Flow focusing through gels as a tool to generate 3D concentration profiles in hydrogel-filled microfluidic chips

Laminar flow patterning is an iconic microfluidic technology used to deliver chemicals to specific regions on a two-dimensional surface with high spatial fidelity. Here we present a novel extension of this technology using Darcy flow within a three-dimensional (3D) hydrogel. Our test device is a simple 3-inlet microfluidic channel, totally filled with collagen, a cured biological hydrogel, where the concentration profiles of solutes are manipulated via the inlet pressures. This method allows solutes to be delivered with 50 micron accuracy within the gel, as we evidence by controlling concentration profiles of 40 kDa and 1 kDa fluorescent polysaccharide dyes. Furthermore, we design and test a 3D-printed version of our device with an extra two inlets for control of the vertical position of the concentration profile, demonstrating that this method is easily extensible to control of the concentration profile in 3D

Configurable gel geometry via flow patterning for angiogenisis assays

Here we introduce a versatile flow patterning technique (Fig1b) for pillar-less patterning of straight (Fig3) and highly curved (Fig2) gel geometries to test a wide range of in vivo-like vessel sprouting conditions. Much current research focuses on implementing angiogenesis on Organ-on-Chip (OOC) platforms for investigation of the role of signaling proteins[1] and the role of angiogenesis in cancer treatment[2]. Our technique avoids using pillars to confine the gel (Fig 1a) and thereby reduces the contact area between the growing spouts and the unnaturally stiff chip material which is known to affect migration rate of adherent cell types[4]

Chipholder-integrated 3d-printed pneumatic logic controllers for microfluidic chips

Here we design, develop and test 3D printed pneumatic controllers which can be easily built into chip holders for microfluidic devices. On-chip pneumatic controllers are used for microfluidic process automation to reduce the complexity of chip interfacing and off-chip control.[1] Commonly, on-chip elastomeric valves are used [2] but these cost chip real-estate, and often require special lithographic techniques and multilayer device design [2,3]. 3Dprinted valves are a recent development.[5] We present an improved fabrication technique for 3D-printed valves and use them to construct logic control platforms which can be used on any chip with the correct fittings

Patterning Biological Gels for 3D Cell Culture inside Microfluidic Devices by Local Surface Modification through Laminar Flow Patterning

Microfluidic devices are used extensively in the development of new in vitro cell culture models like organs-on-chips. A typical feature of such devices is the patterning of biological hydrogels to offer cultured cells and tissues a controlled three-dimensional microenvironment. A key challenge of hydrogel patterning is ensuring geometrical confinement of the gel, which is generally solved by inclusion of micropillars or phaseguides in the channels. Both of these methods often require costly cleanroom fabrication, which needs to be repeated even when only small changes need be made to the gel geometry, and inadvertently expose cultured cells to non-physiological and mechanically stiff structures. Here, we present a technique for facile patterning of hydrogel geometries in microfluidic chips, but without the need for any confining geometry built into the channel. Core to the technique is the use of laminar flow patterning to create a hydrophilic path through an otherwise hydrophobic microfluidic channel. When a liquid hydrogel is injected into the hydrophilic region, it is confined to this path by the surrounding hydrophobic regions. The various surface patterns that are enabled by laminar flow patterning can thereby be rendered into three-dimensional hydrogel structures. We demonstrate that the technique can be used in many different channel geometries while still giving the user control of key geometric parameters of the final hydrogel. Moreover, we show that human umbilical vein endothelial cells can be cultured for multiple days inside the devices with the patterned hydrogels and that they can be stimulated to migrate into the gel under the influence of trans-gel flows. Finally, we demonstrate that the patterned gels can withstand trans-gel flow velocities in excess of physiological interstitial flow velocities without rupturing or detaching. This novel hydrogel-patterning technique addresses fundamental challenges of existing methods for hydrogel patterning inside microfluidic chips, and can therefore be applied to improve design time and the physiological realism of microfluidic cell culture assays and organs-on-chips

Flow Through Gels as a Tool to Generate 3D Concentration Profiles in Hydrogel-Filled Devices

Laminar flow patterning is an iconic microfluidic technology used to locally deliver chemicals to specific regions on a two-dimensional surface with high spatial fidelity. Here we present a novel extension of this technology by flow patterning inside cured, three-dimensional (3D) hydrogels. We control the pressures at the inlets to shape the size and position of a region of high solute concentration in the main channel filled with a cured collagen 1 hydrogel. This method allows solutes to be delivered with 50 micron accuracy within the gel, as evidenced by control of concentration profiles of 40 kDa fluorescent dye

Diffusion from steady-state profile (DSSP) for low cost, low concentration measurement of diffusion

Here we present Diffusion from Steady-State Profile (DSSP), a simple, low-cost technique to measure the diffusivity of labeled proteins in hydrogels that are typically used for 3D cell culture. This is a steady-state technique which allows the use of long-exposure imaging, thereby enabling operation with low protein concentrations without the need for relatively expensive imaging equipment or immunoassays

A Multiplexable Plasmonic Hairpin-DNA Sensor Based On Target-specific Tether Dynamics

The need for measurements of multiple biomarkers simultaneously at subnanomolar concentrations asks for the development of new sensors with high sensitivity, specificity, precision, and accuracy. Currently, multiplexed sensing in single molecule sensors increases the complexity of the system in terms of reagents and sample read-out. In this letter, we propose a novel approach to multiplex hairpin-based single-DNA molecule sensors, which overcomes the limitations of the present approaches for multiplexing. By target-dependent ssDNA hairpin design, we can create DNA tethers that have distinct tether dynamics upon target binding. Our numerical model shows that by changing the stem length of the ssDNA hairpin, significantly different dynamic tether behavior will be observed. By exploiting the distance-dependent coupling of AuNPs to gold films, we can probe this dynamic behavior along the z-axis using a simple laser equipped microscope

A metastasis-on-a-chip approach to explore the sympathetic modulation of breast cancer bone metastasis

Organ-on-a-chip models have emerged as a powerful tool to model cancer metastasis and to decipher specific crosstalk between cancer cells and relevant regulators of this particular niche. Recently, the sympathetic nervous system (SNS) was proposed as an important modulator of breast cancer bone metastasis. However, epidemiological studies concerning the benefits of the SNS targeting drugs on breast cancer survival and recurrence remain controversial. Thus, the role of SNS signaling over bone metastatic cancer cellular processes still requires further clarification. Herein, we present a novel humanized organ-on-a-chip model recapitulating neuro-breast cancer crosstalk in a bone metastatic context. We developed and validated an innovative three-dimensional printing based multi-compartment microfluidic platform, allowing both selective and dynamic multicellular paracrine signaling between sympathetic neurons, bone tropic breast cancer cells and osteoclasts. The selective multicellular crosstalk in combination with biochemical, microscopic and proteomic profiling show that synergistic paracrine signaling from sympathetic neurons and osteoclasts increase breast cancer aggressiveness demonstrated by augmented levels of pro-inflammatory cytokines (e.g. interleukin-6 and macrophage inflammatory protein 1α). Overall, this work introduced a novel and versatile platform that could potentially be used to unravel new mechanisms involved in intracellular communication at the bone metastatic niche