Selective Fluorination of the Surface of Polymeric Materials after Stereolithography 3D Printing.

With the microfluidics community embracing 3D resin printing as a rapid fabrication method, controlling surface chemistry has emerged as a new challenge. Fluorination of 3D-printed surfaces is highly desirable in many applications due to chemical inertness, low friction coefficients, antifouling properties, and the potential for selective hydrophobic patterning. Despite sporadic reports, silanization methods have not been optimized for covalent bonding with polymeric resins. As a case study, we tested the silanization of a commercially available (meth)acrylate-based resin (BV-007A) with a fluoroalkyl trichlorosilane. Interestingly, plasma oxidation was unnecessary for silanization of this resin and indeed was ineffective. Solvent-based deposition in a fluorinated oil (FC-40) generated significantly higher contact angles than deposition in ethanol or gas-phase deposition, yielding hydrophobic surfaces with contact angle >110° under optimized conditions. Attenuated total reflectance-Fourier transform infrared spectroscopy indicated that the increase in the contact angle correlated with consumption of a carbonyl moiety, suggesting covalent bonding of silane without plasma oxidation. Consistent with a covalent bond, silanization was resistant to mechanical damage and hydrolysis in methanol and was stable over long-term storage. When tested on a suite of photocrosslinkable resins, this silanization protocol generated highly hydrophobic surfaces (contact angle > 110°) on three resins and moderate hydrophobicity (90-100°) on the remainder. Selective patterning of hydrophobic regions in an open 3D-printed microchannel was possible in combination with simple masking techniques. Thus, this facile fluorination strategy is expected to be applicable for resin-printed materials in a variety of contexts including micropatterning and multiphase microfluidics.

Harmonic dependence of thermal magnetic particle imaging.

Advances in instrumentation and tracer materials are still required to enable sensitive, accurate, and localized in situ 3D temperature monitoring by magnetic particle imaging (MPI). We have developed a high-resolution magnetic particle imaging instrument and implemented a low-noise multi-harmonic lock-in detection method to observe and quantify temperature variations in iron oxide nanoparticle tracers using the harmonic ratio method for determining temperature. Using isolated harmonics for MPI and temperature imaging revealed an apparent dependence of imaging resolution on harmonic number. Thus, we present experimental and simulation studies to quantify the imaging resolution dependence on temperature and harmonic number, and directly validate the fundamental origin of MPI imaging resolution on harmonic number based on the concept of a harmonic point-spread-function.

Applied tutorial for the design and fabrication of biomicrofluidic devices by resin 3D printing.

Resin 3D printing, especially digital light processing (DLP) printing, is a promising rapid fabrication method for bio-microfluidic applications such as clinical tests, lab-on-a-chip devices, and sensor integrated devices. The benefits of 3D printing lead many to believe this fabrication method will accelerate the use of microfluidics, but there are a number of potential obstacles to overcome for bioanalytical labs to fully utilize this technology. For commercially available printing materials, this includes challenges in producing prints with the print resolution and mechanical stability required for a particular design, along with cytotoxic components within many photopolymerizing resins and low optical compatibility for imaging experiments. Potential solutions to these problems are scattered throughout the literature and rarely available in head-to-head comparisons. Therefore, we present here a concise guide to the principles of resin 3D printing most relevant for fabrication of bioanalytical microfluidic devices. Intended to quickly orient labs that are new to 3D printing, the tutorial includes the results of selected systematic tests to inform resin selection, strategies for design optimization, and improvement of biocompatibility of resin 3D printed bio-microfluidic devices.

Rapid Fabrication by Digital Light Processing 3D Printing of a SlipChip with Movable Ports for Local Delivery to Ex Vivo Organ Cultures.

SlipChips are two-part microfluidic devices that can be reconfigured to change fluidic pathways for a wide range of functions, including tissue stimulation. Currently, fabrication of these devices at the prototype stage requires a skilled microfluidic technician, e.g., for wet etching or alignment steps. In most cases, SlipChip functionality requires an optically clear, smooth, and flat surface that is fluorophilic and hydrophobic. Here, we tested digital light processing (DLP) 3D printing, which is rapid, reproducible, and easily shared, as a solution for fabrication of SlipChips at the prototype stage. As a case study, we sought to fabricate a SlipChip intended for local delivery to live tissue slices through a movable microfluidic port. The device was comprised of two multi-layer components: an enclosed channel with a delivery port and a culture chamber for tissue slices with a permeable support. Once the design was optimized, we demonstrated its function by locally delivering a chemical probe to slices of hydrogel and to living tissue with up to 120 µm spatial resolution. By establishing the design principles for 3D printing of SlipChip devices, this work will enhance the ability to rapidly prototype such devices at mid-scale levels of production.

Spatially resolved measurement of dynamic glucose uptake in live ex vivo tissues.

Highly proliferative cells depend heavily on glycolysis as a source of energy and biological precursor molecules, and glucose uptake is a useful readout of this aspect of metabolic activity. Glucose uptake is commonly quantified by using flow cytometry for cell cultures and positron emission tomography for organs in vivo. However, methods to detect spatiotemporally resolved glucose uptake in intact tissues are far more limited, particularly those that can quantify changes in uptake over time in specific tissue regions and cell types. Using lymph node metabolism as a case study, we developed an optimized method to detect dynamic and spatially resolved glucose uptake in living tissue by combining ex vivo tissue slice culture with a fluorescent glucose analogue. Live slices of murine lymph node were treated with the glucose analogue 2-[N-(7-nitrobenz-2-oxa-1,3-dia-xol-4-yl)amino]-2-deoxyglucose (2-NBDG). Incubation parameters were optimized to differentiate glucose uptake in activated versus naïve lymphocytes. Regional glucose uptake could be imaged at both the tissue level, by widefield microscopy, and at the cellular level, by confocal microscopy. Furthermore, the glucose assay was readily multiplexed with live immunofluorescence labelling to generate maps of 2-NBDG uptake across tissue regions, revealing highest uptake in T cell-dense regions. The signal was predominantly intracellular and localized to lymphocytes rather than stromal cells. Finally, we demonstrated that the assay was repeatable in the same slices, and imaged the dynamic distribution of glucose uptake in response to ex vivo T cell stimulation for the first time. We anticipate that this method will serve as a broadly applicable, user-friendly platform to quantify dynamic metabolic activities in complex tissue microenvironments.

Acute Lymph Node Slices Are a Functional Model System to Study Immunity Ex Vivo.

The lymph node is a highly organized and dynamic structure that is critical for facilitating the intercellular interactions that constitute adaptive immunity. Most ex vivo studies of the lymph node begin by reducing it to a cell suspension, thus losing the spatial organization, or fixing it, thus losing the ability to make repeated measurements. Live murine lymph node tissue slices offer the potential to retain spatial complexity and dynamic accessibility, but their viability, level of immune activation, and retention of antigen-specific functions have not been validated. Here we systematically characterized live murine lymph node slices as a platform to study immunity. Live lymph node slices maintained the expected spatial organization and cell populations while reflecting the 3D spatial complexity of the organ. Slices collected under optimized conditions were comparable to cell suspensions in terms of both 24-h viability and inflammation. Slices responded to T cell receptor cross-linking with increased surface marker expression and cytokine secretion, in some cases more strongly than matched lymphocyte cultures. Furthermore, slices processed protein antigens, and slices from vaccinated animals responded to ex vivo challenge with antigen-specific cytokine secretion. In summary, lymph node slices provide a versatile platform to investigate immune functions in spatially organized tissue, enabling well-defined stimulation, time-course analysis, and parallel read-outs.

User-defined local stimulation of live tissue through a movable microfluidic port.

Many in vivo tissue responses begin locally, yet most in vitro stimuli are delivered globally. Microfluidics has a unique ability to provide focal stimulation to tissue samples with precise control over fluid location, flow rate, and composition. However, previous devices utilizing fixed ports beneath the tissue required manual alignment of the tissue over the ports, increasing the risk of mechanical damage. Here we present a novel microfluidic device that allows the user to define the location of fluid delivery to a living tissue slice without manipulating the tissue itself. The device utilized a two-component SlipChip design to create a mobile port beneath the tissue slice. A culture chamber perforated by an array of ports housed a tissue slice and was separated by a layer of fluorocarbon oil from a single delivery port, fed by a microfluidic channel in the movable layer below. We derived and validated a physical model, based on interfacial tension and flow resistance, to predict the conditions under which fluid delivery occurred without leakage into the gap between layers. Aqueous solution was delivered reproducibly to samples of tissue and gel, and the width of the delivery region was controlled primarily by convection. Tissue slice viability was not affected by stimulation on the device. As a proof-of-principle, we showed that live slices of lymph node tissue could be sequentially targeted for precise stimulation. In the future this device may serve as a platform to study the effects of fluid flow in tissues and to perform local drug screening.