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.

Pneumatic oscillator circuits for timing and control of integrated microfluidics.

Frequency references are fundamental to most digital systems, providing the basis for process synchronization, timing of outputs, and waveform synthesis. Recently, there has been growing interest in digital logic systems that are constructed out of microfluidics rather than electronics, as a possible means toward fully integrated laboratory-on-a-chip systems that do not require any external control apparatus. However, the full realization of this goal has not been possible due to the lack of on-chip frequency references, thus requiring timing signals to be provided from off-chip. Although microfluidic oscillators have been demonstrated, there have been no reported efforts to characterize, model, or optimize timing accuracy, which is the fundamental metric of a clock. Here, we report pneumatic ring oscillator circuits built from microfluidic valves and channels. Further, we present a compressible-flow analysis that differs fundamentally from conventional circuit theory, and we show the utility of this physically based model for the optimization of oscillator stability. Finally, we leverage microfluidic clocks to demonstrate circuits for the generation of phase-shifted waveforms, self-driving peristaltic pumps, and frequency division. Thus, pneumatic oscillators can serve as on-chip frequency references for microfluidic digital logic circuits. On-chip clocks and pumps both constitute critical building blocks on the path toward achieving autonomous laboratory-on-a-chip devices.

A Macro-to-Micro Interface for the Control of Cellular Organization.

The spatial organization of cellular communities plays a fundamental role in determining intercellular communication and emergent behavior. However, few tools exist to modulate tissue organization at the scale of individual cells, particularly in the case of dynamic manipulation. Micromechanical reconfigurable culture achieves dynamic control of tissue organization by culturing adherent cells on microfabricated plates that can be shifted to reorganize the arrangement of the cells. While biological studies utilizing this approach have been previously reported, this paper focuses on the engineering of the device, including the mechanism for translating manual manipulation to precise microscale position control, fault-tolerant design for manufacture, and the synthetic-to-living interface.

Microfluidic serial dilution ladder.

Serial dilution is a fundamental procedure that is common to a large number of laboratory protocols. Automation of serial dilution is thus a valuable component for lab-on-a-chip systems. While a handful of different microfluidic strategies for serial dilution have been reported, approaches based on continuous flow mixing inherently consume larger amounts of sample volume and chip real estate. We employ valve-driven circulatory mixing to address these issues and also introduce a novel device structure to store each stage of the dilution process. The dilution strategy is based on sequentially mixing the rungs of a ladder structure. We demonstrate a 7-stage series of 1 : 1 dilutions with R(2) equal to 0.995 in an active device area of 1 cm(2).

Microfabrication of High-Resolution Porous Membranes for Cell Culture.

Microporous membranes are widely utilized in cell biology to study cell-cell signaling and cell migration. However, the thickness and low porosity of commercial track-etched membranes limit the quality of cell imaging and the degree of cell-cell contact that can be achieved on such devices. We employ photolithography-based microfabrication to achieve porous membranes with pore diameter as small as 0.9 µm, up to 40% porosity, and less than 5% variation in pore size. Through the use of a soap release layer, membranes as thin as 1 µm can be achieved. The thin membranes minimally disrupt contrast enhancement optics, thus allowing good quality imaging of unlabeled cells under white light, unlike commercial membranes. In addition, the polymer membrane materials display low autofluorescence even after patterning, facilitating high quality fluorescence microscopy. Finally, confocal imaging suggests that substantial cell-cell contact is possible through the pores of these thin membranes. This membrane technology can enhance existing uses of porous membranes in cell biology as well as enable new types of experiments.

Reducing Uncertainty Through Mutual Information in Structural and Systems Biology.

Systems biology models are useful models of complex biological systems that may require a large amount of experimental data to fit each model's parameters or to approximate a likelihood function. These models range from a few to thousands of parameters depending on the complexity of the biological system modeled, potentially making the task of fitting parameters to the model difficult - especially when new experimental data cannot be gathered. We demonstrate a method that uses structural biology predictions to augment systems biology models to improve systems biology models' predictions without having to gather more experimental data. Additionally, we show how systems biology models' predictions can help evaluate novel structural biology hypotheses, which may also be expensive or infeasible to validate.

Fibroblasts influence muscle progenitor differentiation and alignment in contact independent and dependent manners in organized co-culture devices.

Myoblasts are precursor muscle cells that lie nascent to mature skeletal muscle. Once muscle is damaged, these cells migrate, fuse, and regenerate the muscle tissue. It is known that skeletal muscle can partially regenerate in vivo after muscle tissue damage. However, this regeneration does not always occur, especially in more severe injuries. Cellular therapy using tissue-engineering approaches has been shown to improve organ repair and function. To exploit potential benefits of using cell therapy as an avenue for skeletal muscle repair, it is important to understand the cellular dynamics underlying skeletal myocyte formation and growth. Cardiac fibroblasts have been shown to have a major influence on cardiomyocyte function, repair, and overall spatial distribution. However, little is known regarding fibroblasts' role on skeletal myocyte function. In this study, we utilized a reconfigurable co-culture device to understand the contact and paracrine effects of fibroblasts on skeletal myocyte alignment and differentiation using murine myoblast and fibroblast cell lines. We demonstrate that myotube alignment is increased by direct contact with fibroblasts, while myotube differentiation is reduced both in the gap and contact configurations with fibroblasts after 6 days of co-culture. Furthermore, neutralizing antibodies to FGF-2 can block these effects of fibroblasts on myotube differentiation and alignment. Finally, bi-directional signaling is critical to the observed myoblast-fibroblast interactions, since conditioned media could not reproduce the same effects observed in the gap configuration. These findings could have direct implications on cell therapies for repairing skeletal muscle, which have only utilized skeletal myoblasts or stem cell populations alone.

Stochastic Gradient Bayesian Optimal Experimental Designs for Simulation-based Inference.

Simulation-based inference (SBI) methods tackle complex scientific models with challenging inverse problems. However, SBI models often face a significant hurdle due to their non-differentiable nature, which hampers the use of gradient-based optimization techniques. Bayesian Optimal Experimental Design (BOED) is a powerful approach that aims to make the most efficient use of experimental resources for improved inferences. While stochastic gradient BOED methods have shown promising results in high-dimensional design problems, they have mostly neglected the integration of BOED with SBI due to the difficult non-differentiable property of many SBI simulators. In this work, we establish a crucial connection between ratio-based SBI inference algorithms and stochastic gradient-based variational inference by leveraging mutual information bounds. This connection allows us to extend BOED to SBI applications, enabling the simultaneous optimization of experimental designs and amortized inference functions. We demonstrate our approach on a simple linear model and offer implementation details for practitioners.

Pneumatic computers for embedded control of microfluidics.

Alternative computing approaches that interface readily with physical systems are well suited for embedded control of those systems. We demonstrate finite state machines implemented as pneumatic circuits of microfluidic valves and use these controllers to direct microfluidic liquid handling procedures on the same chip. These monolithic integrated systems require only power to be supplied externally, in the form of a vacuum source. User input can be provided directly to the chip by covering pneumatic ports with a finger. State machines with up to four bits of state memory are demonstrated, and next-state combinational logic can be fully reprogrammed by changing the hole-punch pattern on a membrane in the chip. These pneumatic computers demonstrate a framework for the embedded control of physical systems and open a path to stand-alone lab-on-a-chip devices capable of highly complex functionality.

PIEZO1-HaloTag hiPSCs: Bridging Molecular, Cellular and Tissue Imaging.

PIEZO1 channels play a critical role in numerous physiological processes by transducing diverse mechanical stimuli into electrical and chemical signals. Recent studies underscore the importance of endogenous PIEZO1 activity and localization in regulating mechanotransduction. To enable physiologically and clinically relevant human-based studies, we genetically engineered human induced pluripotent stem cells (hiPSCs) to express a HaloTag fused to endogenous PIEZO1. Combined with super-resolution imaging, our chemogenetic approach allows precise visualization of PIEZO1 in various cell types. Further, the PIEZO1-HaloTag hiPSC technology allows non-invasive monitoring of channel activity via Ca2+-sensitive HaloTag ligands, with temporal resolution approaching that of patch clamp electrophysiology. Using lightsheet imaging of hiPSC-derived neural organoids, we also achieve molecular scale PIEZO1 imaging in three-dimensional tissue samples. Our advances offer a novel platform for studying PIEZO1 mechanotransduction in human cells and tissues, with potential for elucidating disease mechanisms and development of targeted therapeutics.

Fabrication of Multilayer Molds by Dry Film Photoresist.

Dry film photoresists are widely employed to fabricate high-aspect-ratio microstructures, such as molds for microfluidic devices. Unlike liquid resists, such as SU-8, dry films do not require a cleanroom facility, and it is straightforward to prepare uniform and reproducible films as thick as 500 µm. Multilayer patterning, however, can be problematic with dry film resists even though it is critical for a number of microfluidic devices. Layer-to-layer mask alignment typically requires the first layer to be fully developed, making the pattern visible, before applying and patterning the second layer. While a liquid resist can flow over the topography of previous layers, this is not the case with dry film lamination. We found that post-exposure baking of dry film photoresists can preserve a flat topography while revealing an image of the patterned features that is suitable for alignment to the next layer. We demonstrate the use of this technique with two different types of dry film resist to fabricate master molds for a hydrophoresis size-sorting device and a cell chemotaxis device.

Phase-Optimized Peristaltic Pumping by Integrated Microfluidic Logic.

Microfluidic droplet generation typically entails an initial stabilization period on the order of minutes, exhibiting higher variation in droplet volume until the system reaches monodisperse production. The material lost during this period can be problematic when preparing droplets from limited samples such as patient biopsies. Active droplet generation strategies such as antiphase peristaltic pumping effectively reduce stabilization time but have required off-chip control hardware that reduces system accessibility. We present a fully integrated device that employs on-chip pneumatic logic to control phase-optimized peristaltic pumping. Droplet generation stabilizes in about a second, with only one or two non-uniform droplets produced initially.

High-resolution integrated piezoresistive sensors for microfluidic monitoring.

Microfluidic devices are traditionally monitored by bulky and expensive off-chip sensors. We have developed a soft piezoresistive sensor capable of measuring micron-level strains that can be easily integrated into devices via soft lithography. We apply this sensor to achieve fast and localized monitoring of pressure, flow, and valve actuation.

Microenvironmental regulation of the sinusoidal endothelial cell phenotype in vitro.

UNLABELLED: Liver sinusoidal endothelial cells (LSECs) differ, both structurally and functionally, from endothelial cells (ECs) lining blood vessels of other tissues. For example, in contrast to other ECs, LSECs possess fenestrations, have low detectable levels of platelet endothelial cell adhesion molecule 1 expression, and in rat tissue, they distinctively express a cell surface marker recognized by the SE-1 antibody. These unique phenotypic characteristics seen in hepatic tissue are lost over time upon culture in vitro; therefore, this study sought to systematically examine the effects of microenvironmental stimuli--namely, extracellular matrix and neighboring cells, on the LSEC phenotype in vitro. In probing the role of the underlying extracellular matrix, we identified collagen I and collagen III as well as mixtures of collagen I/collagen IV/fibronectin as having a positive effect on LSEC survival. Furthermore, using a stable hepatocellular model (hepatocyte-fibroblast) we were able to prolong the expression of both SE-1 and phenotypic functions of LSEC such as factor VIII activity and AcLOL uptake in cocultured LSECs through the production of short-range paracrine signals. In the course of these experiments, we identified the antigen recognized by SE-1 as CD32b. CONCLUSION: Collectively, this study has identified several microenvironmental regulators of liver sinusoidal endothelial cells that prolong their phenotypic functions for up to 2 weeks in culture, enabling the development of better in vitro models of liver physiology and disease.

Cell patterning by surface tension pinning in microfluidic channels.

We present a simple method to pattern multiple cell populations inside a microfluidic channel. The microchannel is partially filled with a cell suspension, and the position of the liquid boundary remains pinned by surface tension. Cells then adhere only in the filled portion of the channel, producing a very sharp boundary. The process can be performed in an unmodified microfluidic channel with only a manual syringe and can be repeated multiple times to pattern cocultures or tricultures. We demonstrate the patterning method with two different mammalian cell types, 3T3 fibroblasts and NMuMG epithelial cells, and channel heights of 1.5 mm and 0.5 mm. We anticipate that this method will be useful for studies of cell-cell interactions where precise control of the fluidic microenvironment is required.

Microfluidic filter device with nylon mesh membranes efficiently dissociates cell aggregates and digested tissue into single cells.

Tissues are increasingly being analyzed at the single cell level in order to characterize cellular diversity and identify rare cell types. Single cell analysis efforts are greatly limited, however, by the need to first break down tissues into single cell suspensions. Current dissociation methods are inefficient, leaving a significant portion of the tissue as aggregates that are filtered away or left to confound results. Here, we present a simple and inexpensive microfluidic device that simultaneously filters large tissue fragments and dissociates smaller aggregates into single cells, thereby improving single cell yield and purity. The device incorporates two nylon mesh membranes with well-defined, micron-sized pores that operate on aggregates of different size scales. We also designed the device so that the first filtration could be performed under tangential flow to minimize clogging. Using cancer cell lines, we demonstrated that aggregates were effectively dissociated using high flow rates and pore sizes that were smaller than a single cell. However, pore sizes that were less than half the cell size caused significant damage. We then improved results by passing the sample through two filter devices in series, with single cell yield and purity predominantly determined by the pore size of the second membrane. Next, we optimized performance using minced and digested murine kidney tissue samples, and determined that the combination of 50 and 15 µm membranes was optimal. Finally, we integrated these two membranes into a single filter device and performed validation experiments using minced and digested murine kidney, liver, and mammary tumor tissue samples. The dual membrane microfluidic filter device increased single cell numbers by at least 3-fold for each tissue type, and in some cases by more than 10-fold. These results were obtained in minutes without affecting cell viability, and additional filtering would not be required prior to downstream applications. In future work, we will create complete tissue analysis platforms by integrating the dual membrane microfluidic filter device with additional upstream tissue processing technologies, as well as downstream operations such as cell sorting and detection.

Microfluidic device for rapid digestion of tissues into cellular suspensions.

The ability to harvest single cells from tissues is currently a bottleneck for cell-based diagnostic technologies, and remains crucial in the fields of tissue engineering and regenerative medicine. Tissues are typically broken down using proteolytic digestion and various mechanical treatments, but success has been limited due to long processing times, low yield, and high manual labor burden. Here, we present a novel microfluidic device that utilizes precision fluid flows to improve the speed and efficiency of tissue digestion. The microfluidic channels were designed to apply hydrodynamic shear forces at discrete locations on tissue specimens up to 1 cm in length and 1 mm in diameter, thereby accelerating digestion through hydrodynamic shear forces and improved enzyme-tissue contact. We show using animal organs that our digestion device with hydro-mincing capabilities was superior to conventional scalpel mincing and digestion based on recovery of DNA and viable single cells. Thus, our microfluidic digestion device can eliminate or reduce the need to mince tissue samples with a scalpel, while reducing sample processing time and preserving cell viability. Another advantage is that downstream microfluidic operations could be integrated to enable advanced cell processing and analysis capabilities. We envision our novel device being used in research and clinical settings to promote single cell-based analysis technologies, as well as to isolate primary, progenitor, and stem cells for use in the fields of tissue engineering and regenerative medicine.

Patterning of sharp cellular interfaces with a reconfigurable elastic substrate.

Micropatterned cocultures are a useful experimental tool for the study of cell-cell interactions. Patterning methods often rely on sequential seeding of different cell types or removal of a barrier separating two populations, but it is difficult to pattern sharp interfaces between pure populations with low cross-contamination when using these approaches. Patterning by the use of reconfigurable substrates can overcome these limitations, but such methods can be costly and challenging to employ in a typical biology laboratory. Here, we describe a low-cost and simple-to-use reconfigurable substrate comprised of a transparent elastic material that is partially cut to form a slit that opens when the device is stretched. The slit seals back up when released, allowing two initially separate, adherent cell populations to be brought together to form a contact interface. Fluorescent imaging of patterned cocultures demonstrates the early establishment of a sharp cellular interface. As a proof of principle, we demonstrate the use of this device to study competition at the interface of two stem cell populations.

Compartmentalized Culture of Perivascular Stroma and Endothelial Cells in a Microfluidic Model of the Human Endometrium.

The endometrium is the inner lining of the uterus. Following specific cyclic hormonal stimulation, endometrial stromal fibroblasts (stroma) and vascular endothelial cells exhibit morphological and biochemical changes to support embryo implantation and regulate vascular function, respectively. Herein, we integrated a resin-based porous membrane in a dual chamber microfluidic device in polydimethylsiloxane that allows long term in vitro co-culture of human endometrial stromal and endothelial cells. This transparent, 2-µm porous membrane separates the two chambers, allows for the diffusion of small molecules and enables high resolution bright field and fluorescent imaging. Within our primary human co-culture model of stromal and endothelial cells, we simulated the temporal hormone changes occurring during an idealized 28-day menstrual cycle. We observed the successful differentiation of stroma into functional decidual cells, determined by morphology as well as biochemically as measured by increased production of prolactin. By controlling the microfluidic properties of the device, we additionally found that shear stress forces promoted cytoskeleton alignment and tight junction formation in the endothelial layer. Finally, we demonstrated that the endometrial perivascular stroma model was sustainable for up to 4 weeks, remained sensitive to steroids and is suitable for quantitative biochemical analysis. Future utilization of this device will allow the direct evaluation of paracrine and endocrine crosstalk between these two cell types as well as studies of immunological events associated with normal vs. disease-related endometrial microenvironments.

Scaling of pneumatic digital logic circuits.

The scaling of integrated circuits to smaller dimensions is critical for achieving increased system complexity and speed. Digital logic circuits composed of pneumatic microfluidic components have to this point been limited to a circuit density of 2-4 gates cm(-2), constraining the complexity of the digital systems that can be achieved. We explored the use of precision machining techniques to reduce the size of pneumatic valves and resistors, and to achieve more accurate and efficient placement of ports and vias. In this way, we attained an order of magnitude increase in circuit density, reaching as high as 36 gates cm(-2). A 12-bit binary counter circuit composed of 96 gates was realized in an area of 360 mm(2). The reduction in size also brought an order of magnitude increase in speed. The frequency of a 13-stage ring oscillator increased from 2.6 Hz to 22.1 Hz, and the maximum clock frequency of a binary counter increased from 1/3 Hz to 6 Hz.