A passive pump-assisted microfluidic assay for quantifying endothelial wound healing in response to fluid shear stress

There as an urgent need to quantify the endothelial wound-healing process in response to fluid shear stress to improve the biological and clinical understanding of healing mechanisms, which is of great importance for preventing healing impairment, chronic wounds, and postoperative in-stent restenosis. However, current experimental platforms not only require expensive, cumbersome, and powered pumping devices (to, e.g., generate cell scratches and load shear stress stimulation) but also lack quantitative controls for quantitative analysis. In this paper, a passive pump-assisted microfluidic assay is developed to quantify endothelial wound healing in response to fluid shear stress. Our assay consists of passive constant-flow pumps based on the siphon principle and a three-inlet microfluidic chip for cell wound-healing experiments. We also propose a method for quantitatively adjusting cell scratch size by controlling trypsin flow. Both numerical simulations and fluorescein experiments validate the effectiveness of this method. Moreover, we use the designed microfluidic assay to successfully generate cell scratches, load a 12-h shear stress of 5 dyn/cm² to the cells, and observe wound healing. The results indicate that the healing of a cell scratch is significantly accelerated under the stimulation of shear stress. In conclusion, our passive pump-assisted microfluidic assay shows versatility, applicability, and the potential for quantifying endothelial wound healing in response to fluid shear stress.     

Adhesion behaviors of human trophoblast cells by contact with endothelial cells

Although it is still not clear whether migratory trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by a mechanism of directional cell division/proliferation, this process involves the attachment and adhesion of trophoblasts to endothelial cells lining the blood vessel walls. This raises the possibility that the cell–cell contact with endothelial cells may regulate trophoblast cell adhesion behaviors according to the surrounding flow condition. To test this, the adhesion forces of early gestation human trophoblast cells (TCs) cultured on glass slides coated with type I rat collagen or cultured with human umbilical vein endothelial cells (HUVECs) were measured quantitatively using a micropipette aspiration technique. Then, the resistance of TCs co-cultured with HUVECs to flow-induced shear stress was assessed with a flow chamber technique. The results showed that the adhesion force of TCs to glass slides coated with collagen was positively correlated with the concentration of collagen. By contact with endothelial cells, the adhesion force and the resistance to shear stress for the TCs were significantly enhanced. The interdiction of integrin β1 interaction remarkably reduced the adhesion forces of TCs to endothelial cells, hence their resistance to shear stress. The results therefore suggest that the contacts of TCs with endothelial cells enhance the adhesion forces of human TCs, partially by regulating with the integrin β1 according to the flow condition (i.e., the shear stress) in such a way to prevent the TCs from being carried downstream by flowing blood.     

Patterning cells and shear flow conditions: convenient observation of endothelial cell remoulding, enhanced production of angiogenesis factors and drug response

We present a method that allows patterning cells and shear flow conditions for endothelial cell based assays. This method is novel in combining (1) cell culture on the surface of a substrate both topographically and chemically patterned; (2) multi-shear flow assays after covering the cell substrate with a microfluidic cover plate containing microchannels of different channel widths, and (3) conventional immunostaining assays after removal of the cover plate. This method has the advantage of performing cell cultures and immunoassays in standard cell biology environments with open access, facilitating the formation of confluent cell layers and the observation of cell responses to shear-flow and drug stimulations. To obtain multi-shear stress conditions, a single channel with stepwise increasing channel widths was patterned on the surfaces of both the substrate and the microfluidic cover plate. As results, we observed excellent viability of endothelial cells in the whole range of applied shear stresses (0-25 dyn cm(-2)) and shear stress dependent cytoskeleton remoulding, activation of von Willebrand factor (vWF), and re-organisation of angiogenesis factors such as tetra peptide acetyl-Ser-Asp-Lys-Pro (AcSDKP) of endothelial cells. To validate this approach for drug analysis, we also studied drug effects under shear stress conditions. Our results indicate that the drug effect of combretastatin A-4, an anti-tumour vascular targeting drug, could be significantly enhanced under shear flow conditions.     

Microcirculation within grooved substrates regulates cell positioning and cell docking inside microfluidic channels

Immobilization of cells inside microfluidic devices is a promising approach for enabling studies related to drug screening and cell biology. Despite extensive studies in using grooved substrates for immobilizing cells inside channels, a systematic study of the effects of various parameters that influence cell docking and retention within grooved substrates has not been performed. We demonstrate using computational simulations that the fluid dynamic environment within microgrooves significantly varies with groove width, generating microcirculation areas in smaller microgrooves. Wall shear stress simulation predicted that shear stresses were in the opposite direction in smaller grooves (25 and 50 microm wide) in comparison to those in wider grooves (75 and 100 microm wide). To validate the simulations, cells were seeded within microfluidic devices, where microgrooves of different widths were aligned perpendicularly to the direction of the flow. Experimental results showed that, as predicted, the inversion of the local direction of shear stress within the smaller grooves resulted in alignment of cells on two opposite sides of the grooves under the same flow conditions. Also, the amplitude of shear stress within microgrooved channels significantly influenced cell retainment in the channels. Therefore, our studies suggest that microscale shear stresses greatly influence cellular docking, immobilization, and retention in fluidic systems and should be considered for the design of cell-based microdevices.     

Endothelin-1 and angiotensin II secretion at different lengths of endothelial cell monolayer in the view of tensile stress accumulation in the upper endothelial cell membrane

Theoretical analysis and experimental observations have shown that tensile stress inside an endothelial cell membrane is capable of growing in the direction opposite to blood flow and can accumulate to a level that is three or more orders of magnitude higher than flow-induced shear stress on the membrane surface. This phenomenon is called cell membrane tension accumulation (CMTA). We hypothesize that correlation may exist between the endothelial cell monolayer length or CMTA and secretory function of endothelial cells. To verify this hypothesis, a paired experimental study was devised to measure the secretion of endothelin (ET-1) and angiotensin II (Ang II) by two monolayers of cultured human glomerular vascular endothelial cell (HGVEC) monolayers subjected an identical steady shear stress. After replicate cultured HGVEC monolayer with two kinds of length of 6 cm and 10 cm were subjected to the same steady laminar shear stress of 0.45 N/m² for 24 h, the average secretion rates of ET-1 and Ang II in 6 cm long increased l.7- and 0.5-fold (n=26, P<0.00l) over 10 cm long, respectively. Over 10 h of exposure to 0.65 N/m², the average secretion rate of both ET-1 and Ang II by HGVEC monolayer of 6 cm in length exceeded 0.5-fold (n=26, P<0.0001) over 10 cm in length. All these demonstrated that the close relationship may exist between length of endothelial cell monolayer and secretion of ET-1 and Ang II by endothelial cells, indicating the possible existence of the cumulative effect of the tensile stress in the upper endothelial cell membrane under the shear flow field.     

Examining the lateral displacement of HL60 cells rolling on asymmetric P-selectin patterns

The lateral displacement of cells orthogonal to a flow stream by rolling on asymmetrical receptor patterns presents a new opportunity for the label-free separation and analysis of cells. Understanding the nature of cell rolling trajectories on such substrates is necessary to the engineering of substrates and the design of devices for cell separation and analysis. Here, we investigate the statistical nature of cell rolling and the effect of pattern geometry and flow shear stress on cell rolling trajectories using micrometer-scale patterns of biomolecular receptors with well-defined edges. Leukemic myeloid HL60 cells expressing the PSGL-1 ligand were allowed to flow across a field of patterned lines fabricated using microcontact printing and functionalized with the P-selectin receptor, leveraging both the specific adhesion of this ligand-receptor pair and the asymmetry of the receptor pattern inclination angle with respect to the fluid shear flow direction (α = 5, 10, 15, and 20°). The effects of the fluid shear stress magnitude (τ = 0.5, 1, 1.5, and 2.0 dyn/cm(2)), α, and P-selectin incubation concentration were quantified in terms of the rolling velocity and edge tracking length. Rolling cells tracked along the inclined edges of the patterned lines before detaching and reattaching on another line. The detachment of rolling cells after tracking along the edge was consistent with a Poisson process of history-independent interactions. Increasing the edge inclination angle decreased the edge tracking length in an exponential manner, contrary to the shear stress magnitude and P-selectin incubation concentration, which did not have a significant effect. On the basis of these experimental data, we constructed an empirical model that predicted the occurrence of the maximum lateral displacement at an edge angle of 7.5°. We also used these findings to construct a Monte Carlo simulation for the prediction of rolling trajectories of HL60 cells on P-selectin-patterned substrates with a specified edge inclination angle. The prediction of lateral displacement in the range of 200 μm within a 1 cm separation length supports the feasibility of label-free cell separation via asymmetric receptor patterns in microfluidic devices.     

A microfluidic flow-stretch chip for investigating blood vessel biomechanics

This microfluidic flow-stretch chip integrates fluid shear stress (FSS) and cyclic stretch (CS), two major mechanical stimulations in cardiovascular systems, for cultured cells. The model chip can deliver FSS and CS simultaneously or independently to vascular cells to mimic the haemodynamic microenvironment of blood vessels in vivo. By imposing FSS-only, CS-only, and FSS+CS stimulation on rat mesenchymal stem cells and human umbilical vein endothelial cells, we found the alignment of the cellular stress fibers varied with cell type and the type of stimulation. The flow-stretch chip is a reliable tool for simulating the haemodynamic microenvironment.     

Production of reactive oxygen species in endothelial cells under different pulsatile shear stresses and glucose concentrations

A hemodynamic Lab-on-a-chip system was developed in this study. This system has two unique features: (1) it consists of a microfluidic network with an array of endothelial cell seeding sites for testing them under multiple conditions, and (2) the flow rate and the frequency of the culture medium in the microchannel are controlled by a pulsation free pump to mimic the flow profile of the blood in the blood vessel under different physiological conditions. The investigated physiological conditions were: (1) the resting condition in a normal shear stress of 15 dyne cm(-2) with a normal heart rate of 70 bpm, (2) an exhaustive exercise condition with a high shear stress of 30 dyne cm(-2) and a fast heart rate of 140 bpm, and (3) a constant high shear stress of 30 dyne cm(-2). Two chemical conditions were investigated (10 mM and 20 mM glucose) to mimic hyperglycemic conditions in diabetes patients. The effects of various shear stresses either alone or in combination with different glucose concentrations on endothelial cells were examined using the developed hemodynamic Lab-on-a-chip system by assessing two parameters. One is the intracellular level of reactive oxygen species (ROS) determined by a fluorescent probe, H(2)DCFDA. Another is the mitochondrial morphology revealed with a fluorescent dye, MitoTracker Green FM. The results showed that ROS level was elevated nearly 4-fold after 60 min of exhaustive exercise. We found that the pulsatile nature of the fluid was the determination factor for causing ROS generation in the cells as almost no increase of ROS was detected in the constant shear stress condition. Similarly, much higher level of ROS was detected when 10 mM glucose was applied to the cells under normal or high pulsatile shear stresses compared with under a static condition. These results suggest that it is necessary to use pulsatile shear stress to represent the physiological conditions of the blood flow, and demonstrate the advantage of utilizing this newly developed hemodynamic Lab-on-a-chip system over the conventional non-pulsatile system in the future shear stress related studies.     

微流控芯片流体剪切力和肿瘤坏死因子-α共同作用对大鼠软骨细胞表型的影响

流体剪切力是生物体内普遍存在的一种生物力学形式,是细胞微环境的重要组成部分,对细胞多种生物学行为有重要调节作用。该研究以微流控芯片技术为基础,建立了一种基于流阻原理能同时产生4个不同大小流体剪切力的微流控芯片平台,用以研究低流速的流体剪切力对大鼠原代软骨细胞表型维持的影响。结果表明,流体剪切力可促进软骨细胞的表型维持。还加入了肿瘤坏死因子-α(TNF-α),考察流体剪切力和TNF-α共同作用对软骨细胞表型的影响。结果表明,在剪切力和TNF-α共同作用下,软骨细胞的Ⅱ型胶原和蛋白多糖表达明显下调。该研究为软骨组织工程和骨性关节炎的疾病研究提供有力的研究平台,为骨关节疾病治疗和防治提供了理论依据。     

Micro-bioreactor array for controlling cellular microenvironments

High throughput experiments can be used to spatially and temporally investigate the many factors that regulate cell differentiation. We have developed a micro-bioreactor array (MBA) that is fabricated using soft lithography and contains twelve independent micro-bioreactors perfused with culture medium. The MBA enables cultivation of cells that are either attached to substrates or encapsulated in hydrogels, at variable levels of hydrodynamic shear, and with automated image analysis of the expression of cell differentiation markers. The flow and mass transport in the MBA were characterized by computational fluid dynamic (CFD) modeling. The representative MBA configurations were validated using the C2C12 cell line, primary rat cardiac myocytes and human embryonic stem cells (hESCs) (lines H09 and H13). To illustrate the utility of the MBA for controlled studies of hESCs, we established correlations between the expression of smooth muscle actin and cell density for three different flow configurations.     

Vascular mimetics based on microfluidics for imaging the leukocyte--endothelial inflammatory response

We describe the development, validation, and application of a novel PDMS-based microfluidic device for imaging leukocyte interaction with a biological substrate at defined shear force employing a parallel plate geometry that optimizes experimental throughput while decreasing reagent consumption. The device is vacuum bonded above a standard 6-well tissue culture plate that accommodates a monolayer of endothelial cells, thereby providing a channel to directly observe the kinetics of leukocyte adhesion under defined shear flow. Computational fluid dynamics (CFD) was applied to model the shear stress and the trajectory of leukocytes within the flow channels at a micron length scale. In order to test this model, neutrophil capture, rolling, and deceleration to arrest as a function of time and position was imaged in the transparent channels. Neutrophil recruitment to the substrate proved to be highly sensitive to disturbances in flow streamlines, which enhanced the rate of neutrophil-surface collisions at the entrance to the channels. Downstream from these disturbances, the relationship between receptor mediated deceleration of rolling neutrophils and dose response of stimulation by the chemokine IL-8 was found to provide a functional readout of integrin activation. This microfluidic technique allows detailed kinetic studies of cell adhesion and reveals neutrophil activation within seconds to chemotactic molecules at concentrations in the picoMolar range.     

Peptide-mediated selective adhesion of smooth muscle and endothelial cells in microfluidic shear flow

Microfluidic devices have recently emerged as effective tools for cell separation compared to traditional techniques. These devices offer the advantages of small sample volumes, low cost, and high purity. Adhesion-based separation of cells from heterogeneous suspensions can be achieved by taking advantage of specific ligand-receptor interactions. The peptide sequences Arg-Glu-Asp-Val (REDV) and Val-Ala-Pro-Gly (VAPG) are known to bind preferentially to endothelial cells (ECs) and smooth muscle cells (SMCs), respectively. This article examines the roles of REDV and VAPG and fluid shear stress in achieving selective capture of ECs and SMCs in microfluidic devices. The adhesion of ECs in REDV-coated devices and SMCs in VAPG-coated devices increases significantly compared to that of the nontargeted cells with decreasing shear stress. Furthermore, the adhesion of these cells is shown to be independent of whether these cells flow through the devices as suspensions of only one cell type or as a heterogeneous suspension containing ECs, SMCs, and fibroblasts. Whereas the overall adhesion of cells in the devices is determined mainly by shear stress, the selectivity of adhesion depends on the type of peptide and on the device surface as well as on the shear stress.     

A multichannel dampened flow system for studies on shear stress-mediated mechanotransduction

Shear stresses are powerful regulators of cellular function and potent mediators of the development of vascular disease. We have designed and optimized a system allowing the application of flow to cultured cells in a multichannel format. By using a multichannel peristaltic pump, flow can be driven continuously in the system for long-term studies in multiple isolated flow loops. A key component of the system is a dual-chamber pulse dampener that removes the pulsatility of the flow without the need for having an open system or elevated reservoir. We optimized the design parameters of the pulse dampening chambers for the maximum reduction in flow pulsation while minimizing the fluid needed for each isolated flow channel. Human umbilical vein endothelial cells (HUVECs) were exposed to steady and pulsatile shear stress using the system. We found that cells under steady flow had a marked increased production of eNOS and formation of actin stress fibers in comparison to those under pulsatile flow conditions. Overall, the results confirm the utility of the device as a practical means to apply shear stress to cultured cells in the multichannel format and provide steady, long term flow to microfluidic devices.     

Using a co-culture microsystem for cell migration under fluid shear stress

We have successfully developed a microsystem to co-cultivate two types of cells with a minimum defined gap of 50 μm, and to quantitatively study the impact of fluid shear stress on the mutual influence of cell migration velocity and distance. We used the hydrostatic pressure to seed two different cells, endothelial cells (ECs) and smooth muscle cells (SMCs), on opposite sides of various gap sizes (500 μm, 200 μm, 100 μm, and 50 μm). After cultivating the cells for 12 h and peeling the co-culture microchip from the culture dish, we studied the impacts of gap size on the migration of either cell type in the absence or presence of fluid shear stress (7 dyne cm(-2) and 12 dyne cm(-2)) influence. We found that both gap size and shear stress have profound influence on cell migration. Smaller gap sizes (100 μm and 50 μm) significantly enhanced cell migration, suggesting a requirement of an effective concentration of released factor(s) by either cell type in the gap region. Flow-induced shear stress delayed the migration onset of either cell type in a dose-dependent manner regardless of the gap size. Moreover, shear stress-induced decrease of cell migration becomes evident when the gap size was 500 μm. We have developed a co-culture microsystem for two kinds of cells and overcome the conventional difficulties in observation and mixed culture, and it would have more application for bio-manipulation and tissue repair engineering.     

A microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow

Biological cells in vivo typically reside in a dynamic flowing microenvironment with extensive biomechanical and biochemical cues varying in time and space. These dynamic biomechanical and biochemical signals together act to regulate cellular behaviors and functions. Microfluidic technology is an important experimental platform for mimicking extracellular flowing microenvironment in vitro. However, most existing microfluidic chips for generating dynamic shear stress and biochemical signals require expensive, large peripheral pumps and external control systems, unsuitable for being placed inside cell incubators to conduct cell biology experiments. This study has developed a microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow. Further, based on the lumped-parameter and distributed-parameter models of multiscale fluid dynamics, the oscillatory flow field and the concentration field of biochemical factors has been simulated at the cell culture region within the designed microfluidic chip. Using the constructed experimental system, the feasibility of the designed microfluidic chip has been validated by simulating biochemical factors with red dye. The simulation results demonstrate that dynamic shear stress and biochemical signals with adjustable period and amplitude can be generated at the cell culture chamber within the microfluidic chip. The amplitudes of dynamic shear stress and biochemical signals is proportional to the pressure difference and inversely proportional to the flow resistance, while their periods are correlated positively with the flow capacity and the flow resistance. The experimental results reveal the feasibility of the designed microfluidic chip. Conclusively, the proposed microfluidic generator based on autonomously oscillatory flow can generate dynamic shear stress and biochemical signals without peripheral pumps and external control systems. In addition to reducing the experimental cost, due to the tiny volume, it is beneficial to be integrated into cell incubators for cell biology experiments. Thus, the proposed microfluidic chip provides a novel experimental platform for cell biology investigations.     

Fabrication of Bonded Monolithic Porous Layer Open Tubular (monoPLOT) Columns in Wide Bore Capillary by Laminar Flow Thermal Initiation

A novel scalable procedure for the thermally initiated polymerisation of bonded monolithic porous layers of controlled thickness within open tubular fused silica capillaries (monoPLOT columns) is presented. Porous polymer layers of either polystyrene-divinylbenzene or butyl methacrylate-ethylene dimethacrylate, of variable thickness and morphology were polymerised inside fused silica capillaries utilising combined thermal initiation and laminar flow of the polymerisation mixture. The procedure enables the production through thermal initiation of monoPLOT columns of varying length, internal diameter, user defined morphology and layer thickness for potential use in both liquid and gas chromatography. The morphology and thickness of the bonded polymer layer on the capillary wall is strongly dependent on the laminar flow properties of the polymerisation mixture and the changing shear stress within the fluid across the inner diameter of the open capillary. Owing to the highly controlled rate of polymerisation and its dependence on fluid shear stress at the capillary wall, the procedure was demonstrably scalable, as illustrated by the polymerisation of identical layers within different capillary diameters.     

Fabrication of Bonded Monolithic Porous Layer Open Tubular (monoPLOT) Columns in Wide Bore Capillary by Laminar Flow Thermal Initiation

A novel scalable procedure for the thermally initiated polymerisation of bonded monolithic porous layers of controlled thickness within open tubular fused silica capillaries (monoPLOT columns) is presented. Porous polymer layers of either polystyrene-divinylbenzene or butyl methacrylate-ethylene dimethacrylate, of variable thickness and morphology were polymerised inside fused silica capillaries utilising combined thermal initiation and laminar flow of the polymerisation mixture. The procedure enables the production through thermal initiation of monoPLOT columns of varying length, internal diameter, user defined morphology and layer thickness for potential use in both liquid and gas chromatography. The morphology and thickness of the bonded polymer layer on the capillary wall is strongly dependent on the laminar flow properties of the polymerisation mixture and the changing shear stress within the fluid across the inner diameter of the open capillary. Owing to the highly controlled rate of polymerisation and its dependence on fluid shear stress at the capillary wall, the procedure was demonstrably scalable, as illustrated by the polymerisation of identical layers within different capillary diameters.

     

The phenomenon of cell membrane tensile stress accumulation and its effect on endothelin-1 secretion by vascular endothelial cells

Theoretical analysis has shown that that the tensile stress in the upper cell membrane of the vascular endothelium could accumulate upstream to a very high level despite of the identical shear environments. This phenomenon is called cell membrane tension accumulation (CMTA). To verify the theoretical analysis, the secretion of endothelin-1 (ET-1) by a paired human umbilical vein segments with different lengths (10 and 15 cm, respectively) were measured. The results clearly showed highly significant differences in the secretion rates of ET-1 between the 10 cm-long vein (segment A) and the 15 cm-long vein (segment B) under the same shear stress level of 0.48 N/m². When exposed to a shear stress of 0.48 N/m² for 24 h, segment B secreted ET-1 at an average rate of 34.9154±0.9830 pg/cm² h, almost 14% higher than the average rate of 30.6274±0.4912 pg/cm² h recorded by segment A (P<0.01). The present study, therefore, confirms that CMTA does in fact occur in the blood vessel. This phenomenon affects the secretion of ET-1 by vascular endothelial cells, and may be more important than shear stress in its effect on the metabolism and biological function of endothelial cells.     

Self-organization in the flow of complex fluids (colloid and polymer systems). Part 2: Theoretical models

Flow induced transitions in complex fluids are usually accompanied by changes in the internal media structure and the flow symmetry. In this review paper, we discuss the theoretical models and approaches that have been used for the analysis of different types of flow instabilities and flow patterns. The main attention is focused on the basic fluid models which reveal vortex and banding flow structures at high shear rates. The Oldroyd-B fluid is one of such models. The Reynolds and the Weissenberg (or Deborah) numbers are the parameters governing its flow behavior. For this model, the secondary flow patterns arising in viscometric flows of different geometries at the bifurcation point are described. Complex fluids which are able to exist in multiple states can form coexisting bands of different structures with different rheological properties and flowing with different shear rates at the same shear stress. Shear banding is typical for fluids demonstrating non-monotonous flow curves described by such models as the diffusive Johnson-Segalman fluid model, for example. Recent progress in exploring this phenomenon is discussed.     

Spatially selective reagent delivery into cancer cells using a two-layer microfluidic culture system

In this work, we demonstrate a two-layer microfluidic system capable of spatially selective delivery of drugs and other reagents under low shear stress. Loading occurs by hydrodynamically focusing a reagent stream over a particular region of the cell culture. The system consisted of a cell culture chamber and fluid flow channel, which were located in different layers to reduce shear stress on cells. Cells in the center of the culture chamber were exposed to parallel streams of laminar flow, which allowed fast changes to be made to the cellular environment. The shear force was reduced to 2.7 dyn cm⁻² in the two-layer device (vs. 6.0 dyn cm⁻² in a one-layer device). Cells in the side of the culture chamber were exposed to the side streams of buffer; the shear force was further reduced to a greater extent since the sides of the culture chamber were separated from the main fluid path. The channel shape and flow rate of the multiple streams were optimized for spatially controlled reagent delivery. The boundaries between streams were well controlled at a flow rate of 0.1 mL h⁻¹, which was optimized for all streams. We demonstrated multi-reagent delivery to different regions of the same culture well, as well as selective treatment of cancer cells with a built in control group in the same well. In the case of apoptosis induction using staurosporine, 10% of cells remained viable after 24 h of exposure. Cells in the same chamber, but not exposed to staurosporine, had a viability of 90%. This chip allows dynamic observation of cellular behavior immediately after drug delivery, as well as long-term drug treatment with the benefit of large cell numbers, device simplicity, and low shear stress.