Partitioning of hydrogels in 3D-printed microchannels.

Hydrogels allow for controlling the diffusion rate and amount of solute according to the hydrogel network and thus have found many applications in drug delivery, biomaterials, toxicology, and tissue engineering. This paper describes a 3D-printed microfluidic chip for the straightforward partitioning of hydrogel barriers between microchannels. We use a previously-reported 3-channel architecture whereby the middle channel is filled with a hydrogel - acting like a porous barrier for diffusive transport - and the two side channels act as sink and source; the middle channel communicates with the side channels via orthogonal, small capillary channels that are also responsible for partitioning the hydrogel during filling. Our 3D-printed microfluidic chip is simple to fabricate by stereolithography (SL), inexpensive, reproducible, and convenient, so it is more adequate for transport studies than a microchip fabricated by photolithographic procedures. The chip was fabricated in a resin made of poly(ethylene glycol) diacrylate (PEG-DA) (MW = 258) (PEG-DA-258). The SL process allowed us to print high aspect ratio (37 : 1) capillary channels (27 µm-width and 1 mm-height) and enable the trapping of liquid-phase hydrogels in the hydrogel barrier middle channel. We studied the permeability of hydrogel barriers made of PEG-DA (MW = 700) (PEG-DA-700, 10% polymer content by wt. in water) - as a model of photopolymerizable barriers - and agarose (MW = 120 000, 2% polymer content by wt. in water) - as a model of thermally-gelled barriers. We measured the diffusion of fluorescein, 10k-dextran-Alexa 680 and BSA-Texas Red through these barriers. Fluorescein diffusion was observed through both 10% PEG-DA-700 and 2% agarose barriers while 10k-dextran-Alexa 680 and BSA-Texas Red diffused appreciably only through the 2% agarose hydrogel barrier. Our microfluidic chip facilitates the tuning of such barriers simply by altering the hydrogel materials. The straightforward trapping of selective barriers in 3D-printed microchannels should find wide applicability in drug delivery, tissue engineering, cell separation, and organ-on-a-chip platforms.

Digital Manufacturing for Microfluidics.

The microfluidics field is at a critical crossroads. The vast majority of microfluidic devices are presently manufactured using micromolding processes that work very well for a reduced set of biocompatible materials, but the time, cost, and design constraints of micromolding hinder the commercialization of many devices. As a result, the dissemination of microfluidic technology-and its impact on society-is in jeopardy. Digital manufacturing (DM) refers to a family of computer-centered processes that integrate digital three-dimensional (3D) designs, automated (additive or subtractive) fabrication, and device testing in order to increase fabrication efficiency. Importantly, DM enables the inexpensive realization of 3D designs that are impossible or very difficult to mold. The adoption of DM by microfluidic engineers has been slow, likely due to concerns over the resolution of the printers and the biocompatibility of the resins. In this article, we review and discuss the various printer types, resolution, biocompatibility issues, DM microfluidic designs, and the bright future ahead for this promising, fertile field.

High-Precision Stereolithography of Biomicrofluidic Devices.

Stereolithography (SL) is emerging as an attractive alternative to soft lithography for fabricating microfluidic devices due to its low cost and high design efficiency. Low molecular weight poly(ethylene glycol)diacrylate (MW = 258) (PEG-DA-258) has been used for SL 3D-printing of biocompatible microdevices at submillimeter resolution. However, 3D-printing resins that simultaneously feature high transparency, high biocompatibility, and high resolution are still lacking. It is found that photosensitizer isopropyl thioxanthone can, in a concentration-dependent manner, increase the absorbance of the resin (containing PEG-DA-258 and photoinitator Irgacure-819) by over an order of magnitude. This increase in absorbance allows for SL printing of microdevices at sub pixel resolution with commercially available desktop printers and without compromising transparency or biocompatibility. The assembly-free, rapid (<15 h) 3D-printing of a variety of complex 3D microfluidic devices such as a 3D-fluid router, a passive chaotic micro-mixer, an active micro-mixer with pneumatic microvalves, and high-aspect ratio (37:1) microchannels of single pixel width is demonstrated. These manufacturing capabilities are unavailable in conventional microfluidic rapid prototyping techniques. The low absorption of small hydrophobic molecules and microfluidic labeling of cultured mammalian cells in 3D-printed PEG-DA-258 microdevices is demonstrated, indicating the potential of PEG-DA-based fabrication of cell-based assays, drug discovery, and organ-on-chip platforms.

Digital Manufacturing of Selective Porous Barriers in Microchannels Using Multi-Material Stereolithography.

We have developed a sequential stereolithographic co-printing process using two different resins for fabricating porous barriers in microfluidic devices. We 3D-printed microfluidic channels with a resin made of poly(ethylene glycol) diacrylate (MW = 258) (PEG-DA-258), a UV photoinitiator, and a UV sensitizer. The porous barriers were created within the microchannels in a different resin made of either PEG-DA (MW = 575) (PEG-DA-575) or 40% (w/w in water) PEG-DA (MW = 700) (40% PEG-DA-700). We showed selective hydrogen ion diffusion across a 3D-printed PEG-DA-575 porous barrier in a cross-channel diffusion chip by observing color changes in phenol red, a pH indicator. We also demonstrated the diffusion of fluorescein across a 3D-printed 40% PEG-DA-700 porous barrier in a symmetric-channel diffusion chip by measuring fluorescence intensity changes across the porous barrier. Creating microfluidic chips with integrated porous barriers using a semi-automated 3D printing process shortens the design and processing time, avoids assembly and bonding complications, and reduces manufacturing costs compared to micromolding processes. We believe that our digital manufacturing method for fabricating selective porous barriers provides an inexpensive, simple, convenient and reproducible route to molecule delivery in the fields of molecular filtration and cell-based microdevices.

Desktop-Stereolithography 3D-Printing of a Poly(dimethylsiloxane)-Based Material with Sylgard-184 Properties.

The advantageous physiochemical properties of poly(dimethylsiloxane) (PDMS) have made it an extremely useful material for prototyping in various technological, scientific, and clinical areas. However, PDMS molding is a manual procedure and requires tedious assembly steps, especially for 3D designs, thereby limiting its access and usability. On the other hand, automated digital manufacturing processes such as stereolithography (SL) enable true 3D design and fabrication. Here the formulation, characterization, and SL application of a 3D-printable PDMS resin (3DP-PDMS) based on commercially available PDMS-methacrylate macromers, a high-efficiency photoinitiator and a high-absorbance photosensitizer, is reported. Using a desktop SL-printer, optically transparent submillimeter structures and microfluidic channels are demonstrated. An optimized blend of PDMS-methacrylate macromers is also used to SL-print structures with mechanical properties similar to conventional thermally cured PDMS (Sylgard-184). Furthermore, it is shown that SL-printed 3DP-PDMS substrates can be rendered suitable for mammalian cell culture. The 3DP-PDMS resin enables assembly-free, automated, digital manufacturing of PDMS, which should facilitate the prototyping of devices for microfluidics, organ-on-chip platforms, soft robotics, flexible electronics, and sensors, among others.

3D-printed Quake-style microvalves and micropumps.

Here we demonstrate a 3D-printable microvalve that is transparent, built with a biocompatible resin, and has a simple architecture that can be easily scaled up into large arrays. The open-at-rest valve design is derived from Quake's PDMS valve design. We used a stereolithographic (SL) 3D printer to print a thin (25 or 10 µm-thick) membrane (1200 or 500 µm-diam.) that is pneumatically pressed (∼3-6 psi) over a bowl-shaped seat to close the valve. We used poly(ethylene diacrylate) (MW = 258) (PEG-DA-258) as the resin because it yields transparent cytocompatible prints. Although the flexibility of PEG-DA-258 is inferior to that of other microvalve fabrication materials such as PDMS, the valve benefits from the bowl design and the membrane's high restoring force since it does not need a negative pressure to re-open. We also 3D-printed a micropump by combining three Quake-style valves in series. The micropump only requires positive pressure for its operation and profits from the fast return to the valves' open states. Moreover, we printed a 64-valve array constructed with 500 µm-diam. valves to demonstrate the reliability and scalability of the valves. Overall, we demonstrate the 3D-printing of compact microvalves and micropumps using a process that precludes the need for specialized, time-consuming labor.

Large-scale microfluidic gradient arrays reveal axon guidance behaviors in hippocampal neurons.

High-throughput quantitative approaches to study axon growth behaviors have remained a challenge. We have developed a 1024-chamber microfluidic gradient generator array that enables large-scale investigations of axon guidance and growth dynamics from individual primary mammalian neurons, which are exposed to gradients of diffusible molecules. Our microfluidic method (a) generates statistically rich data sets, (b) produces a stable, reproducible gradient with negligible shear stresses on the culture surface, (c) is amenable to the long-term culture of primary neurons without any unconventional protocol, and (d) eliminates the confounding influence of cell-secreted factors. Using this platform, we demonstrate that hippocampal axon guidance in response to a netrin-1 gradient is concentration-dependent-attractive at higher concentrations and repulsive at lower concentrations. We also show that the turning of the growth cone depends on the angle of incidence of the gradient. Our study highlights the potential of microfluidic devices in producing large amounts of data from morphogen and chemokine gradients that play essential roles not only in axonal navigation but also in stem cell differentiation, cell migration, and immune response.

An open-chamber flow-focusing device for focal stimulation of micropatterned cells.

Microfluidic devices can deliver soluble factors to cell and tissue culture microenvironments with precise spatiotemporal control. However, enclosed microfluidic environments often have drawbacks such as the need for continuous culture medium perfusion which limits the duration of experiments, incongruity between microculture and macroculture, difficulty in introducing cells and tissues, and high shear stress on cells. Here, we present an open-chamber microfluidic device that delivers hydrodynamically focused streams of soluble reagents to cells over long time periods (i.e., several hours). We demonstrate the advantage of the open chamber by using conventional cell culture techniques to induce the differentiation of myoblasts into myotubes, a process that occurs in 7-10 days and is difficult to achieve in closed chamber microfluidic devices. By controlling the flow rates and altering the device geometry, we produced sharp focal streams with widths ranging from 36 µm to 187 µm. The focal streams were reproducible (∼12% variation between units) and stable (∼20% increase in stream width over 10 h of operation). Furthermore, we integrated trenches for micropatterning myoblasts and microtraps for confining single primary myofibers into the device. We demonstrate with finite element method (FEM) simulations that shear stresses within the cell trench are well below values known to be deleterious to cells, while local concentrations are maintained at ∼22% of the input concentration. Finally, we demonstrated focused delivery of cytoplasmic and nuclear dyes to micropatterned myoblasts and myofibers. The open-chamber microfluidic flow-focusing concept combined with micropatterning may be generalized to other microfluidic applications that require stringent long-term cell culture conditions.

3D-printing of transparent bio-microfluidic devices in PEG-DA.

The vast majority of microfluidic systems are molded in poly(dimethylsiloxane) (PDMS) by soft lithography due to the favorable properties of PDMS: biocompatible, elastomeric, transparent, gas-permeable, inexpensive, and copyright-free. However, PDMS molding involves tedious manual labor, which makes PDMS devices prone to assembly failures and difficult to disseminate to research and clinical settings. Furthermore, the fabrication procedures limit the 3D complexity of the devices to layered designs. Stereolithography (SL), a form of 3D-printing, has recently attracted attention as a way to customize the fabrication of biomedical devices due to its automated, assembly-free 3D fabrication, rapidly decreasing costs, and fast-improving resolution and throughput. However, existing SL resins are not biocompatible and patterning transparent resins at high resolution remains difficult. Here we report procedures for the preparation and patterning of a transparent resin based on low-MW poly(ethylene glycol) diacrylate (MW 250) (PEG-DA-250). The 3D-printed devices are highly transparent and cells can be cultured on PEG-DA-250 prints for several days. This biocompatible SL resin and printing process solves some of the main drawbacks of 3D-printed microfluidic devices: biocompatibility and transparency. In addition, it should also enable the production of non-microfluidic biomedical devices.

The upcoming 3D-printing revolution in microfluidics.

In the last two decades, the vast majority of microfluidic systems have been built in poly(dimethylsiloxane) (PDMS) by soft lithography, a technique based on PDMS micromolding. A long list of key PDMS properties have contributed to the success of soft lithography: PDMS is biocompatible, elastomeric, transparent, gas-permeable, water-impermeable, fairly inexpensive, copyright-free, and rapidly prototyped with high precision using simple procedures. However, the fabrication process typically involves substantial human labor, which tends to make PDMS devices difficult to disseminate outside of research labs, and the layered molding limits the 3D complexity of the devices that can be produced. 3D-printing has recently attracted attention as a way to fabricate microfluidic systems due to its automated, assembly-free 3D fabrication, rapidly decreasing costs, and fast-improving resolution and throughput. Resins with properties approaching those of PDMS are being developed. Here we review past and recent efforts in 3D-printing of microfluidic systems. We compare the salient features of PDMS molding with those of 3D-printing and we give an overview of the critical barriers that have prevented the adoption of 3D-printing by microfluidic developers, namely resolution, throughput, and resin biocompatibility. We also evaluate the various forces that are persuading researchers to abandon PDMS molding in favor of 3D-printing in growing numbers.

3D-printed microfluidic automation.

Microfluidic automation - the automated routing, dispensing, mixing, and/or separation of fluids through microchannels - generally remains a slowly-spreading technology because device fabrication requires sophisticated facilities and the technology's use demands expert operators. Integrating microfluidic automation in devices has involved specialized multi-layering and bonding approaches. Stereolithography is an assembly-free, 3D-printing technique that is emerging as an efficient alternative for rapid prototyping of biomedical devices. Here we describe fluidic valves and pumps that can be stereolithographically printed in optically-clear, biocompatible plastic and integrated within microfluidic devices at low cost. User-friendly fluid automation devices can be printed and used by non-engineers as replacement for costly robotic pipettors or tedious manual pipetting. Engineers can manipulate the designs as digital modules into new devices of expanded functionality. Printing these devices only requires the digital file and electronic access to a printer.

Utilization of extracellular information before ligand-receptor binding reaches equilibrium expands and shifts the input dynamic range.

Cell signaling systems sense and respond to ligands that bind cell surface receptors. These systems often respond to changes in the concentration of extracellular ligand more rapidly than the ligand equilibrates with its receptor. We demonstrate, by modeling and experiment, a general "systems level" mechanism cells use to take advantage of the information present in the early signal, before receptor binding reaches a new steady state. This mechanism, pre-equilibrium sensing and signaling (PRESS), operates in signaling systems in which the kinetics of ligand-receptor binding are slower than the downstream signaling steps, and it typically involves transient activation of a downstream step. In the systems where it operates, PRESS expands and shifts the input dynamic range, allowing cells to make different responses to ligand concentrations so high as to be otherwise indistinguishable. Specifically, we show that PRESS applies to the yeast directional polarization in response to pheromone gradients. Consideration of preexisting kinetic data for ligand-receptor interactions suggests that PRESS operates in many cell signaling systems throughout biology. The same mechanism may also operate at other levels in signaling systems in which a slow activation step couples to a faster downstream step.

Microfluidic transwell inserts for generation of tissue culture-friendly gradients in well plates.

Gradients of biochemical molecules play a key role in many physiological processes such as axon growth, tissue morphogenesis, and trans-epithelium nutrient transport, as well as in pathophysiological phenomena such as wound healing, immune response, bacterial invasion, and cancer metastasis. In this paper, we report a microfluidic transwell insert for generating quantifiable concentration gradients in a user-friendly and modular format that is compatible with conventional cell cultures and with tissue explant cultures. The device is simply inserted into a standard 6-well plate, where it hangs self-supported at a distance of ~250 µm above the cell culture surface. The gradient is created by small microflows from the device, through an integrated track-etched porous membrane, into the cell culture well. The microfluidic transwell can deliver stable, quantifiable gradients over a large area with extremely low fluid shear stress to dissociated cells or tissue explants cultured independently on the surface of a 6-well plate. We used finite-element modeling to describe the porous membrane flow and molecular transport and to predict gradients generated by the device. Using the device, we applied a gradient of the chemotactic peptide N-formyl-met-leu-phe (fMLP) to a large population of HL-60 cells (a neutrophil cell line) and directly observed the migration with time-lapse microscopy. On quantification of the chemotactic response with an automated tracking algorithm, we found 74% of the cells moving towards the gradient. Additionally, the modular design and low fluid shear stress made it possible to apply gradients of growth factors and second messengers to mouse retinal explant cultures. With a simplified interface and well-defined gradients, the microfluidic transwell device has potential for broad applications to gradient-sensing biology.

A modular cell culture device for generating arrays of gradients using stacked microfluidic flows.

Microfluidics has become increasingly important for the study of biochemical cues because it enables exquisite spatiotemporal control of the microenvironment. Well-characterized, stable, and reproducible generation of biochemical gradients is critical for understanding the complex behaviors involved in many biological phenomena. Although many microfluidic devices have been developed which achieve these criteria, the ongoing challenge for these platforms is to provide a suitably benign and physiologically relevant environment for cell culture in a user-friendly format. To achieve this paradigm, microfluidic designs must consider the full scope of cell culture from substrate preparation, cell seeding, and long-term maintenance to properly observe gradient sensing behavior. In addition, designs must address the challenges associated with altered culture conditions and shear forces in flow-based devices. With this consideration, we have designed and characterized a microfluidic device based on the principle of stacked flows to achieve highly stable gradients of diffusible molecules over large areas with extremely low shear forces. The device utilizes a benign vacuum sealing strategy for reversible application to pre-established cell cultures. We apply this device to an existing culture of breast cancer cells to demonstrate the negligible effect of its shear flow on migratory behavior. Lastly, we extend the stacked-flow design to demonstrate its scalable architecture with a prototype device for generating an array of combinatorial gradients.

A neuron-benign microfluidic gradient generator for studying the response of mammalian neurons towards axon guidance factors.

Investigation of biochemical cues in isolation or in combinations in cell culture systems is crucial for unraveling the mechanisms that govern neural development and repair. The most widely used experimental paradigms that elicit axon guidance in vitro utilize as the source of the gradient a pulsatile pipette, transfected cells, or a loaded gel, producing time-varying gradients of poor reproducibility which are not well suited for studying slow-growing mammalian cells. Although microfluidic device design have allowed for generating stable, complex gradients of diffusible molecules, the flow-induced shear forces in a microchannel has made it impossible to maintain viable mammalian neuronal cultures for sufficiently long times. In this paper, we describe axonal responses of mouse cortical neurons in a "neuron-benign" gradient-generator device based on an open chamber that can establish highly stable gradients of diffusible molecules for at least 6 h with negligible shear stress, and also allows the neurons to thrive for at least 2 weeks. Except for the period when the gradient is on, the cells in the gradient are under the same conditions as the cells on the control surfaces, which ensure a consistent set of micro-environmental variables. The gradient stability and uniformity over the cell culture surface achieved by the device, together with our software platform for acquiring, post-processing and quantitatively analyzing the large number of images allowed us to extract valuable information even from small datasets. We report a directed response of primary mammalian neurons (from E14 embryonic mice cortex) to a diffusible gradient of netrin in vitro. We infer from our studies that a large majority (∼73%) of the neurons that extend axons during the gradient application grow towards the netrin source, and our data analysis also indicates that netrin acts as a growth factor for this same population of neurons.

Electrochemically programmed release of biomolecules and nanoparticles.

The controlled release of molecules or nanoparticle conjugates is an important tool for a wide range of applications in science and engineering. Here we demonstrate electrochemically programmed release of biomolecules and nanoparticles immobilized on patterned gold electrodes using the thiol-gold linkage. This technique exploits the reductive desorption of self-assembled monolayers and allows both spatially controlled release and regeneration of small molecules (e.g., drugs), biopolymers (e.g., peptides, proteins, DNA), protein assemblies (e.g., viruses), and nanoparticles (e.g., particle-DNA conjugates). Fluorescence microscopy is used to image the release of avidin and nanoparticles in phosphate-buffered saline and to determine the kinetics of desorption. We also demonstrate that the electrodes can be regenerated using the same conjugation scheme.