Microtubule transport, concentration and alignment in enclosed microfluidic channels

The kinesin-microtubule system has emerged as a versatile model system for biologically-derived microscale transport. While kinesin motors in cells transport cargo along static microtubule tracks, for in vitro transport applications it is preferable to invert the system and transport cargo-functionalized microtubules along immobilized kinesin motors. However, for efficient cargo transport and to enable this novel transport system to be interfaced with traditional microfluidics, it is important to fabricate enclosed microchannels that are compatible with kinesin motors and microtubules, that enable fluorescence imaging of microtubule movement, and that provide fluidic connections for sample introduction. Here we construct a three-tier hierarchical system of microfluidic channels that links microscale transport channels to macroscopic fluid connections. Shallow microchannels (5 μm wide and 1 μm deep) are etched in a glass substrate and bonded to a cover glass using PMMA as an adhesive, while intermediate channels (∼100 μm wide) serve as reservoirs and connect to 250 μm deep microchannels that hold fine gauge tubing for fluid injection. To demonstrate the utility of this device, we first show the performance of a directional rectifier that redirects 96% of moving microtubules and, because any microtubules that detach rapidly rebind to the motor-coated surface, suffers no microtubule loss over time. Second, we develop an approach, using a headless kinesin construct, to eliminate gradients in motor adsorption and microtubule binding in the enclosed channels, which enables precise control of kinesin density in the microchannels. Finally, we show that a 60 μm diameter circular ring functionalized with motors concentrates and aligns bundles of ∼3000 uniformly oriented microtubules, while suffering negligible ATP depletion. These aligned isopolar microtubules are an important tool for microscale transport applications and can be employed as a model in vitro system for studying kinesin-driven microtubule organization in cells. Electronic Supplementary Material Supplementary material is available in the online version of this article at Ying-Ming Huang and Maruti Uppalapati contributed equally to this work.     

A microfluidic device for separation of amniotic fluid mesenchymal stem cells utilizing louver-array structures

Human mesenchymal stem cells can differentiate into multiple lineages for cell therapy and, therefore, have attracted considerable research interest recently. This study presents a new microfluidic device for bead and cell separation utilizing a combination of T-junction focusing and tilted louver-like structures. For the first time, a microfluidic device is used for continuous separation of amniotic stem cells from amniotic fluids. An experimental separation efficiency as high as 82.8% for amniotic fluid mesenchymal stem cells is achieved. Furthermore, a two-step separation process is performed to improve the separation efficiency to 97.1%. These results are based on characterization experiments that show that this microfluidic chip is capable of separating beads with diameters of 5, 10, 20, and 40 μm by adjusting the volume-flow-rate ratio between the flows in the main and side channels of the T-junction focusing structure. An optimal volume-flow-rate ratio of 0.5 can lead to high separation efficiencies of 87.8% and 85.7% for 5-μm and 10-μm beads, respectively, in a one-step separation process. The development of this microfluidic chip may be promising for future research into stem cells and for cell therapy.     

Design, Microfabrication and Analysis of a Microfluidic Chamber for the Perfusion of Brain Tissue Slices

Successful perfusion and survival of brain slices using a microfabricated fluidic interface chamber is demonstrated. Up to three chambers are fabricated on the same glass substrate using a standard photolithography process. Their base is filled with arrays of micropillars to replace the nylon mesh used in classical interface chambers. These micropillars confine the flow and also uphold slices at the interface between perfusate and oxygen. Enhanced exposure of the neural tissue to oxygen and to the nutritive substances is reached. Computational fluid dynamics and empirical tests are used to study the flow properties of the chambers. The influence of various micropillar arrangements on the flow is as well analyzed. In these flat perfusion chambers, the flow is laminar and remains confined within the chamber even though the side-walls are not higher than the micropillars (400 m). At comparable flow rate this small volume microfluidic chamber (about 100 l) has a perfusate exchange rate at least 4 times faster than conventional perfusion chambers, making experiments with dynamic control of the perfusion medium possible. Using a zero-Mg²⁺ model of epileptiform activity, spontaneous single and multi-spike bursts in the CA3 region of a rat hippocampal brain slice have been observed for more than 5 hours. Compatibility of brain slice perfusion chambers with micro- and nanotechnology is expected to open new avenues in neurophysiology research using multifunction perfusion systems with important integrated features (e.g., microfluidic channels for drug delivery, electrode or sensor arrays).     

An agarose-based microfluidic platform with a gradient buffer for 3D chemotaxis studies

The current state-of-art in 3D microfluidic chemotaxis device (μFCD) is limited by the inherent coupling of the fluid flow and chemical concentration gradients. Here, we present an agarose-based 3D μFCD that decouples these two important parameters, in that the flow control channels are separated from the cell compartment by an agarose gel wall. This decoupling is enabled by the transport property of the agarose gel, which—in contrast to the conventional microfabrication material such as polydimethylsiloxane (PDMS)—provides an adequate physical barrier for convective fluid flow while at the same time readily allowing protein diffusion. We demonstrate that in this device, a gradient can be pre-established in an agarose layer above the cell compartment (a gradient buffer) before adding the 3D cell-containing matrix, and the dextran (10 kDa) concentration gradients can be re-established within 10 min across the cell-containing matrix and remain stable indefinitely. We successfully quantified the chemotactic response of murine dendritic cells to a gradient of CCL19, an 8.8 kDa lymphoid chemokine, within a type I collagen matrix. This model system is easy to set up, highly reproducible, and will benefit research on 3D chemoinvasion studies, for example with cancer cells or immune cells. Because of its gradient buffering capacity, it is particularly suitable for studying rapidly migrating cells like mature dendritic cells and neutrophils. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Ulrike Haessler and Yevgeniy Kalinin have equal contribution.     

Microfluidic self-assembly of tumor spheroids for anticancer drug discovery

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since it may provide a better model than monolayer culture of in vivo tumors. Moreover, continuous dynamic perfusion allows the establishment of physiologically relevant drug profiles to exposed spheroids. Here we present a physiologically inspired design allowing microfluidic self-assembly of spheroids, formation of uniform spheroid arrays, and characterizations of spheroid dynamics all in one platform. Our microfluidic device is based on hydrodynamic trapping of cancer cells in controlled geometries and the formation of spheroids is enhanced by maintaining compact groups of the trapped cells due to continuous perfusion. It was found that spheroid formation speed (average of 7 h) and size uniformity increased with increased flow rate (up to 10 μl min⁻¹). A large amount of tumor spheroids (7,500 spheroids per square centimeter) with a narrow size distribution (10 ± 1 cells per spheroid) can be formed in the device to provide a good platform for anticancer drug assays. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Liz Y. Wu and Dino Di Carlo contributed equally to this work.     

Microfluidic self-assembly of live <Emphasis Type="Italic">Drosophila</Emphasis> embryos for versatile high-throughput analysis of embryonic morphogenesis

A method for assembling Drosophila embryos in a microfluidic device was developed for studies of thermal perturbation of early embryonic development. Environmental perturbation is a complimentary method to injection of membrane-impermeable macromolecules for assaying genetic function and investigating robustness in complex biochemical networks. The development of a high throughput method for perturbing embryos would facilitate the isolation and mapping of signaling pathways. We immobilize Drosophila embryos inside a microfluidic device on minimal potential-energy wells created through surface modification, and thermally perturb these embryos using binary laminar flows of warm and cold solutions. We self-assemble embryos onto oil adhesive pads with an alcohol surfactant carrier fluid (detachment: 0.1 mL/min), and when the surfactant is removed, the embryo-oil adhesion increases to ∼25 mL/min flow rates, which allows for high velocities required for sharp gradients of thermal binary flows. The microfluidic thermal profile was numerically characterized by simulation and experimentally characterized by fluorescence thermometry. The effects of thermal perturbation were observed to induce abnormal morphogenetic movements in live embryos by using time-lapse differential interference contrast (DIC) microscopy.     

Microfluidic array for three-dimensional perfusion culture of human mammary epithelial cells

The ability to culture cells in three dimensional extracellular matrix (3D ECM) has proven to be an important tool for laboratory biology. Here, we demonstrate a microfluidic perfusion array on a 96-well plate format capable of long term 3D ECM culture within biomimetic microchambers. The array consists of 32 independent flow units, each with a 4 μl open-top culture chamber, and 350 μl inlet and outlet wells. Perfusion is generated using gravity and surface tension forces, allowing the array to be operated without any external pumps. MCF-10A mammary epithelial cells cultured in Matrigel in the microfluidic array exhibit acinus morphology over 9 days consistent with previous literature. We further demonstrated the application of the microfluidic array for in vitro anti-cancer drug screening.     

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 μl 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.     

Flexible microfluidic devices supported by biodegradable insertion scaffolds for convection-enhanced neural drug delivery

Convection enhanced delivery (CED) can improve the spatial distribution of drugs delivered directly to the brain. In CED, drugs are infused locally into tissue through a needle or catheter inserted into brain parenchyma. Transport of the infused material is dominated by convection, which enhances drug penetration into tissue compared with diffusion mediated delivery. We have fabricated and characterized an implantable microfluidic device for chronic convection enhanced delivery protocols. The device consists of a flexible parylene-C microfluidic channel that is supported during its insertion into tissue by a biodegradable poly(DL-lactide-co-glycolide) scaffold. The scaffold is designed to enable tissue penetration and then erode over time, leaving only the flexible channel implanted in the tissue. The device was able to reproducibly inject fluid into neural tissue in acute experiments with final infusate distributions that closely approximate delivery from an ideal point source. This system shows promise as a tool for chronic CED protocols.     

<Emphasis Type="Italic">In situ</Emphasis> micropatterning technique by cell crushing for co-cultures inside microfluidic biochips

To perform dynamic cell co-culture on micropatterned areas, we have developed a new type of “on chip and in situ” micropatterning technique. The microchip is composed of a 200 μm thick PDMS (polydimethylsiloxane) chamber at the top of which are located 100 μm thick microstamps. The PDMS chamber is bonded to a glass slide. After sterilization and cell adhesion processes, a controlled force is applied on the top of the PDMS chamber. Mechanically, the microstamps come into contact of the cells. Due to the applied force, the cells located under the microstamps are crushed. Then, a microfluidic perfusion is applied to rinse the microchip and remove the detached cells. To demonstrate the potential of this technique, it was applied successfully to mouse fibroblasts (Swiss 3T3) and liver hepatocarcinoma (HepG2/C3a) cell lines. Micropatterned areas were arrays of octagons of 150, 300 and 500 μm mean diameter. The force was applied during 30 to 60s depending on the cell types. After cell crushing, when perfusion was applied, the cells could successfully grow over the patterned areas. Cultures were successfully performed during 72 h of perfusion. In addition, monolayers of HepG2/C3a were micropatterned and then co cultured with mouse fibroblasts. Numerical simulations have demonstrated that the presence of the microstamps at the top of the PDMS chamber create non uniform flow and shear stress applied on the cells. Once fabricated, the main advantage of this technique is the possibility to use the same microchip several times for cell micropatterning and microfluidic co-cultures. This protocol avoids complex and numerous microfabrication steps that are usually required for micropatterning and microfluidic cell culture in the same time.     

A computational and experimental study inside microfluidic systems: the role of shear stress and flow recirculation in cell docking

In this paper, microfluidic devices containing microwells that enabled cell docking were investigated. We theoretically assessed the effect of geometry on recirculation areas and wall shear stress patterns within microwells and studied the relationship between the computational predictions and experimental cell docking. We used microchannels with 150 μm diameter microwells that had either 20 or 80 μm thickness. Flow within 80 μm deep microwells was subject to extensive recirculation areas and low shear stresses (<0.5 mPa) near the well base; whilst these were only presented within a 10 μm peripheral ring in 20 μm thick microwells. We also experimentally demonstrated that cell docking was significantly higher (p < 0.01) in 80 μm thick microwells as compared to 20 μm thick microwells. Finally, a computational tool which correlated physical and geometrical parameters of microwells with their fluid dynamic environment was developed and was also experimentally confirmed.     

Perfused drop microfluidic device for brain slice culture-based drug discovery

Living slices of brain tissue are widely used to model brain processes in vitro. In addition to basic neurophysiology studies, brain slices are also extensively used for pharmacology, toxicology, and drug discovery research. In these experiments, high parallelism and throughput are critical. Capability to conduct long-term electrical recording experiments may also be necessary to address disease processes that require protein synthesis and neural circuit rewiring. We developed a novel perfused drop microfluidic device for use with long term cultures of brain slices (organotypic cultures). Slices of hippocampus were placed into wells cut in polydimethylsiloxane (PDMS) film. Fluid level in the wells was hydrostatically controlled such that a drop was formed around each slice. The drops were continuously perfused with culture medium through microchannels. We found that viable organotypic hippocampal slice cultures could be maintained for at least 9 days in vitro. PDMS microfluidic network could be readily integrated with substrate-printed microelectrodes for parallel electrical recordings of multiple perfused organotypic cultures on a single MEA chip. We expect that this highly scalable perfused drop microfluidic device will facilitate high-throughput drug discovery and toxicology.     

A microperfused incubator for tissue mimetic 3D cultures

High density, three-dimensional (3D) cultures present physical similarities to in vivo tissue and are invaluable tools for pre-clinical therapeutic discoveries and development of tissue engineered constructs. Unfortunately, the use of dense cultures is hindered by intra-culture transport limits allowing just a few layer thick cultures for reproducible studies. In order to overcome diffusion limits in intra-culture nutrient and gas availability, a simple scalable microfluidic perfusion platform was developed and validated. A novel perfusion approach maintained laminar flow of nutrients through the culture to meet metabolic need, while removing depleted medium and catabolites. Velocity distributions and 3D flow patterns were measured using microscopic particle image velocimetry. The effectiveness of forced convection laminar perfusion was confirmed by culturing 700 μm thick neural-astrocytic (1:1) constructs at cell density approaching that of the brain (50,000 cells/mm³). At the optimized flow rate of the nutrient medium, the culture viability reached 90% through the full construct thickness at 2 days of perfusion while unperfused controls exhibited widespread cell death. The membrane aerated perfusion platform was integrated within a miniature, imaging accessible enclosure enabling temperature and gas control of the culture environment. Temperature measurements demonstrated fast feedback response to environmental changes resulting in the maintenance of the physiological temperature within 37 ± 0.2°C. Reproducible culturing of tissue equivalents within dynamically controlled environments will provide higher fidelity to in vivo function in an in vitro accessible format for cell-based assays and regenerative medicine.     

Automatic microfluidic platform for cell separation and nucleus collection

This study reports a new biochip capable of cell separation and nucleus collection utilizing dielectrophoresis (DEP) forces in a microfluidic system comprising of micropumps and microvalves, operating in an automatic format. DEP forces operated at a low voltage (15 V_p–p) and at a specific frequency (16 MHz) can be used to separate cells in a continuous flow, which can be subsequently collected. In order to transport the cell samples continuously, a serpentine-shape (S-shape) pneumatic micropump device was constructed onto the chip device to drive the samples flow through the microchannel, which was activated by the pressurized air injection. The mixed cell samples were first injected into an inlet reservoir and driven through the DEP electrodes to separate specific samples. Finally, separated cell samples were collected individually in two outlet reservoirs controlled by microvalves. With the same operation principle, the nucleus of the specific cells can be collected after the cell lysis procedure. The pumping rate of the micropump was measured to be 39.8 μl/min at a pressure of 25 psi and a driving frequency of 28 Hz. For the cell separation process, the initial flow rate was 3 μl/min provided by the micropump. A throughput of 240 cells/min can be obtained by using the developed device. The DEP electrode array, microchannels, micropumps and microvalves are integrated on a microfluidic chip using micro-electro-mechanical-systems (MEMS) technology to perform several crucial procedures including cell transportation, separation and collection. The dimensions of the integrated chip device were measured to be 6 × 7 cm. By integrating an S-shape pump and pneumatic microvalves, different cells are automatically transported in the microchannel, separated by the DEP forces, and finally sorted to specific chambers. Experimental data show that viable and non-viable cells (human lung cancer cell, A549-luc-C8) can be successfully separated and collected using the developed microfluidic platform. The separation accuracy, depending on the DEP operating mode used, of the viable and non-viable cells are measured to be 84 and 81%, respectively. In addition, after cell lysis, the nucleus can be also collected using a similar scheme. The developed automatic microfluidic platform is useful for extracting nuclear proteins from living cells. The extracted nuclear proteins are ready for nuclear binding assays or the study of nuclear proteins.     

Effect of Flow on Complex Biological Macromolecules in Microfluidic Devices

Understanding the transport, orientation, and deformation of biological macromolecules by flow is important in designing microfluidic devices. In this study, epi-fluorescence microscopy was used to characterize the behavior of macromolecules in flow in a microfluidic device, particularly how the flow affects the conformation of the molecules. The microfluidic flow path consists of a large, inlet reservoir connected to a long, rectangular channel followed by a large downstream reservoir. The flow contains both regions of high elongation (along the centerline as the fluid converges from the upstream reservoir into the channel) and shear (in the channel near the walls). Solutions of -DNA labeled with a fluorescent probe were first characterized rheologically to determine fluid relaxation times, then introduced into the microfluidic device. Images of the DNA conformation in the device were captured through an epi-fluorescent microscope. The conformation of DNA molecules under flow showed tremendous heterogeneity, as observed by Chu [7,12] and co-workers in pure shear and pure elongational flows. Histograms of the distribution of conformations were measured along the channel centerline as a function of axial position and revealed dramatic stretching of the molecules due to the converging flow followed by an eventual return to equilibrium coil size far downstream of the channel entry. The importance of shear was probed via a series of measurements near the channel centerline and near the channel wall. High shear rates near the channel wall also resulted in dramatic stretching of the molecules, and may result in chain scission of the macromolecules.     

Development of high throughput optical sensor array for on-line pH monitoring in micro-scale cell culture environment

On-line pH detection of cell culture environment is necessary in a bioprocess or tissue engineering. Devices by means of electrochemical mechanisms for this purpose have been reported to be less suitable compared with optical-based sensing principles. More recently, some non-invasive optical sensing systems have been proposed for online pH monitoring of cell culture environment. However, these devices are not for multi-target pH monitoring purpose, and are large in scale and thus not appropriate for the pH monitoring at a micro scale such as in microbioreactor or microfluidic-based cell culture platform. To tackle these issues, an optical fiber sensor array for on-line pH monitoring was proposed using microfluidic technology. The working principle is based on the optical absorption of phenol red normally contained in culture medium. Different from other device of the similar working principle, the proposed device requires less liquid volume (less than 0.8 μl), is non-invasive, and particularly can be configured as an array for high throughput pH monitoring. The present device has been optimized for the shape of detection chamber in a microfluidic chip with the aid of computational fluid dynamics (CFD) simulation, to avoid flow dead zone and thus to reduce the response time of detection. Both simulation and experimental results revealed that the design of oval detection chamber (axis, 1.5 and 2.0 mm) can considerably reduce the response time. Preliminary test has proved that the optical pH detection device is able to detect pH with average detection sensitivity of 0.83 V/pH in the pH range of 6.8–7.8, which is normally experienced in mammalian cell culture.     

Membrane-activated microfluidic rotary devices for pumping and mixing

Microfluidic devices are operated at a low-Reynolds-number flow regime such that the transportation and mixing of fluids are naturally challenging. There is still a great need to integrate fluid control systems such as pumps, valves and mixers with other functional microfluidic devices to form a micro-total-analysis-system. This study presents a new pneumatic microfluidic rotary device capable of transporting and mixing two different kinds of samples in an annular microchannel by using MEMS (Micro-electro-mechanical-systems) technology. Pumping and mixing can be achieved using a single device with different operation modes. The micropump has four membranes with an annular layout and is compact in size. The new device has a maximum pumping rate of 165.7 μL/min at a driving frequency of 17 Hz and an air pressure of 30 psi. Experimental data show that the pumping rate increases as higher air pressure and driving frequency are applied. In addition, not only can the microfluidic rotary device work as a peristaltic pumping device, but it also is an effective mixing device. The performance of the micromixer is extensively characterized. Experimental data indicate that a mixing index as high as 96.3% can be achieved. The developed microfluidic rotary device can be easily integrated with other microfluidic devices due to its simple and reliable PDMS fabrication process. The development of the microfluidic rotary device can be promising for micro-total-analysis-systems.     

Rapid detection of pathogens using antibody-coated microbeads with bioluminescence in microfluidic chips

Detection of pathogens was demonstrated in a polydimethylsiloxane (PDMS)/glass microfluidic chip with which microbead-based immunoseparation platform and the bioluminescence technology were integrated. Escherichia coli (E. coli) O157:H7 was used as the model bacteria. The microchamber in microfluidic chip was filled with glass beads coated with antibodies which could capture specific organism, and the capture efficiency of the chip for the bacteria was about 91.75%∼95.62%. Then the concentration of bacteria was determined by detecting adenosine triphosphate (ATP) employing bioluminescence reaction of firefly luciferin-lucifera-ATP on chip. The method allowed reliable detection of E. coli O157:H7 concentrations from 3.2 × 10¹ cfu/μL to 3.2 × 10⁵ cfu/μL within 20 min. This research demonstrated excellent reproducibility, stability, and specificity, and could accurately detect the pathogenic bacteria in food samples. The microfluidic chip and the equipments used in this method are easy to miniaturize, thus the method has great potential to be developed to a portable device for rapid detection of pathogens.     

Design Considerations for a Microfluidic Device to Quantify the Platelet Adhesion to Collagen at Physiological Shear Rates

Accurate assessment of blood platelet function is essential in understanding thrombus formation which plays a central role in cardiovascular disease. Parallel plate flow chambers have been widely used as they allow for platelet adhesion on a collagen surface at physiologically relevant fluid mechanical forces. Standard parallel plate flow chambers typically need several milliliters of blood, which is substantially more than can be obtained from small animals. We designed, fabricated, and assessed the functionality of a microfluidic channel with a width of 500 μm and a height of 50 μm in which a wall shear rate of 1000 s⁻¹ can be achieved with a flow rate of 15 μL/min. The velocity distribution in the microchannel predicted from the equations of motion was compared to experimentally measured velocities of fluorescent beads. This analysis showed that the motion of beads was quite similar to the predicted motion. Adhesion of platelets from whole blood at a shear rate of 1000 s⁻¹ onto a collagen surface using the microfluidic flow channel was qualitatively similar to platelet adhesion observed with a standard sized parallel plate flow chamber. After 5 min flow the surface coverage of platelets in the microfluidic device was about 55% while in a traditional size flow chamber the surface coverage was about 75%. This suggests that the microfluidic flow chamber can be used to quantify platelet adhesion for system where only very small amounts of blood are available.     

Microfluidics/CMOS orthogonal capabilities for cell biology

The study of individual cells and cellular networks can greatly benefit from the capabilities of microfabricated devices for the stimulation and the recording of electrical cellular events. In this contribution, we describe the development of a device, which combines capabilities for both electrical and pharmacological cell stimulation, and the subsequent recording of electrical cellular activity. The device combines the unique advantages of integrated circuitry (CMOS technology) for signal processing and microfluidics for drug delivery. Both techniques are ideally suited to study electrogenic mammalian cells, because feature sizes are of the same order as the cell diameter, ∼ 50 μm. Despite these attractive features, we observe a size mismatch between microfluidic devices, with bulky fluidic connections to the outside world, and highly miniaturized CMOS chips. To overcome this problem, we developed a microfluidic flow cell that accommodates a small CMOS chip. We simulated the performances of a flow cell based on a 3-D microfluidic system, and then fabricated the device to experimentally verify the nutrient delivery and localized drug delivery performance. The flow-cell has a constant nutrient flow, and six drug inlets that can individually deliver a drug to the cells. The experimental analysis of the nutrient and drug flow mass transfer properties in the flowcell are in good agreement with our simulations. For an experimental proof-of-principle, we successfully delivered, in a spatially resolved manner, a ‘drug’ to a culture of HL-1 cardiac myocytes.