluidic-Based Multi-Shear Device for Investigating the Effects of Low Fluid-Induced Stresses on Osteoblasts

Interstitial fluid flow (IFF) within the extracellular matrix (ECM) produces low magnitude shear stresses on cells. Fluid flow-induced stress (FSS) plays an important role during tissue morphogenesis. To investigate the effect of low FSS generated by IFF on cells, we developed a microfluidic-based cell culture device that can generate multiple low shear stresses. By changing the length and width of the flow-in channels, different continuous low level shear stresses could be generated in individual cell culture chambers. Numerical calculations demonstrate uniform shear stress distributions of the major cell culture area of each chamber. This calculation is further confirmed by the wall shear stress curves. The effects of low FSS on MC3T3-E1 proliferation and differentiation were studied using this device. It was found that FSS ranging from 1.5 to 52.6 µPa promoted MC3T3-E1 proliferation and differentiation, but FSS over 412 µPa inhibited the proliferation and differentiation of MC3T3-E1 cells. FSS ranging from 1.5 to 52.6 µPa also increased the expression of Runx2, a key transcription factor regulating osteoblast differentiation. It is suggested that Runx2 might be an important regulator in low FSS-induced MC3T3-E1 differentiation. This device allows for detailed study of the effect of low FSS on the behaviors of cells; thus, it would be a useful tool for analysis of the effects of IFF-induced shear stresses on cells.

Induced Current Pharmacological Split Stimulation System for Neuronal Networks

Magnetic stimulation noninvasively modulates neuronal activity through a magnetically induced current. However, despite the usefulness and popularity of this method, the effects of neuronal activity in the nonstimulated regions on the stimulus responses are unknown. Here, we report that the induced current-evoked responses were affected by neuronal activities in the nonstimulated regions. Our experiment used a Mu-metal-based localized induced current stimulation (LICS) system combined with the microfabricated cell culture chamber system and a microelectrode array (MEA). The cell culture chamber system has radiating microtunnels connecting one central and eight outer chambers, which were fabricated using soft lithography and a replica modeling technique with SU-8 photoresist and polydimethylsiloxane (PDMS). Rat cortical neurons were separately cultured in the chambers and formed functional synaptic connections through the microtunnels. By applying a biphasic alternating pulsed magnetic field to the Mu-metal located in the central chamber, induced currents were mainly generated near the cultured neurons and modified the neuronal activities, which were recorded through MEA. Furthermore, we confirmed that the evoked responses were modified by localized pharmacological stimulation (LPS) in the outer chambers. These results suggest that our system would be promising tool for analyzing the effect of magnetic stimulation on interacting neuronal activity.

A microfluidic cell co-culture platform with a liquid fluorocarbon separator

A microfluidic cell co-culture platform that uses a liquid fluorocarbon oil barrier to separate cells into different culture chambers has been developed. Characterization indicates that the oil barrier could be effective for multiple days, and a maximum pressure difference between the oil barrier and aqueous media in the cell culture chamber could be as large as ~3.43 kPa before the oil barrier fails. Biological applications have been demonstrated with the separate transfection of two groups of primary hippocampal neurons with two different fluorescent proteins and subsequent observation of synaptic contacts between the neurons. In addition, the quality of the fluidic seal provided by the oil barrier is shown to be greater than that of an alternative solid-PDMS valve barrier design by testing the ability of each device to block low molecular weight CellTracker dyes used to stain cells in the culture chambers.

Cell culture monitoring for drug screening and cancer research: a transparent, microfluidic, multi-sensor microsystem

We present a novel, multiparametric microphysiometry system for the dynamic online monitoring of human cancer cell metabolism. The optically transparent, modular, hybrid microsystem is based on a glass chip and combines a cell cultivation chamber, microfluidics and metabolic monitoring with fully integrated chemo- and biosensors. pH and oxygen are measured in the cell culture area, and biosensors for lactate and glucose are connected downstream by microfluidics. The wafer-level fabrication features thin-film platinum and iridium oxide microelectrodes on a glass chip, microfluidics in an epoxy resist, a hybrid assembly and an on-chip reference electrode. The reliable analytical performance of the sensors in cell culture medium was demonstrated. The pH sensors exhibit a long-term stable, linear response. The oxygen sensors show a linear behaviour, which is also observed for low oxygen concentrations. Glucose and lactate measurements show a linear, long-term stable, selective and reversible behaviour in the desired range. T98G human brain cancer cells were cultivated and cell culture metabolism was measured on-chip. Stop/flow cycles were applied and extracellular acidification, respiration, glucose consumption and lactate production were quantified. Long-term metabolic rates were determined and all parameters could be measured in the outlet channel. A placement downstream of the cell cultivation area for biosensors was realised. A highly effective medium exchange and undiluted sampling from the cell culture chamber with low flow rates (2 μl min(-1)) and low volumes (15 μl per cycle) were achieved. The drug screening application was demonstrated by detecting alteration and recovery effects of cellular metabolism induced by the addition of substances to the medium.

A Novel Platform for Simultanoues Mechanical Stimulation and Characterization of Single Cells Based on Dielectric Elastomers and Atomic Force Microscopy

We have developed a novel set-up to simultaneously 1) apply static and dynamic deformations to adherent cells in culture 2) optically image cells under fluorescence microscopy and 3) assay the near-membrane mechanical properties with atomic force microscopy. In this system, the cell culture substrate is formed by a film of dielectric elastomer which can be eletro-actuated. The geometry and position of the actuating electrodes and the applied potential can be manipulated to obtain specific strain fields over the cell culture chamber. We have modeled the electro-mechanical behavior of the actuated elastomer film and using optical markers we have established an experimental procedure to optimize and quantify the strain at the adherent cells. This cell culture device has been integrated together with a commercial atomic force microscope coupled with an inverted optical microscope equipped for fluorescence. This novel set-up allows us to temporally assess, with sub-micron spatial resolution, single cell topography and elasticity, as well as ion fluxes, all during static or cyclically applied deformations. Preliminary results on fibrobalsts (3T3 NIH) show reproducible and reversible increase in cell elastic modulus as a response to 4% applied uni-axial stretch; additionally high resolution elasticity maps of an area of 40x40 μm on a single fibroblast could be obtained while stretching a single cell. When measuring cardiomyocites from mouse embryo, profiles of Ca2+ intracellular concentration could be also monitored while applying static and dynamic stretches. This study provides proof-of-concept for this set-up as a flexible experimental platform to investigate mechano-transduction mechanisms at the single cell level.

Inhibitory effects of dobutamine on human gastric adenocarcinoma.

To explore the inhibitory effects of dobutamine on gastric adenocarcinoma cells. Dobutamine was used to treat gastric adenocarcinoma cells (SGC-7901) and cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of dobutamine combined with cisplatin on cell viability were also analyzed. Cell migration was studied using the wound healing assay, and cell proliferation was analyzed using the colony formation assay. A cell invasion assay was carried out using Transwell cell culture chambers. The cell cycle and cell apoptosis were analyzed by flow cytometry. Western blot and immunocytochemistry were performed to determine the expression of Yes-associated protein (YAP) in treated cells. Dobutamine significantly inhibited cell growth, migration, cell colony formation, and cell invasion into Matrigel. Dobutamine also arrested the cell cycle at G1/S phase, and increased the rate of apoptosis of gastric adenocarcinoma cells. The expression of YAP was detected mainly in the nucleus in the absence of dobutamine. However, reduced expression of phosphorylated YAP was mainly found in the cytosol following treatment with dobutamine. Dobutamine has significant inhibitory effects on gastric adenocarcinoma cells and may be used in neoadjuvant therapy not only for gastric cancer, but also for other tumors.

Patient-Specific iPSCs-Based Liver-On-A-Chip

Successful advent of future medicine (e.g., personalized, preventive, predictive, participating medicine) will be strongly bonded to the availability of human-physiology-describing models. The combination of advanced researches in induced personalized stem cells (iPSCs) and the state-of-the-art microengineering is considered to create in-vitro models of organ functions in a near future; the first can generate patient-specific human cell lines, and the latter enables physiologically relevant microenvironments for recapitulating organ-level functions. Here we present a patient-specific iPSC-derived hepatocytes in an integrated microphysiological analysis platform (iMAP). The iMAP for iPSC-derived hepatocyte model is designed to culture the hepatocytes using endothelial-like physical barriers, which separate cell culture chambers and perfusion channels with Peclet number <<1. The physiological architecture supports enhanced hepatic functions of the iPSCs-derived hepatocytes, especially protein synthesis and drug metabolism. Compared to a conventional sandwich culture method, the liver-on-a-chip supports three-dimensional sinusoidal organization, enhanced junction protein and continuously refreshed media around the hepatic tissue to express higher albumin secretion and specifically enhanced drug metabolism (e.g., phenacetin is more metabolized to acetaminophen and N-acetyl-p-benzoquinone imine). Also, iPSCs-derived hepatocytes, by being matured from hepatoblast in the liver-on-a-chip platform, can increase the structural integrity due to avoiding dissociation of polarized hepatocytes and organogenesis-like tissue development in the microenvironment. The iMAP for iPSC-derived hepatocyte model will be tested for patient-specific pharmacokinetics/pharmacodynamics by involving various patient-derived iPSCs, and also other organ models will be integrated to estimate organ-organ interactions, which will provide various human-physiology-describing in-vitro models.

In situ fiber‐optical monitoring of cytosolic calcium in tissue explant cultures

We present a fluorescence‐lifetime based method for monitoring cell and tissue activity in situ, during cell culturing and in the presence of a strong autofluorescence background. The miniature fiber‐optic probes are easily incorporated in the tight space of a cell culture chamber or in an endoscope. As a first application we monitored the cytosolic calcium levels in porcine tracheal explant cultures using the Calcium Green‐5N (CG5N) indicator. Despite the simplicity of the optical setup we are able to detect changes of calcium concentration as small as 2.5 nM, with a monitoring time resolution of less than 1 s. (© 2013 WILEY‐VCH Verlag GmbH &amp;Co. KGaA, Weinheim)

Microgravity Cell Culture Systems and Bioreactors: Current Status and Future Developments

The International Space Station (ISS) offers remarkable opportunities for scientists to carry out experiments. However, the schedule for astronauts on board is tight, leaving not much time for science. This is one reason why the currently developed space hardwares such as cell culture systems and bioreactors have to demonstrate high levels of automation that save precious crew time. Life-science hardware for space applications has to provide the optimal conditions for biological samples and to ensure proper functionality under microgravity conditions. Furthermore, the hardware has to pass the stringent safety requirements for manned space flights. Before space life-science hardware is ready for the flight, numerous test runs have to be performed to verify its flawless functionality and safety. In this chapter we introduce state-of-the-art instruments as examples of currently used cell culture hardware for space applications. The “PADIAC” blood cell culture chamber was used recently in space to further investigate the behavior of T-lymphocytes to microgravity. The experiences with the “PADIAC” hardware combined with one of our previous studies such as “SACESTRE” are currently being used to develop and build a new piece of space hardware called “YEAST BIOREACTOR”. This instrument will allow yeast cultivation during an extended period of time as well as the exposure of samples periodically to other stressors in addition to microgravity. An analytical tool called “OoClamp” is also introduced to enable the measurement of the electrical properties of living cells under microgravity conditions. This tool is intended as an integrated part of future bioreactors for the on-site verification of the health status of cells, for example. Such a system would be ideal for life-science experiments in space because, without having to bring back the cells, substantial data on cellular processes could be gathered in space.

Cardiac Cell Culture Model As a Left Ventricle Mimic for Cardiac Tissue Generation

A major challenge in cardiac tissue engineering is the delivery of hemodynamic mechanical cues that play a critical role in the early development and maturation of cardiomyocytes. Generation of functional cardiac tissue capable of replacing or augmenting cardiac function therefore requires physiologically relevant environments that can deliver complex mechanical cues for cardiomyocyte functional maturation. The goal of this work is the development and validation of a cardiac cell culture model (CCCM) microenvironment that accurately mimics pressure-volume changes seen in the left ventricle and to use this system to achieve cardiac cell maturation under conditions where mechanical loads such as pressure and stretch are gradually increased from the unloaded state to conditions seen in vivo. The CCCM platform, consisting of a cell culture chamber integrated within a flow loop was created to accomplish culture of 10 day chick embryonic ventricular cardiomyocytes subject to 4 days of stimulation (10 mmHg, ∼13% stretch at a frequency of 2 Hz). Results clearly show that CCCM conditioned cardiomyocytes accelerate cardiomyocyte structural and functional maturation in comparison to static unloaded controls as evidenced by increased proliferation, alignment of actin cytoskeleton, bundle-like sarcomeric α-actinin expression, higher pacing beat rate at lower threshold voltages, and increased shortening. These results confirm the CCCM microenvironment can accelerate immature cardiac cell structural and functional maturation for potential cardiac regenerative applications.

Localized Induced Current Stimulation to Neuronal Culture Using Soft Magnetic Material

SUMMARY To establish precisely focused magnetic stimulation, we developed a Mu-metal-based low-frequency localized induced current (LIC) stimulation system with microfabricated dual cell-culture chamber. The dual cell-culture chamber was arranged in a concentric circle manner. Between the inner and outer chambers, four or eight connecting microchannels were fabricated using polydimethylsiloxane (PDMS). Rat cortical neurons were separately cultured in outer and inner chambers. Through the micro-channels, functional synaptic connections were formed. Mu-metal with very high magnetic permeability was aligned along the outer circle, which allowed the application of LIC stimulation to the cells in the outer chamber. Applying low-frequency magnetic fields to the Mu-metal, induced currents were generated and the electrical activity of the cells in the outer chamber was modified depending on the stimulation intensity. Following the modified activity in the outer circles, the cells in the inner chamber also showed slightly depressed activity patterns. These results suggested that our system would be promising for localized stimulation of neuronal networks and highly effective regulation of network activities. © 2013 Wiley Periodicals, Inc. Electron Comm Jpn, 96(8): 9–17, 2013; Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/ecj.11473

Generating arbitrary chemical patterns for multipoint dosing of single cells.

Living cells reside within anisotropic microenvironments that orchestrate a broad range of polarized responses through physical and chemical cues. To unravel how localized chemical signals influence complex behaviors, tools must be developed for establishing patterns of chemical gradients that vary over subcellular dimensions. Here, we present a strategy for addressing this critical need in which an arbitrary number of chemically distinct, subcellular dosing streams are created in real time within a microfluidic environment. In this approach, cells are cultured on a thin polymer membrane that serves as a barrier between the cell-culture environment and a reagent chamber containing multiple reagent species flowing in parallel under low Reynolds number conditions. Focal ablation of the membrane creates pores that allow solution to flow from desired regions within this reagent pattern into the cell-culture chamber, resulting in narrow, chemically distinct dosing streams. Unlike previous dosing strategies, this system provides the capacity to tailor arbitrary patterns of reagents on the fly to suit the geometry and orientation of specific cells.

A novel microfluidic co-culture system for investigation of bacterial cancer targeting

Although bacterial cancer targeting in animal models has been previously demonstrated and suggested as a possible therapeutic tool, a thorough understanding of the mechanisms responsible for cancer specificity would be required prior to clinical applications. To visualize bacterial preference for cancer cells over normal cells and to elucidate the cancer-targeting mechanism, a simple microfluidic platform has been developed for in vitro studies. This platform allows simultaneous cultures of multiple cell types in independent culture environments in isolated chambers, and creates a stable chemical gradient across a collagen-filled passage between each of these cell culture chambers and the central channel. The established chemical gradient induces chemotactic preferential migration of bacteria toward a particular cell type for quantitative analysis. As a demonstration, we tested differential bacterial behavior on a two-chamber device where we quantified bacterial preference based on the difference in fluorescence intensities of green fluorescence protein (GFP)-expressing bacteria at two exits of the collagen-filled passages. Analysis of the chemotactic behavior of Salmonella typhimurium toward normal versus cancer hepatocytes using the developed platform revealed an apparent preference for cancer hepatocytes. We also demonstrate that alpha-fetoprotein (AFP) is one of the key chemo-attractants for S. typhimurium in targeting liver cancer.

Cell culture using centrifugal microfluidic platform with demonstration on Pichia pastoris

This paper discusses the vortical flow, mixing and cell culture of Pichia pastoris using a centrifugal microfluidic (CM) chamber. The resultant "spiral toroidal vortex" in the chamber is made up of a primary vortex induced from inertial acceleration/deceleration of the chamber superposed by a secondary toroidal vortex due to Coriolis acceleration acting on the primary vortex. A validated numerical fluid-flow model with minimized numerical diffusion effect has been developed to investigate the flow and consequently mixing of two-color liquids through cyclic constant acceleration-and-deceleration in the same rotation direction until homogeneous mixing of the two liquids in the CM chamber has been established. The specific mixing time is found to improve with increase in acceleration/deceleration and angular span of the chamber. An experimental CM platform with three cell-culture chambers of different angular spans has been built and Pichia pastoris cell culture has been successfully demonstrated. Cell growth can be monitored over time on the extracted samples by measuring the optical density at 600-nm wave-length. Comparing with conventional cell culture, Pichia pastoris cultured on CM platform exhibits a very short lag (cell preparation/budding) phase prior to the log phase (cell growth). While it takes 8 to 12 h for the conventional shake flask in the lag phase, it takes only 2 h for the CM platform irrespective of initial cell concentration (8.1 × 10(4) to 8.1 × 10(5)/ml), acceleration/deceleration (10 to 32/s(2)) and angular span of the culture chamber (π/12 to π/4), representing significant time reduction. This is largely attributed to better growth conditions due to enhanced mixing and appropriate shear-stress stimulation from the efficient spiral toroidal vortex.

Associations between high levels of Notch1 expression and high invasion and poor overall survival in hepatocellular carcinoma

Although Notch1 expression has been associated with progression or prognosis in various tumors, the role of Notch1 in hepatocellular carcinoma (HCC) remains unknown. This study sought to investigate the clinicopathological and prognostic relevance of Notch1 expression in HCC as well as the underlying mechanisms responsible. HCC tissues were stained with an anti-Notch1 antibody. The invasion capacities of cells were measured using Transwell cell culture chambers. Reverse transcription PCR and/or western blot were used to evaluate the expression levels of Notch1, matrix metalloproteinase (MMP)-2, and MMP-9. Notch1 expression was downregulated by RNA interference. The activity of MMP-2/MMP-9 was quantified by enzyme-linked immunosorbent assay, and cellular apoptosis was analyzed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Notch1 expression was mainly localized within the cytoplasm and at the cell membrane. High Notch1 expression correlated with tumor size, tumor grade, metastasis, venous invasion, and American Joint Committee on Cancer TNM stage (P < 0.05), and patients with high levels of Notch1 expression were at a significantly increased risk for shortened survival time (P < 0.05). In vitro, the downregulation of Notch1 expression decreased the invasion capacity of HCC cells via the regulation of MMP-2 and MMP-9. The results of the MTT assay showed that downregulation of Notch1 did not affect HCC cell viability. Notch1 may represent a novel candidate marker for patient prognosis as well a molecular target for HCC therapy.

HIV‐infected patients show functionally defective high‐density lipoprotein (HDL) paralleled with changes in HDL‐associated proteins

Purpose of the studyEpidemiological studies have consistently demonstrated an inverse association between plasma HDL concentrations and cardiovascular risk. Although this cardioprotective role has been mainly attributed to its role in promoting cellular cholesterol efflux, there is an emerging interest in the anti‐inflammatory and antioxidant properties of HDL. The aim of the study is to investigate the anti‐inflammatory properties of HDL isolated from HIV‐infected patients.MethodsCross‐sectional study of 113 HIV‐infected patients and 70 non‐infected control subjects without evident CVD. From each subject, HDL was isolated by ultracentrifugation and its anti‐inflammatory status was tested as its ability to inhibit MCP‐1‐induced migration of the monocytic cell line THP‐1 using transwell cell culture chamber inserts with micropore filters of 5 microns pore size. HDL‐associated proteins were measured by commercial ELISAs.ResultsTwenty‐three HIV‐infected patients were ART‐naïve (32±15 years, 66.7% male) and ninety were currently on ART (46±11 years, 78.9 % male). Most patients on ART (91.1%) had undetectable viral load (&lt;50 copies/mL). When compared to healthy subjects, both naïve and treated HIV‐infected patients had lower plasma HDL‐cholesterol levels (naïve: 45±12, ART: 50±10, controls: 55±10 mg/dL, p&lt;0.05). HDL isolated from HIV naïve patients showed a significantly reduced anti‐inflammatory activity (THP‐1 monocyte migration capacity was 203% times higher in HIV patients than in control subjects). The anti‐inflammatory activity of HDL from ART‐treated HIV‐infected patients was significantly improved when compared to naïve patients, although it remained significantly lower than controls (130% THP‐1 monocyte migration vs controls). HDL from HIV‐infected patients had a decreased concentration of the anti‐inflammatory proteins Apo A1 (controls: 2.1±0.3, naïve: 1.4±0.2, ART: 1.6±0.1 mg/ml) and LCAT (controls: 0.53±0.09, naïve: 0.34±0.06, ART: 0.48±0.05 μg/ml), and increased of the pro‐inflammatory protein serum amiloid (controls: 1.42±0.53, naïve: 3.23±1.29, ART: 2.72±0.70 μg/ml) than healthy controls.ConclusionsOur data demonstrate HIV‐infected patients, even with effective therapy, showed a marked reduction of HDL anti‐inflammatory properties. This dysfunctional HDL seems to be associated with changes HDL composition and may contribute to the increased CVD risk observed in HIV infection.

Evaluation of the Absorption of Methotrexate on Cells and Its Cytotoxicity Assay by Using an Integrated Microfluidic Device Coupled to a Mass Spectrometer

An integrated microfluidic device was developed for high-throughput drug screening with an online electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-Q-TOF MS). The multiple gradient generator followed by an array of microscale cell culture chambers and on-chip solid-phase extraction (SPE) columns for sample pretreatment prior to mass analysis was integrated in the device which was fabricated in one single step. By using the combination system, the process for characterization of drug absorption and evaluation of cytotoxicity could be simultaneously realized. To validate the feasibility, the absorption of methotrexate and its effects on HepG2 and Caco-2 cells were investigated. With the increasing concentration of drugs, the percentage of apoptotic cells appeared in a dose-dependent fashion. By comparison with the results obtained from ESI-Q-TOF MS analysis and cytotoxicity assay, we found that higher intracellular drug concentration resulted in increased cell cytotoxicity. The technique presented herein provides an easy protocol to screen drugs rapidly with low drug consumption, high throughput, and high sensitivity.