Transforming Static Barrier Tissue Models into Dynamic Microphysiological Systems.

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. However, these platforms have not gained widespread adoption in bioscience laboratories where open-well, membrane-based approaches serve as the gold standard for mimicking tissue barriers, despite lacking fluid flow capabilities. This issue can be primarily attributed to the incompatibility of existing microphysiological systems with standard protocols and tools developed for open-well systems. Here, we present a protocol for creating a reconfigurable membrane-based platform with an open-well structure, flow enhancement capability, and compatibility with conventional protocols. This system utilizes a magnetic assembly approach that enables reversible switching between open-well and microfluidic modes. With this approach, users have the flexibility to begin an experiment in the open-well format using standard protocols and add or remove flow capabilities as needed. To demonstrate the practical usage of this system and its compatibility with standard techniques, an endothelial cell monolayer was established in an open-well format. The system was reconfigured to introduce fluid flow and then switched to the open-well format to conduct immunostaining and RNA extraction. Due to its compatibility with conventional open-well protocols and flow enhancement capability, this reconfigurable design is expected to be adopted by both engineering and bioscience laboratories.

Microengineering 3D Collagen Matrices with Tumor-Mimetic Gradients in Fiber Alignment.

In the tumor microenvironment (TME), collagen fibers facilitate tumor cell migration through the extracellular matrix. Previous studies have focused on studying the responses of cells on uniformly aligned or randomly aligned collagen fibers. However, the in vivo environment also features spatial gradients in alignment, which arise from the local reorganization of the matrix architecture due to cell-induced traction forces. Although there has been extensive research on how cells respond to graded biophysical cues, such as stiffness, porosity, and ligand density, the cellular responses to physiological fiber alignment gradients have been largely unexplored. This is due, in part, to a lack of robust experimental techniques to create controlled alignment gradients in natural materials. In this study, we image tumor biopsy samples and characterize the alignment gradients present in the TME. To replicate physiological gradients, we introduce a first-of-its-kind biofabrication technique that utilizes a microfluidic channel with constricting and expanding geometry to engineer 3D collagen hydrogels with tunable fiber alignment gradients that range from sub-millimeter to millimeter length scales. Our modular approach allows easy access to the microengineered gradient gels, and we demonstrate that HUVECs migrate in response to the fiber architecture. We provide preliminary evidence suggesting that MDA-MB-231 cell aggregates, patterned onto a specific location on the alignment gradient, exhibit preferential migration towards increasing alignment. This finding suggests that alignment gradients could serve as an additional taxis cue in the ECM. Importantly, our study represents the first successful engineering of continuous gradients of fiber alignment in soft, natural materials. We anticipate that our user-friendly platform, which needs no specialized equipment, will offer new experimental capabilities to study the impact of fiber-based contact guidance on directed cell migration.

Electropatterning-Contemporary developments for selective particle arrangements employing electrokinetics.

The selective positioning and arrangement of distinct types of multiscale particles can be used in numerous applications in microfluidics, including integrated circuits, sensors and biochips. Electrokinetic (EK) techniques offer an extensive range of options for label-free manipulation and patterning of colloidal particles by exploiting the intrinsic electrical properties of the target of interest. EK-based techniques have been widely implemented in many recent studies, and various methodologies and microfluidic device designs have been developed to achieve patterning two- and three-dimensional (3D) patterned structures. This review provides an overview of the progress in electropatterning research during the last 5 years in the microfluidics arena. This article discusses the advances in the electropatterning of colloids, droplets, synthetic particles, cells, and gels. Each subsection analyzes the manipulation of the particles of interest via EK techniques such as electrophoresis and dielectrophoresis. The conclusions summarize recent advances and provide an outlook on the future of electropatterning in various fields of application, especially those with 3D arrangements as their end goal.

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment.

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (<40 µm in thickness) of COL1 fibers that extend over distances less than 500 µm and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 µm in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

The Modular µSiM Reconfigured: Integration of Microfluidic Capabilities to Study In Vitro Barrier Tissue Models under Flow.

Microfluidic tissue barrier models have emerged to address the lack of physiological fluid flow in conventional "open-well" Transwell-like devices. However, microfluidic techniques have not achieved widespread usage in bioscience laboratories because they are not fully compatible with traditional experimental protocols. To advance barrier tissue research, there is a need for a platform that combines the key advantages of both conventional open-well and microfluidic systems. Here, a plug-and-play flow module is developed to introduce on-demand microfluidic flow capabilities to an open-well device that features a nanoporous membrane and live-cell imaging capabilities. The magnetic latching assembly of this design enables bi-directional reconfiguration and allows users to conduct an experiment in an open-well format with established protocols and then add or remove microfluidic capabilities as desired. This work also provides an experimentally-validated flow model to select flow conditions based on the experimental needs. As a proof-of-concept, flow-induced alignment of endothelial cells and the expression of shear-sensitive gene targets are demonstrated, and the different phases of neutrophil transmigration across a chemically stimulated endothelial monolayer under flow conditions are visualized. With these experimental capabilities, it is anticipated that both engineering and bioscience laboratories will adopt this reconfigurable design due to the compatibility with standard open-well protocols.

The Modular µSiM: A Mass Produced, Rapidly Assembled, and Reconfigurable Platform for the Study of Barrier Tissue Models In Vitro.

Advanced in vitro tissue chip models can reduce and replace animal experimentation and may eventually support "on-chip" clinical trials. To realize this potential, however, tissue chip platforms must be both mass-produced and reconfigurable to allow for customized design. To address these unmet needs, an extension of the µSiM (microdevice featuring a silicon-nitride membrane) platform is introduced. The modular µSiM (m-µSiM) uses mass-produced components to enable rapid assembly and reconfiguration by laboratories without knowledge of microfabrication. The utility of the m-µSiM is demonstrated by establishing an hiPSC-derived blood-brain barrier (BBB) in bioengineering and nonengineering, brain barriers focused laboratories. In situ and sampling-based assays of small molecule diffusion are developed and validated as a measure of barrier function. BBB properties show excellent interlaboratory agreement and match expectations from literature, validating the m-µSiM as a platform for barrier models and demonstrating successful dissemination of components and protocols. The ability to quickly reconfigure the m-µSiM for coculture and immune cell transmigration studies through addition of accessories and/or quick exchange of components is then demonstrated. Because the development of modified components and accessories is easily achieved, custom designs of the m-µSiM shall be accessible to any laboratory desiring a barrier-style tissue chip platform.

Local extensional flows promote long-range fiber alignment in 3D collagen hydrogels.

Randomly oriented type I collagen (COL1) fibers in the extracellular matrix are reorganized by biophysical forces into aligned domains extending several millimeters and with varying degrees of fiber alignment. These aligned fibers can transmit traction forces, guide tumor cell migration, facilitate angiogenesis, and influence tissue morphogenesis. To create aligned COL1 domains in microfluidic cell culture models, shear flows have been used to align thin COL1 matrices (<50µm in height) in a microchannel. However, there has been limited investigation into the role of shear flows in aligning 3D hydrogels (>130µm). Here, we show that pure shear flows do not induce fiber alignment in 3D atelo COL1 hydrogels, but the simple addition of local extensional flow promotes alignment that is maintained across several millimeters, with a degree of alignment directly related to the extensional strain rate. We further advance experimental capabilities by addressing the practical challenge of accessing a 3D hydrogel formed within a microchannel by introducing a magnetically coupled modular platform that can be released to expose the microengineered hydrogel. We demonstrate the platform's capability to pattern cells and fabricate multi-layered COL1 matrices using layer-by-layer fabrication and specialized modules. Our approach provides an easy-to-use fabrication method to achieve advanced hydrogel microengineering capabilities that combine fiber alignment with biofabrication capabilities.

A miniaturized 3D printed pressure regulator (µPR) for microfluidic cell culture applications.

Well-defined fluid flows are the hallmark feature of microfluidic culture systems and enable precise control over biophysical and biochemical cues at the cellular scale. Microfluidic flow control is generally achieved using displacement-based (e.g., syringe or peristaltic pumps) or pressure-controlled techniques that provide numerous perfusion options, including constant, ramped, and pulsed flows. However, it can be challenging to integrate these large form-factor devices and accompanying peripherals into incubators or other confined environments. In addition, microfluidic culture studies are primarily carried out under constant perfusion conditions and more complex flow capabilities are often unused. Thus, there is a need for a simplified flow control platform that provides standard perfusion capabilities and can be easily integrated into incubated environments. To this end, we introduce a tunable, 3D printed micro pressure regulator (µPR) and show that it can provide robust flow control capabilities when combined with a battery-powered miniature air pump to support microfluidic applications. We detail the design and fabrication of the µPR and: (i) demonstrate a tunable outlet pressure range relevant for microfluidic applications (1-10 kPa), (ii) highlight dynamic control capabilities in a microfluidic network, (iii) and maintain human umbilical vein endothelial cells (HUVECs) in a multi-compartment culture device under continuous perfusion conditions. We anticipate that our 3D printed fabrication approach and open-access designs will enable customized µPRs that can support a broad range of microfluidic applications.

Microengineered 3D Collagen Gels with Independently Tunable Fiber Anisotropy and Directionality.

Cellular processes, including differentiation, proliferation, and migration, have been linked to the alignment (anisotropy) and orientation (directionality) of collagen fibers in the native extracellular matrix (ECM). Given the critical role that biophysical cell-matrix interactions play in regulating biological functions, several microfluidic-based methods have been used to establish 3D collagen gels with defined fiber properties; these gels have helped to establish quantitative relationships between structural ECM cues and observed cell responses. Although existing microfluidic fabrication methods provide excellent definition over collagen fiber anisotropy, they have not demonstrated the independent control over fiber anisotropy and directionality necessary to replicate in vivo collagen architecture. Therefore, to advance collagen microengineering capabilities, we present a user-friendly technology platform that uses controlled fluid flows within a non-uniform microfluidic channel network to create collagen landscapes that can be tuned as a function of extensional strain rate. Herein, we demonstrate capabilities to i) control the degree of fiber anisotropy, ii) create spatial gradients in fiber anisotropy, iii) independently define fiber directionality, and iv) generate multi-material interfaces within a 3D environment. We then address the practical issue of integrating cells into microfluidic systems by using a peel-off template technique to provide direct access to microengineered collagen gels, and demonstrate that cells respond to the defined properties of the landscape. Finally, the platform's modular capability is highlighted by integrating a sub-micrometer thick porous parylene membrane onto the microengineered collagen as a method to define cell-substrate interactions.

Engineering fiber anisotropy within natural collagen hydrogels.

It is well known that biophysical properties of the extracellular matrix (ECM), including stiffness, porosity, composition, and fiber alignment (anisotropy), play a crucial role in controlling cell behavior in vivo. Type I collagen (collagen I) is a ubiquitous structural component in the ECM and has become a popular hydrogel material that can be tuned to replicate the mechanical properties found in vivo. In this review article, we describe popular methods to create 2-D and 3-D collagen I hydrogels with anisotropic fiber architectures. We focus on methods that can be readily translated from engineering and materials science laboratories to the life-science community with the overall goal of helping to increase the physiological relevance of cell culture assays.

A Low-Cost, Rapidly Integrated Debubbler (RID) Module for Microfluidic Cell Culture Applications.

Microfluidic platforms use controlled fluid flows to provide physiologically relevant biochemical and biophysical cues to cultured cells in a well-defined and reproducible manner. Undisturbed flows are critical in these systems, and air bubbles entering microfluidic channels can lead to device delamination or cell damage. To prevent bubble entry into microfluidic channels, we report a low-cost, Rapidly Integrated Debubbler (RID) module that is simple to fabricate, inexpensive, and easily combined with existing experimental systems. We demonstrate successful removal of air bubbles spanning three orders of magnitude with a maximum removal rate (dV/dt)max = 1.5 mL min-1, at flow rates required to apply physiological wall shear stress (1-200 dyne cm-2) to mammalian cells cultured in microfluidic channels.

New experimental models of the blood-brain barrier for CNS drug discovery.

INTRODUCTION: The blood-brain barrier (BBB) is a dynamic biological interface which actively controls the passage of substances between the blood and the central nervous system (CNS). From a biological and functional standpoint, the BBB plays a crucial role in maintaining brain homeostasis inasmuch that deterioration of BBB functions are prodromal to many CNS disorders. Conversely, the BBB hinders the delivery of drugs targeting the brain to treat a variety of neurological diseases. Area covered: This article reviews recent technological improvements and innovation in the field of BBB modeling including static and dynamic cell-based platforms, microfluidic systems and the use of stem cells and 3D printing technologies. Additionally, the authors laid out a roadmap for the integration of microfluidics and stem cell biology as a holistic approach for the development of novel in vitro BBB platforms. Expert opinion: Development of effective CNS drugs has been hindered by the lack of reliable strategies to mimic the BBB and cerebrovascular impairments in vitro. Technological advancements in BBB modeling have fostered the development of highly integrative and quasi- physiological in vitro platforms to support the process of drug discovery. These advanced in vitro tools are likely to further current understanding of the cerebrovascular modulatory mechanisms.

A Reversibly Sealed, Easy Access, Modular (SEAM) Microfluidic Architecture to Establish In Vitro Tissue Interfaces.

Microfluidic barrier tissue models have emerged as advanced in vitro tools to explore interactions with external stimuli such as drug candidates, pathogens, or toxins. However, the procedures required to establish and maintain these systems can be challenging to implement for end users, particularly those without significant in-house engineering expertise. Here we present a module-based approach that provides an easy-to-use workflow to establish, maintain, and analyze microscale tissue constructs. Our approach begins with a removable culture insert that is magnetically coupled, decoupled, and transferred between standalone, prefabricated microfluidic modules for simplified cell seeding, culture, and downstream analysis. The modular approach allows several options for perfusion including standard syringe pumps or integration with a self-contained gravity-fed module for simple cell maintenance. As proof of concept, we establish a culture of primary human microvascular endothelial cells (HMVEC) and report combined surface protein imaging and gene expression after controlled apical stimulation with the bacterial endotoxin lipopolysaccharide (LPS). We also demonstrate the feasibility of incorporating hydrated biomaterial interfaces into the microfluidic architecture by integrating an ultra-thin (< 1 µm), self-assembled hyaluronic acid/peptide amphiphile culture membrane with brain-specific Young's modulus (~ 1kPa). To highlight the importance of including biomimetic interfaces into microscale models we report multi-tiered readouts from primary rat cortical cells cultured on the self-assembled membrane and compare a panel of mRNA targets with primary brain tissue signatures. We anticipate that the modular approach and simplified operational workflows presented here will enable a wide range of research groups to incorporate microfluidic barrier tissue models into their work.

Rapid detection of trace bacteria in biofluids using porous monoliths in microchannels.

We present advancements in microfluidic technology for rapid detection of as few as 10 rickettsial organisms in complex biological samples. An immuno-reactive filter, macroporous polyacrylamide monolith (PAM), fabricated within a microfluidic channel enhances solid-phase immuno-capture, staining and detection of targeted bacteria. Bacterial cells in samples flowing through the channel are forced to interact with the PAM filter surface due to size exclusion, overcoming common transport and kinetic limitations for rapid (min), high-efficiency (~100%) capture. In the process, targeted cells in sample volumes of 10 µl to >100 µl are concentrated within a sub-50 nl region at the PAM filter edge in the microchannel, thus concentrating them over 1000-fold. This significantly increases sensitivity, as the hydrophilic PAM also yields low non-specific immuno-fluorescence backgrounds with samples including serum, blood and non-targeted bacteria. The concentrated target cells are detected using fluorescently-labeled antibodies. With a single 2.0×2.0×0.3 mm PAM filter, as few as 10 rickettsial organisms per 100 µl of lysed blood sample can be analyzed within 60 min, as compared to hours or even days needed for conventional detection methods. This method is highly relevant to rapid, multiplexed, low-cost point of care diagnostics at early stages of infection where diagnostics providing more immediate and actionable test results are needed to improve patient outcomes and mitigate potential natural and non-natural outbreaks or epidemics of rickettsial diseases.

Microfluidic platforms for RNA interference screening of virus-host interactions.

RNA interference (RNAi) is a powerful tool for functional genomics with the capacity to comprehensively analyze host-pathogen interactions. High-throughput RNAi screening is used to systematically perturb cellular pathways and discover therapeutic targets, but the method can be tedious and requires extensive capital equipment and expensive reagents. To aid in the development of an inexpensive miniaturized RNAi screening platform, we have developed a two part microfluidic system for patterning and screening gene targets on-chip to examine cellular pathways involved in virus entry and infection. First, a multilayer polydimethylsiloxane (PDMS)-based spotting device was used to array siRNA molecules into 96 microwells targeting markers of endocytosis, along with siRNA controls. By using a PDMS-based spotting device, we remove the need for a microarray printer necessary to perform previously described small scale (e.g. cellular microarrays) and microchip-based RNAi screening, while still minimizing reagent usage tenfold compared to conventional screening. Second, the siRNA spotted array was transferred to a reversibly sealed PDMS-based screening platform containing microchannels designed to enable efficient cell loading and transfection of mammalian cells while preventing cross-contamination between experimental conditions. Validation of the screening platform was examined using Vesicular stomatitis virus and emerging pathogen Rift Valley fever virus, which demonstrated virus entry pathways of clathrin-mediated endocytosis and caveolae-mediated endocytosis, respectively. The techniques here are adaptable to other well-characterized infection pathways with a potential for large scale screening in high containment biosafety laboratories.

Thirty-minute total synthesis of microfluidic systems and functionalized porous elements via "living" radical photo-polymerization.

A "living" radical photo-polymerization (LRPP) technique is used to rapidly fabricate microfluidic channels and micro-patterned porous polymer monoliths. Surface-initiated LRPP is then used to functionalize porous elements in a robust one-step surface modification process. Assay-ready platforms can be fully realized in less than 30 minutes. An application relevant to clinical diagnostics is presented.

A platform for assessing chemotactic migration within a spatiotemporally defined 3D microenvironment.

While the quantification of cell movement within defined biochemical gradients is now possible with microfluidic approaches, translating this capability to biologically relevant three-dimensional microenvironments remains a challenge. We introduce an accessible platform, requiring only standard tools (e.g. pipettes), that provides robust soluble factor control within a three-dimensional biological matrix. We demonstrate long-lasting linear and non-linear concentration profiles that were maintained for up to ten days using 34.5 muL solute volume. We also demonstrate the ability to superimpose local soluble factor pulses onto existing gradients via defined dosing windows. The combination of long-term and transient gradient characteristics within a three-dimensional environment opens the door for signaling studies that investigate the migratory behavior of cells within a biologically representative matrix. To this end, we apply temporally evolving and long-lasting gradients to study the chemotactic responses of human neutrophils and the invasion of metastatic rat mammary adenocarcinoma cells (MtLN3) within three-dimensional collagen matrices.

Microfluidic logic gates and timers.

We use surface tension-based passive pumping and fluidic resistance to create a number of microfluidic analogs to electronic circuit components. Three classes of components are demonstrated: (1) OR/AND, NOR/NAND, and XNOR digital microfluidic logic gates; (2) programmable, autonomous timers; and (3) slow, perfusive flow rheostats. The components can be implemented with standard pipettes and provide a means of non-electronic and autonomous preprogrammed control with potential utility in cell studies and high throughput screening applications.

Spatiotemporal micropatterning of cells on arbitrary substrates.

This paper presents a readily accessible patterning platform--based upon geometric constraints, discrete cell suspension droplets, and controlled cell settling--that provides both temporal and spatial patterning capabilities. As a demonstration, single-cell (and bead) suspensions as well as multicellular human embryonic stem cell colonies are spatiotemporally patterned onto arbitrary substrates. These substrates include tissue culture surfaces, cell monolayers, protein-coated surfaces, and 3D gel matrices. The generation of soluble factor gradients is also demonstrated. This method is completely passive and does not require external power sources. Spatiotemporal patterning provides a foundation for future biological studies that explore the time-dependent relationships between cell-cell signaling and cellular responses.

Characterization of a membrane-based gradient generator for use in cell-signaling studies.

This paper describes a method to create stable chemical gradients without requiring fluid flow. The absence of fluid flow makes this device amenable to cell signaling applications where soluble factors can impact cell behavior. This device consists of a membrane-covered source region and a large volume sink region connected by a microfluidic channel. The high fluidic resistance of the membrane limits fluid flow caused by pressure differences in the system, but allows diffusive transport of a chemical species through the membrane and into the channel. The large volume sink region at the end of the microfluidic channel helps to maintain spatial and temporal stability of the gradient. The chemical gradient in a 0.5 mm region near the sink region experiences a maximum of 10 percent change between the 6 and 24 h data points. We present the theory, design, and characterization of this device and provide an example of neutrophil chemotaxis as proof of concept for future quantitative cell-signaling applications.