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.

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.

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.

Microfluidic array with integrated oxygenation control for real-time live-cell imaging: effect of hypoxia on physiology of microencapsulated pancreatic islets.

In this article, we present a novel microfluidic islet array based on a hydrodynamic trapping principle. The lab-on-a-chip studies with live-cell multiparametric imaging allow understanding of physiological and pathophysiological changes of microencapsulated islets under hypoxic conditions. Using this microfluidic array and imaging analysis techniques, we demonstrate that hypoxia impairs the function of microencapsulated islets at the single islet level, showing a heterogeneous pattern reflected in intracellular calcium signaling, mitochondrial energetic, and redox activity. Our approach demonstrates an improvement over conventional hypoxia chambers that is able to rapidly equilibrate to true hypoxia levels through the integration of dynamic oxygenation. This work demonstrates the feasibility of array-based cellular analysis and opens up new modality to conduct informative analysis and cell-based screening for microencapsulated pancreatic islets.

A microfabricated platform for establishing oxygen gradients in 3-D constructs.

Oxygen gradients are increasingly implicated in a number of biological processes, including stem cell differentiation and cancer metastasis. Unfortunately, the current in vitro tools designed to mimic conditions found in vivo lack application flexibility, simplicity in operation, and precise spatial control that most researchers require for widespread dissemination. The novel microfluidic-based device presented here addresses all the above concerns, offering a simple platform for enhanced control over the oxygen microenvironment exposed to three-dimensional cell-seeded constructs. The device utilizes an oxygen diffusion membrane approach to establish a gradient across a construct sandwiched between two continually perfused microfluidic networks. The device is capable of forming steady-state gradients at both the conditions tested-0 % to 5 % O2 and 0 % to 21 % O2-but a wide variety of profiles within the construct are possible. Cell viability with two model cell lines was also tested, with no adverse effects relative to the control.

Microfluidic wound bandage: localized oxygen modulation of collagen maturation.

Restoring tissue oxygenation has the potential to improve poorly healing wounds with impaired microvasculature. Compared with more established wound therapy using hyperbaric oxygen chambers, topical oxygen therapy has lower cost and better patient comfort, although topical devices have provided inconsistent results. To provide controlled topical oxygen while minimizing moisture loss, a major issue for topical oxygen, we have devised a novel wound bandage based on microfluidic diffusion delivery of oxygen. In addition to modulating oxygen from 0 to 100% in 60 seconds rise time, the microfluidic oxygen bandage provides a conformal seal around the wound. When 100% oxygen is delivered, it penetrates wound tissues as measured in agar phantom and in vivo wounds. Using this microfluidic bandage, we applied the oxygen modulation to 8 mm excisional wounds prepared on diabetic mice. Treatment with the microfluidic bandage demonstrated improved collagen maturity in the wound bed, although only marginal differences were observed in total collagen, microvasculature, and external closure rates. Our results show that proper topical oxygen can improve wound parameters underneath the surface. Because of the ease of fabrication, the oxygen bandage represents an economical yet practical method for oxygen wound research.

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.

Channel surface patterning of alternating biomimetic protein combinations for enhanced microfluidic tumor cell isolation.

Here, we report a new method for multicomponent protein patterning in a microchannel and also a technique for improving immunoaffinity-based circulating tumor cell (CTC) capture by patterning regions of alternating adhesive proteins using the new method. The first of two proteins, antiepithelial cell adhesion molecule (anti-EpCAM), provides the specificity for CTC capture. The second, E-selectin, increases CTC capture under shear. Patterning regions with and without E-selectin allows captured leukocytes, which also bind E-selectin and are unwanted impurities in CTC isolation, to roll a short distance and detach from the capture surface. This reduces leukocyte capture by up to 82%. The patterning is combined with a leukocyte elution step in which a calcium chelating buffer effectively deactivates E-selectin so that leukocytes may be rinsed away 60% more efficiently than with a buffer containing calcium. The alternating patterning of this biomimetic protein combination, used in conjunction with the elution step, reduces capture of leukocytes while maintaining a high tumor cell capture efficiency that is up to 1.9 times higher than the tumor cell capture efficiency of a surface with only anti-EpCAM. The new patterning technique described here does not require mask alignment and can be used to spatially control the immobilization of any two proteins or protein mixtures inside a sealed microfluidic channel.

Islet preconditioning via multimodal microfluidic modulation of intermittent hypoxia.

Simultaneous stimulation of ex vivo pancreatic islets with dynamic oxygen and glucose is a critical technique for studying how hypoxia alters glucose-stimulated response, especially in transplant environments. Standard techniques using a hypoxic chamber cannot provide both oxygen and glucose modulations, while monitoring stimulus-secretion coupling factors in real-time. Using novel microfluidic device with integrated glucose and oxygen modulations, we quantified hypoxic impairment of islet response by calcium influx, mitochondrial potentials, and insulin secretion. Glucose-induced calcium response magnitude and phase were suppressed by hypoxia, while mitochondrial hyperpolarization and insulin secretion decreased in coordination. More importantly, hypoxic response was improved by preconditioning islets to intermittent hypoxia (IH, 1 min/1 min 5-21% cycling for 1 h), translating to improved insulin secretion. Moreover, blocking mitochondrial K(ATP) channels removed preconditioning benefits of IH, similar to mechanisms in preconditioned cardiomyocytes. Additionally, the multimodal device can be applied to a variety of dynamic oxygen-metabolic studies in other ex vivo tissues.

Dual microfluidic perifusion networks for concurrent islet perifusion and optical imaging.

This study explores a new class of duplex microfluidic device which utilizes a dual perifusion network to simultaneously perform live-cell optical imaging of physiological activities and study insulin release kinetics on two islet populations. This device also incorporates on-chip staggered herringbone mixers (SHMs) to increase mixing efficiency and facilitate the generation of user-defined chemical gradients. Mouse islets are used to simultaneously measure dynamic insulin release, changes in mitochondrial potentials, and calcium influx in response to insulin secretagogues (glucose and tolbutamide), and show a high signal-to-noise ratio and spatiotemporal resolution of all measured parameters for both perifusion chambers. This system has many potential applications for studying ß-cell physiology and pathophysiology, as well as for therapeutic drug screening. This dual perifusion device is not limited to islet studies and could easily be applied to other tissues and cells without major modifications.

Systematic prevention of bubble formation and accumulation for long-term culture of pancreatic islet cells in microfluidic device.

Reliable long-term cell culture in microfluidic system is limited by air bubble formation and accumulation. In this study, we developed a bubble removal system capable of both trapping and discharging air bubbles in a consistent and reliable manner. Combined with PDMS (Polydimethylsiloxane) hydrophilic surface treatment and vacuum filling, a microfluidic perifusion system equipped with the bubble trap was successfully applied for long-term culture of mouse pancreatic islets with no bubble formation and no flow interruption. In addition to demonstrating normal cell viability and islet morphology, post-cultured islets exhibited normal insulin secretion kinetics, intracellular calcium signaling, and changes in mitochondrial potentials in response to glucose challenge. This design could be easily adapted by other microfluidic systems due to its simple design, ease of fabrication, and portability.

Direct measurements on CD24-mediated rolling of human breast cancer MCF-7 cells on E-selectin.

Tumor cell rolling on the endothelium plays a key role in the initial steps of cancer metastasis, i.e., extravasation of circulating tumor cells (CTCs). Identification of the ligands that induce the rolling of cells is thus critical to understanding how cancers metastasize. We have previously demonstrated that MCF-7 cells, human breast cancer cells, exhibit the rolling response selectively on E-selectin-immobilized surfaces. However, the ligand that induces rolling of MCF-7 cells on E-selectin has not yet been identified, as these cells lack commonly known E-selectin ligands. Here we report, for the first time to our knowledge, a set of quantitative and direct evidence demonstrating that CD24 expressed on MCF-7 cell membranes is responsible for rolling of the cells on E-selectin. The binding kinetics between CD24 and E-selectin was directly measured using surface plasmon resonance (SPR), which revealed that CD24 has a binding affinity against E-selectin (K(D) = 3.4 ± 0.7 nM). The involvement of CD24 in MCF-7 cell rolling was confirmed by the rolling behavior that was completely blocked when cells were treated with anti-CD24. A simulated study by flowing microspheres coated with CD24 onto E-selectin-immobilized surfaces further revealed that the binding is Ca(2+)-dependent. Additionally, we have found that actin filaments are involved in the CD24-mediated cell rolling, as observed by the decreased rolling velocities of the MCF-7 cells upon treatment with cytochalasin D (an inhibitor of actin-filament dynamics) and the stationary binding of CD24-coated microspheres (the lack of actins) on the E-selectin-immobilized slides. Given that CD24 is known to be directly related to enhanced invasiveness of cancer cells, our results imply that CD24-based cell rolling on E-selectin mediates, at least partially, cancer cell extravasation, resulting in metastasis.

Controlling flow in microfluidic channels with a manually actuated pin valve.

There is a need for a simple method to control fluid flow within microfluidic channels. To meet this need, a simple push pin with a polydimethylsiloxane (PDMS) tip has been integrated into microfluidic networks to be placed within the microchannel to obstruct flow. This new valve design can attain on/off control of fluid flow without an external power source using readily-available, low-cost materials. The valve consists of a 14 gauge (1.6 mm) one inch piece of metal tubing with a PDMS pad at the tip to achieve a fluidic seal when pressed against a microfluidic channel's substrate. The metal tubing or pin is then either manually pushed down to block or pulled up to allow fluid flow. The valve was validated using a pressure transducer and fluorescent dye to determine the breakthrough pressure the valve can withstand over multiple cycles. In the first cycle, the median value for pressure withstood by the valve was 8.8 psi with a range of 17.5-2.7 psi. The pressure the valves were able to withstand during each successive trial was lower suggesting they may be most valuable as a method to control the initial introduction of fluids into a microfluidic device. These valves can achieve flow regulation within microfluidic devices, have a small dead volume, and are simple to fabricate and use, making this technique widely suitable for a range of applications.

Rheologically biomimetic cell suspensions for decreased cell settling in microfluidic devices.

Many microfluidic devices operate with cells suspended in buffer solutions. Researchers who work with large cell types in such devices often run into problems with gravitational cell settling in the injection equipment and in the device itself. A method for reducing this problematic settling is discussed in this paper using tumor cell lines as an example. Microfluidic circulating tumor cell (CTC) isolation devices (MCIDs) are benchmarked using buffer solutions spiked with in-vitro tumor cell lines prior to validation with clinical samples (i.e. whole blood). However, buffer solutions have different rheological properties than whole blood. Here we describe the use of alginate in PBS buffer solutions to mimic blood rheology and reduce cell settling during preliminary validation experiments. Because alginate increases the viscosity of a solution, it helps to maintain cells in suspension. We report that vertical equipment configurations are important to further mitigate the effects of cell settling for MDA-MB-468 carcinoma cells. We also report that alginate does not disrupt the specific binding interactions that are the basis of carcinoma cell capture in MCIDs. These results indicate that vertical equipment configurations and the addition of alginates can be used to reduce cell settling in buffer based MCID testing and other applications involving large cells suspended in buffer solution.

Oxygen sensitive microwells.

Oxygen tension is critical in a number of cell pathways but is often overlooked in cell culture. One reason for this is the difficulty in modulating and assessing oxygen tensions without disturbing the culture conditions. Toward this end, a simple method to generate oxygen-sensitive microwells was developed through embossing polystyrene (PS) and platinum(ii) octaethylporphyrin ketone (PtOEPK) thin films. In addition to monitoring the oxygen tension, microwells were employed in order to isolate uniform clusters of cells in microwells. The depth and width of the microwells can be adapted to different experimental parameters easily by altering the thin film processing or embossing stamp geometries. The thin oxygen sensitive microwell substrate is also compatible with high magnification modalities such as confocal imaging. The incorporation of the oxygen sensor into the microwells produces measurements of the oxygen tension near the cell surface. The oxygen sensitive microwells were calibrated and used to monitor oxygen tensions of Madin-Darby Canine Kidney Cells (MDCKs) cultured at high and low densities as a proof of concept. Wells 500 µm in diameter seeded with an average of 330 cells exhibited an oxygen level of 12.6% whereas wells seeded with an average of 20 cells per well exhibited an oxygen level of 19.5%, a 35.7% difference. This platform represents a new tool for culturing cells in microwells in a format amenable to high magnification imaging while monitoring the oxygen state of the culture media.

Oxygen gradients for open well cellular cultures via microfluidic substrates.

Controlling oxygen concentration at a microscale level can benefit experimental investigations involving oxidative stress, ischemia, and reactive oxygen species (ROS) mediated cellular pathways. Here, we report the application of microfluidic gradient generation in an open-well culture model, in which a gradient of gas is delivered via diffusion through a gas permeable substrate that separates cells from the gas microchannels below. By using diffusion to localize oxygen delivery, microgradients of oxygen concentrations can be rapidly and controllably applied without exposing cells to mechanical stresses or reducing culture volumes inside microfluidic culture chambers. Furthermore, we demonstrate the modulation of intracellular ROS levels in Madin-Darby Canine Kidney (MDCK) cells by applying these oxygen microgradients. Increases in ROS levels consistent with both oxidative stress and hypoxic exposures were observed in MDCK cells. The measured ROS increases were comparable to 100 microM hydrogen peroxide exposure in a control comparison, which is within the range of standard ROS induction methods. Incubation with 200 microM vitamin C was able to demodulate the ROS response at both hypoxic and hyperoxic exposures. By providing microfluidic controlled gradients, constant ROS exposure, and a shear-free open well design, the devices introduced here greatly improve upon standard oxygen-based culturing methods.

Precise control over the oxygen conditions within the Boyden chamber using a microfabricated insert.

Cell migration is a hallmark of cancer cell metastasis and is highly correlated with hypoxia in tumors. The Boyden chamber is a porous membrane-based migration platform that has seen a great deal of use for both in vitro migration and invasion assays due to its adaptability to common culture vessels and relative ease of use. The hypoxic chamber is a current tool that can be implemented to investigate the cellular response to oxygen paradigms. Unfortunately, this method lacks the spatial and temporal precision to accurately model a number of physiological phenomena. In this article, we present a newly developed microfabricated polydimethylsiloxane (PDMS) device that easily adapts to the Boyden chamber, and provides more control over the oxygenation conditions exposed to cells. The device equilibrates to 1% oxygen in about 20 min, thus demonstrating the capabilities of a system for researchers to establish both short-term continuous and intermittent hypoxia regimes. A Parylene-C thin-film coating was used to prevent ambient air penetration through the bulk PDMS and was found to yield improved equilibration times and end-point concentrations. MDA-MD-231 cells, an invasive breast cancer line, were used as a model cell type to demonstrate the effect of oxygen concentration on cell migration through the Boyden chamber porous membrane. Continuous hypoxia downregulated migration of cells relative to the normoxic control, as did an intermittent hypoxia regime (IH) cycling between 0% and 21% oxygen (0-21% IH). However, cells exposed to 5-21% IH exhibited increased migration compared to the other conditions, as well as relative to the normoxic control. The results presented here show the device can be utilized for experiments implementing the Boyden chamber for in vitro hypoxic studies, allowing experiments to be conducted faster and with more precision than currently possible.