Emulating clinical pressure waveforms in cell culture using an Arduino-controlled millifluidic 3D-printed platform for 96-well plates.

High blood pressure is the primary risk factor for heart disease, the leading cause of death globally. Despite this, current methods to replicate physiological pressures in vitro remain limited in sophistication and throughput. Single-chamber exposure systems allow for only one pressure condition to be studied at a time and the application of dynamic pressure waveforms is currently limited to simple sine, triangular, or square waves. Here, we introduce a high-throughput hydrostatic pressure exposure system for 96-well plates. The platform can deliver a fully-customizable pressure waveform to each column of the plate, for a total of 12 simultaneous conditions. Using clinical waveform data, we are able to replicate real patients' blood pressures as well as other medically-relevant pressures within the body and have assembled a small patient-derived waveform library of some key physiological locations. As a proof of concept, human umbilical vein endothelial cells (HUVECs) survived and proliferated for 3 days under a wide range of static and dynamic physiologic pressures ranging from 10 mm Hg to 400 mm Hg. Interestingly, pathologic and supraphysiologic pressure exposures did not inhibit cell proliferation. By integrating with, rather than replacing, ubiquitous lab cultureware it is our hope that this device will facilitate the incorporation of hydrostatic pressure into standard cell culture practice.

Spatially selective cell treatment and collection for integrative drug testing using hydrodynamic flow focusing and shifting.

Hydrodynamic focusing capable of readily producing and controlling laminar flow facilitates drug treatment of cells in existing microfluidic culture devices. However, to expand applications of such devices to multiparameter drug testing, critical limitations in current hydrodynamic focusing microfluidics must be addressed. Here we describe hydrodynamic focusing and shifting as an advanced microfluidics tool for spatially selective drug delivery and integrative cell-based drug testing. We designed and fabricated a co-flow focusing, three-channel microfluidic device with a wide cell culture chamber. By controlling inlet flow rates of sample and two side solutions, we could generate hydrodynamic focusing and shifting that mediated precise regulation of the path and width of reagent and drug stream in the microfluidic device. We successfully validated a hydrodynamic focusing and shifting approach for spatially selective delivery of DiI, a lipophilic fluorophore, and doxorubicin, a chemotherapeutic agent, to tumor cells in our device. Moreover, subsequent flowing of a trypsin EDTA solution over the cells that were exposed to doxorubicin flow allowed us to selectively collect the treated cells. Our approach enabled downstream high-resolution microscopy of the cell suspension to confirm the nuclear delivery of doxorubicin into the tumor cells. In the device, we could also evaluate in situ the cytotoxic effect of doxorubicin to the tumor cells that were selectively treated by hydrodynamic flow focusing and shifting. These results show that hydrodynamic focusing and shifting enable a fast and robust approach to spatially treat and then collect cells in an optimized microfluidic device, offering an integrative assay tool for efficient drug screening and discovery.

A Monolithic 3D Printed Axisymmetric Co-Flow Single and Compound Emulsion Generator.

We report a microfluidic droplet generator which can produce single and compound droplets using a 3D axisymmetric co-flow structure. The design considered for the fabrication of the device integrated a user-friendly and cost-effective 3D printing process. To verify the performance of the device, single and compound emulsions of deionized water and mineral oil were generated and their features such as size, generation frequency, and emulsion structures were successfully characterized. In addition, the generation of bio emulsions such as alginate and collagen aqueous droplets in mineral oil was demonstrated in this study. Overall, the monolithic 3D printed axisymmetric droplet generator could offer any user an accessible and easy-to-utilize device for the generation of single and compound emulsions.

3D Printed Biomimetic Soft Robot with Multimodal Locomotion and Multifunctionality.

Soft robots can outperform traditional rigid robots in terms of structural compliance, enhanced safety, and efficient locomotion. However, it is still a grand challenge to design and efficiently manufacture soft robots with multimodal locomotion capability together with multifunctionality for navigating in dynamic environments and meanwhile performing diverse tasks in real-life applications. This study presents a 3D-printed soft robot, which has spatially varied material compositions (0-50% particle-polymer weight ratio), multiscale hierarchical surface structures (10 nm, 1 µm, and 70 µm features on 5 mm wide robot footpads), and consists of functional components for multifunctionality. A novel additive manufacturing process, magnetic-field-assisted projection stereolithography (M-SL), is innovated to fabricate the proposed robot with prescribed material heterogeneity and structural hierarchy, and hence locally engineered flexibility and preprogrammed functionality. The robot incorporates untethered magnetic actuation with superior multimodal locomotion capabilities for completing tasks in harsh environments, including effective load carrying (up to ∼30 times of its own weight) and obstacle removing (up to 6.5 times of its own weight) in congested spaces (e.g., 5 mm diameter glass tube, gastric folds of a pig stomach) by gripping or pushing objects (e.g., 0.3-8 times of its own weight with a velocity up to 31 mm/s). Furthermore, the robot footpads are covered by multiscale hierarchical spike structures with features spanning from nanometers (e.g., 10 nm) to millimeters. Such high structural hierarchy enables multiple superior functions, including changing a naturally hydrophilic surface to hydrophobic, hairy adhesion, and excellent cell attaching and growth properties. It is found that the hairy adhesion and the engineered hydrophobicity of the robot footpad enable robust navigation in wet and slippery environments. The multimaterial multiscale robot design and the direct digital manufacturing method enable complex and versatile robot behaviors in sophisticated environments, facilitating a wide spectrum of real-life applications.

Competence pili in Streptococcus pneumoniae are highly dynamic structures that retract to promote DNA uptake.

The competence pili of transformable Gram-positive species are phylogenetically related to the diverse and widespread class of extracellular filamentous organelles known as type IV pili. In Gram-negative bacteria, type IV pili act through dynamic cycles of extension and retraction to carry out diverse activities including attachment, motility, protein secretion, and DNA uptake. It remains unclear whether competence pili in Gram-positive species exhibit similar dynamic activity, and their mechanism of action for DNA uptake remains unclear. They are hypothesized to either (1) leave transient cavities in the cell wall that facilitate DNA passage, (2) form static adhesins to enrich DNA near the cell surface for subsequent uptake by membrane-embedded transporters, or (3) play an active role in translocating bound DNA via dynamic activity. Here, we use a recently described pilus labeling approach to demonstrate that competence pili in Streptococcus pneumoniae are highly dynamic structures that rapidly extend and retract from the cell surface. By labeling the principal pilus monomer, ComGC, with bulky adducts, we further demonstrate that pilus retraction is essential for natural transformation. Together, our results suggest that Gram-positive competence pili in other species may also be dynamic and retractile structures that play an active role in DNA uptake.

96-Well Oxygen Control Using a 3D-Printed Device.

Oxygen concentration varies tremendously within the body and has proven to be a critical variable in cell differentiation, proliferation, and drug metabolism among many other physiological processes. Currently, researchers study the gas's role in biology using low-throughput gas control incubators or hypoxia chambers in which all cells in a vessel are exposed to a single oxygen concentration. Here, we introduce a device that can simultaneously deliver 12 unique oxygen concentrations to cells in a 96-well plate and seamlessly integrate into biomedical research workflows. The device inserts into 96-well plates and delivers gas to the headspace, thus avoiding undesirable contact with media. This simple approach isolates each well using gas-tight pressure-resistant gaskets effectively creating 96 "mini-incubators". Each of the 12 columns of the plate is supplied by a distinct oxygen concentration from a gas-mixing gradient generator supplied by two feed gases. The wells within each column are then supplied by an equal flow-splitting distribution network. Using equal feed flow rates, concentrations ranging from 0.6 to 20.5% were generated within a single plate. A549 lung carcinoma cells were then used to show that O2 levels below 9% caused a stepwise increase in cell death for cells treated with the hypoxia-activated anticancer drug tirapirizamine (TPZ). Additionally, the 96-well plate was further leveraged to simultaneously test multiple TPZ concentrations over an oxygen gradient and generate a three-dimensional (3D) dose-response landscape. The results presented here show how microfluidic technologies can be integrated into, rather than replace, ubiquitous biomedical labware allowing for increased throughput oxygen studies.

Magnetic Field-Assisted Stereolithography for Productions of Multimaterial Hierarchical Surface Structures.

Natural organisms provide inspirations for various functional structures and surfaces with significant applications in multidisciplinary fields. These biological systems are generally composed of multiscale surface structures with high geometric complexity and a variety of materials, making it challenging to replicate their characteristics in engineering. This study presents a novel multiscale multimaterial 3D printing method, magnetic field-assisted stereolithography (M-SL), for fabricating hierarchical particle-polymer structures with surface features ranging from a few nanometers to millimeters or even centimeters. Taking inspiration from nature, this study describes the design and fabrication of a bioinspired multiscale hierarchical surface structure, which is characterized of microscale cones, nanoscale pores, and surface wrinkles at a few nanometers. To understand the fundamental physics underlying the hierarchical surface structure fabrication in the proposed M-SL process, the complexities among the M-SL process parameters, material parameters, and printed geometries are discussed. The accuracy of the developed printing method is investigated by comparing the printed geometries and digital designs. Effects of the printed hierarchical surface structure on hydrophobicity and cell viability were characterized and discussed. It was found that the highly hierarchical surface structure changed the polymer composite surface from hydrophilic (contact angle: ∼38°) to hydrophobic (∼146°). In addition, the hierarchical surface structure also created a better environment for cell attachment and growth, with 900% more living cells at 72 h after cell seeding, compared with cells on the nonstructured smooth surface. Local and selective cell seeding can also be enabled by the surface structure design. Experimental results validated the effectiveness of the M-SL 3D printing method on fabricating multimaterial functional objects with hierarchically structured surfaces for a wide spectrum of applications.

Autonomous Temporal Control of Hydrogen Sulfide Delivery.

Hydrogen sulfide (H2S) is dichotomous in nature as it is lethal at higher concentrations, but at lower concentrations it shows a more cytoprotective nature. Due to its involvement in many physiological processes, it has recently risen to significance as a gasotransmitter alongside carbon monoxide (CO) and nitric oxide (NO). Experimentation using H2S is thus important to generate in vitro and in vivo models for this ubiquitous gasotransmitter. However, laboratory studies involving H2S are challenging due to the special handling conditions required to work with such a toxic gas. The use of chemical donors in the cell culture also show temporally varying release profiles, which are not optimal when trying to control H2S in cell cultures lasting several days. Previously we applied microfluidics to deliver stable concentrations of H2S, and in this work, we demonstrate a novel experimental method incorporating feedback control to precisely deliver H2S that accounts for donor solution concentration decay.

Femtoliter droplet confinement of Streptococcus pneumoniae: bacterial genetic transformation by cell-cell interaction in droplets.

Streptococcus pneumoniae (pneumococcus), a deadly bacterial human pathogen, uses genetic transformation to gain antibiotic resistance. Genetic transformation begins when a pneumococcal strain in a transient specialized physiological state called competence, attacks and lyses another strain, releasing DNA, taking up fragments of the liberated DNA, and integrating divergent genes into its genome. While many steps of the process are known and generally understood, the precise mechanism of this natural genetic transformation is not fully understood and the current standard strategies to study it have limitations in specifically controlling and observing the process in detail. To overcome these limitations, we have developed a droplet microfluidic system for isolating individual episodes of bacterial transformation between two confined cells of pneumococcus. By encapsulating the cells in a 10 µm diameter aqueous droplet, we provide an improved experimental model of genetic transformation, as both participating cells can be identified, and the released DNA is spatially restricted near the attacking strain. Specifically, the bacterial cells, one rifampicin (R) resistant, the other novobiocin (N) and spectinomycin (S) resistant were encapsulated in droplets carried by the fluorinated oil FC-40 with 5% surfactant and allowed to carry out competence-specific attack and DNA uptake (and consequently gain antibiotic resistances) within the droplets. The droplets were then broken, and recombinants were recovered by selective plating with antibiotics. The new droplet system encapsulated 2 or more cells in a droplet with a probability up to 71%, supporting gene transfer rates comparable to standard mixtures of unconfined cells. Thus, confinement in droplets allows characterization of natural genetic transformation during a strictly defined interaction between two confined cells.

Multiplex gene transfer by genetic transformation between isolated S. pneumoniae cells confined in microfluidic droplets.

Gene exchange via genetic transformation makes major contributions to antibiotic resistance of the human pathogen, Streptococcus pneumoniae (pneumococcus). The transfers begin when a pneumococcal cell, in a transient specialized physiological state called competence, attacks and lyses another cell, takes up fragments of the liberated DNA, and integrates divergent genes into its genome. Recently, it has been demonstrated that the pneumococcal cells can be enclosed in femtoliter-scale droplets for study of the transformation mechanism, offering the ability to characterize individual cell-cell interactions and overcome the limitations of current methods involving bulk mixed cultures. To determine the relevance and reliability of this new method for study of bacterial genetic transformation, we compared recombination events occurring in 44 recombinants recovered after competence-mediated gene exchange between pairs of cells confined in femtoliter-scale droplets vs. those occurring in exchanges in parallel bulk culture mixtures. The pattern of recombination events in both contexts exhibited the hallmarks of the macro-recombination exchanges previously observed within the more complex natural contexts of biofilms and long-term evolution in the human host.

Open Design 3D-Printable Adjustable Micropipette that Meets the ISO Standard for Accuracy.

Scientific communities are drawn to the open source model as an increasingly utilitarian method to produce and share work. Initially used as a means to develop freely-available software, open source projects have been applied to hardware including scientific tools. Increasing convenience of 3D printing has fueled the proliferation of open labware projects aiming to develop and share designs for scientific tools that can be produced in-house as inexpensive alternatives to commercial products. We present our design of a micropipette that is assembled from 3D-printable parts and some hardware that works by actuating a disposable syringe to a user-adjustable limit. Graduations on the syringe are used to accurately adjust the set point to the desired volume. Our open design printed micropipette is assessed in comparison with a commercial pipette and meets the ISO 8655 standards.

Generation of controllable gaseous H2S concentrations using microfluidics.

Hydrogen sulfide (H2S) plays an important role as an intercellular and intracellular signaling molecule, yet its targets are not well understood. As a molecule it easily evaporates and it is hard to acquire stable concentration for in vitro studies, constituting a major problem for the field to identify its downstream targets and function. Here we develop a microfluidic system that can provide consistent and controllable H2S levels in contrast to the current method of delivering large bolus doses to cells. The system relies on the permeability of H2S gas through a polydimethylsiloxane thin membrane. A hydrogen sulfide donor, sodium hydrosulfide, is perfused in the microchannels below the gas permeable membrane and gaseous H2S diffuses across the membrane, providing a stable concentration for up to 5 hours. Using electrochemical sensors within 3 ppm range, we found that H2S concentration was dependent on two parameters, the concentration of H2S donor, sodium hydrosulfide and the flow rate of the solution in the microchannels. Additionally, different H2S concentration profiles can be obtained by alternating the flow rate, providing an easy means to control the H2S concentration. Our approach constitutes a unique method for H2S delivery for in vitro and ex vivo studies and is ideally suited to identify novel biological processes and cellular mechanisms regulated by H2S.

Proatherogenic Flow Increases Endothelial Stiffness via Enhanced CD36-Mediated Uptake of Oxidized Low-Density Lipoproteins.

OBJECTIVE: Disturbed flow (DF) is well-known to induce endothelial dysfunction and synergistically with plasma dyslipidemia facilitate plaque formation. Little is known, however, about the synergistic impact of DF and dyslipidemia on endothelial biomechanics. Our goal was to determine the impact of DF on endothelial stiffness and evaluate the role of dyslipidemia/oxLDL (oxidized low-density lipoprotein) in this process. APPROACH AND RESULTS: Endothelial elastic modulus of intact mouse aortas ex vivo and of human aortic endothelial cells exposed to laminar flow or DF was measured using atomic force microscopy. Endothelial monolayer of the aortic arch is found to be significantly stiffer than the descending aorta (4.2+1.1 versus 2.5+0.2 kPa for aortic arch versus descending aorta) in mice maintained on low-fat diet. This effect is significantly exacerbated by short-term high-fat diet (8.7+2.5 versus 4.5+1.2 kPa for aortic arch versus descending aorta). Exposure of human aortic endothelial cells to DF in vitro resulted in 50% increase in oxLDL uptake and significant endothelial stiffening in the presence but not in the absence of oxLDL. DF also increased the expression of oxLDL receptor CD36 (cluster of differentiation 36), whereas downregulation of CD36 abrogated DF-induced endothelial oxLDL uptake and stiffening. Furthermore, genetic deficiency of CD36 abrogated endothelial stiffening in the aortic arch in vivo in mice fed either low-fat diet or high-fat diet. We also show that the loss of endothelial stiffening in CD36 knockout aortas is not mediated by the loss of CD36 in circulating cells. CONCLUSIONS: DF facilitates endothelial CD36-dependent uptake of oxidized lipids resulting in local increase of endothelial stiffness in proatherogenic areas of the aorta.

Effect of localized hypoxia on Drosophila embryo development.

Environmental stress, such as oxygen deprivation, affects various cellular activities and developmental processes. In this study, we directly investigated Drosophila embryo development in vivo while cultured on a microfluidic device, which imposed an oxygen gradient on the developing embryos. The designed microfluidic device enabled both temporal and spatial control of the local oxygen gradient applied to the live embryos. Time-lapse live cell imaging was used to monitor the morphology and cellular migration patterns as embryos were placed in various geometries relative to the oxygen gradient. Results show that pole cell movement and tail retraction during Drosophila embryogenesis are highly sensitive to oxygen concentrations. Through modeling, we also estimated the oxygen permeability across the Drosophila embryonic layers for the first time using parameters measured on our oxygen control device.

A microfluidic oxygen gradient demonstrates differential activation of the hypoxia-regulated transcription factors HIF-1α and HIF-2α.

Gas-perfused microchannels generated a linear oxygen gradient via diffusion across a 100 µm polydimethylsiloxane (PDMS) membrane. The device enabled exposure of a single monolayer of cells sharing culture media to a heterogeneous oxygen landscape, thus reflecting the oxygen gradients found at the microscale in the physiological setting and allowing for the real-time exchange of paracrine factors and metabolites between cells exposed to varying oxygen levels. By tuning the distance between two gas supply channels, the slope of the oxygen gradient was controlled. We studied the hypoxic activation of the transcription factors HIF-1α and HIF-2α in human endothelial cells within a spatial linear gradient of oxygen. Quantification of the nuclear to cytosolic ratio of HIF immunofluorescent staining demonstrated that the threshold for HIF-1α activation was below 2.5% O2 while HIF-2α was activated throughout the entire linear gradient. We show for the first time HIF-2α is subject to hyproxya, hypoxia by proxy, wherein hypoxic cells activate HIF in close-proximity normoxic cells. These results underscore the differences between HIF-1α and HIF-2α regulation and suggest that a microfluidic oxygen gradient is a novel tool for identifying distinct hypoxic signaling activation and interactions between differentially oxygenated cells.

Bubble removal with the use of a vacuum pressure generated by a converging-diverging nozzle.

Bubbles are an intrinsic problem in microfluidic devices and they can appear during the initial filling of the device or during operation. This report presents a generalizable technique to extract bubbles from microfluidic networks using an adjacent microfluidic negative pressure network over the entire microfluidic channel network design. We implement this technique by superimposing a network of parallel microchannels with a vacuum microfluidic channel and characterize the bubble extraction rates as a function of negative pressure applied. In addition, we generate negative pressure via a converging-diverging (CD) nozzle, which only requires inlet gas pressure to operate. Air bubbles generated during the initial liquid filling of the microfluidic network are removed within seconds and their volume extraction rate is calculated. This miniaturized vacuum source can achieve a vacuum pressure of 7.23 psi which corresponds to a bubble extraction rate of 9.84 pL/s, in the microfluidic channels we characterized. Finally, as proof of concept it is shown that the bubble removal system enables bubble removal on difficult to fill microfluidic channels such as circular or triangular shaped channels. This method can be easily integrated into many microfluidic experimental protocols.

Vacuum pressure generation via microfabricated converging-diverging nozzles for operation of automated pneumatic logic.

Microfluidic devices with integrated pneumatic logic enable automated fluid handling without requiring external control instruments. These chips offer the additional advantage that they may be powered by vacuum and do not require an electricity source. This work describes a microfluidic converging-diverging (CD) nozzle optimized to generate vacuum at low input pressures, making it suitable for microfluidic applications including powering integrated pneumatic logic. It was found that efficient vacuum pressure was generated for high aspect ratios of the CD nozzle constriction (or throat) width to height and diverging angle of 3.6(o). In specific, for an inlet pressure of 42.2 psia (290.8 kPa) and a volumetric flow rate of approximately 1700 sccm, a vacuum pressure of 8.03 psia (55.3 kPa) was generated. To demonstrate the capabilities of our converging - diverging nozzle device, we connected it to a vacuum powered peristaltic pump driven by integrated pneumatic logic and obtained tunable flow rates from 0 to 130 µL/min. Finally, we demonstrate a proof of concept system for use where electricity and vacuum pressure are not readily available by powering a CD nozzle with a bicycle tire pump and pressure regulator. This system is able to produce a stable vacuum sufficient to drive pneumatic logic, and could be applied to power automated microfluidics in limited resource settings.

A microfluidic array for real-time live-cell imaging of human and rodent pancreatic islets.

In this study, we present a microfluidic array for high-resolution imaging of individual pancreatic islets. The device is based on hydrodynamic trapping principle and enables real-time analysis of islet cellular responses to insulin secretagogues. This device has significant advantages over our previously published perifusion chamber device including significantly increased analytical power and assay sensitivity, as well as improved spatiotemporal resolution. The islet array, with live-cell multiparametric imaging integration, provides a better tool to understand the physiological and pathophysiological changes of pancreatic islets through the analysis of single islet responses. This platform demonstrates the feasibility of array-based islet cellular analysis and opens up a new modality to conduct informative and quantitive evaluation of islets and cell-based screening for new diabetes treatments.

A 3D-Printed Oxygen Control Insert for a 24-Well Plate.

3D printing has emerged as a method for directly printing complete microfluidic devices, although printing materials have been limited to oxygen-impermeable materials. We demonstrate the addition of gas permeable PDMS (Polydimethylsiloxane) membranes to 3D-printed microfluidic devices as a means to enable oxygen control cell culture studies. The incorporation of a 3D-printed device and gas-permeable membranes was demonstrated on a 24-well oxygen control device for standard multiwell plates. The direct printing allows integrated distribution channels and device geometries not possible with traditional planar lithography. With this device, four different oxygen conditions were able to be controlled, and six wells were maintained under each oxygen condition. We demonstrate enhanced transcription of the gene VEGFA (vascular endothelial growth factor A) with decreasing oxygen levels in human lung adenocarcinoma cells. This is the first 3D-printed device incorporating gas permeable membranes to facilitate oxygen control in cell culture.

Microfluidic platform generates oxygen landscapes for localized hypoxic activation.

An open-well microfluidic platform generates an oxygen landscape using gas-perfused networks which diffuse across a membrane. The device enables real-time analysis of cellular and tissue responses to oxygen tension to define how cells adapt to heterogeneous oxygen conditions found in the physiological setting. We demonstrate that localized hypoxic activation of cells elicited specific metabolic and gene responses in human microvascular endothelial cells and bone marrow-derived mesenchymal stem cells. A robust demonstration of the compatibility of the device with standard laboratory techniques demonstrates the wide utility of the method. This platform is ideally suited to study real-time cell responses and cell-cell interactions within physiologically relevant oxygen landscapes.