Confinement dependent chemotaxis in two-photon polymerized linear migration constructs with highly definable concentration gradients.

Dendritic cell chemotaxis is known to follow chemoattractant concentration gradients through tissue of heterogeneous pore sizes, but the dependence of migration velocity on pore size and gradient steepness is not fully understood. We enabled chemotaxis studies for at least 42 hours at confinements relevant to tissue models by two-photon polymerization of linear channel constructs with cross-sections from 10 × 10 µm(2) to 20 × 20 µm(2) inside commercially available chemotaxis analysis chips. Faster directed migration was observed with decreasing channel dimensions despite substantial cell deformation in the narrower channels. Finite element modeling of a cell either partly or fully obstructing chemokine diffusion in the narrow channels revealed strong local accentuation of the chemokine concentration gradients. The modeled concentration differences across a cell correlated well with the observed velocity dependence on channel cross-section. However, added effects due to spatial confinement could not be excluded. The design freedom offered by two-photon polymerization was exploited to minimize the accentuated concentration gradients in cell-blocked channels by introducing "venting slits" to the surrounding medium at a length scale too small (≤500 nm) for the cells to explore, thereby decoupling effects of concentration gradients and spatial confinement. Studies in slitted 10 × 10 µm(2) channels showed significantly reduced migration speeds indistinguishable from speeds observed in unslitted 20 × 20 µm(2) channel. This result agrees with model predictions of very small concentration gradient variations in slitted channels, thus indicating a strong influence of the concentration gradient steepness, not the channel size, on the directed migration velocity.

Facile photoimmobilization of proteins onto low-binding PEG-coated polymer surfaces.

Immobilization of proteins onto polymer surfaces usually requires specific reactive functional groups. Here, we show an easy one-step method to conjugate protein covalently onto almost any polymer surface, including low protein-binding poly(ethylene glycol) (PEG), without the requirement for the presence of specific functional groups. Several types of proteins, including alkaline phosphatase, bovine serum albumin, and polyclonal antibodies, were photoimmobilized onto a PEG-coated polymer surface using a water-soluble benzophenone as photosensitizer. Protein functionality after immobilization was verified for both enzymes and antibodies, and their presence on the surface was confirmed by X-ray photoelectron spectroscopy (XPS) and confocal fluorescence microscopy. Conjugation of capture antibody onto the PEG coating was employed for a simplified ELISA protocol without the need for blocking uncoated surface areas, showing ng/mL sensitivity to a cytokine antigen target. Moreover, spatially patterned attachment of fluorescently labeled protein onto the low-binding PEG-coated surface was achieved with a projection lithography system that enabled the creation of micrometer-sized protein features.

Human cytomegalovirus chemokine receptor US28 induces migration of cells on a CX3CL1-presenting surface.

Human cytomegalovirus (HCMV)-encoded G protein-coupled-receptor US28 is believed to participate in virus dissemination through modulation of cell migration and immune evasion. US28 binds different CC chemokines and the CX3C chemokine CX3CL1. Membrane-anchored CX3CL1 is expressed by immune-activated endothelial cells, causing redirection of CX3CR1-expressing leukocytes in the blood to sites of infection. Here, we used stable transfected cell lines to examine how US28 expression affects cell migration on immobilized full-length CX3CL1, to model how HCMV-infected leukocytes interact with inflamed endothelium. We observed that US28-expressing cells migrated more than CX3CR1-expressing cells when adhering to immobilized CX3CL1. US28-induced migration was G protein-signalling dependent and was blocked by the phospholipase Cß inhibitor U73122 and the intracellular calcium chelator BAPTA-AM. In addition, migration was inhibited in a dose-dependent manner by competition from CCL2 and CCL5, whereas CCL3 had little effect. Instead of migrating, CX3CR1-expressing cells performed 'dancing-on-the-spot' movements, demonstrating that anchored CX3CL1 acts as a strong tether for these cells. At low receptor expression levels, however, no significant difference in migration potential was observed when comparing the migration of CX3CR1- and US28-expressing cells. Thus, these data showed that, in contrast to CX3CR1, which promotes efficient cell capture upon binding to anchored CX3CL1, US28 acts to increase the migration of cells upon binding to the same ligand. Overall, this indicates that infected cells probably move more than uninfected cells in inflamed tissues with high CX3CL1 expression, with soluble chemokines affecting the final migration.

Exploration of two methods for quantitative Mitomycin C measurement in tumor tissue in vitro and in vivo.

: Two methods of quantifying Mitomycin C in tumor tissue are explored. A method of ultraviolet-visible absorption microscopy is developed and applied to measure the concentration of Mitomycin C in preserved mouse tumor tissue, as well as in gelatin samples. Concentrations as low as 60 µM can be resolved using this technique in samples that do not strongly scatter light. A novel method for monitoring the Mitomycin C concentrations inside a tumor is developed, based on microdialysis and ultraviolet-visible spectroscopy. A pump is used to perfuse a microdialysis probe with Ringer's solution, which is fed to a flow cell to determine intratumor concentrations in real time to within a few µM. The success and limitations of these techniques are identified, and suggestions are made as to further development. To the authors' knowledge these are the first attempts made to quantify Mitomycin C concentrations in tumor tissue.

Single-molecule denaturation mapping of DNA in nanofluidic channels.

Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent "barcode" corresponding to a series of local dips and peaks in the intensity trace along the extended molecule. We demonstrate that this structure arises from the physics of local denaturation: statistical mechanical calculations of sequence-dependent melting probability can predict the barcode to be observed experimentally for a given sequence. Consequently, the technique is sensitive to sequence variation without requiring enzymatic labeling or a restriction step. This technique may serve as the basis for a new mapping technology ideally suited for investigating the long-range structure of entire genomes extracted from single cells.

Micro-drilling of polymer tubular ultramicroelectrode arrays for electrochemical sensors.

We present a reproducible fast prototyping procedure based on micro-drilling to produce homogeneous tubular ultramicroelectrode arrays made from poly(3,4-ethylenedioxythiophene) (PEDOT), a conductive polymer. Arrays of Ø 100 µm tubular electrodes each having a height of 0.37 ± 0.06 µm were reproducibly fabricated. The electrode dimensions were analyzed by SEM after deposition of silver dendrites to visualize the electroactive electrode area. The electrochemical applicability of the electrodes was demonstrated by voltammetric and amperometric detection of ferri-/ferrocyanide. Recorded signals were in agreement with results from finite element modelling of the system. The tubular PEDOT ultramicroelectrode arrays were modified by prussian blue to enable the detection of hydrogen peroxide. A linear sensor response was demonstrated for hydrogen peroxide concentrations from 0.1 mM to 1 mM.

3D microperfusion of mesoscale human microphysiological liver models improves functionality and recapitulates hepatic zonation.

Hepatic in vitro models that accurately replicate phenotypes and functionality of the human liver are needed for applications in toxicology, pharmacology and biomedicine. Notably, it has become clear that liver function can only be sustained in 3D culture systems at physiologically relevant cell densities. Additionally, drug metabolism and drug-induced cellular toxicity often follow distinct spatial micropatterns of the metabolic zones in the liver acinus, calling for models that capture this zonation. We demonstrate the manufacture of accurate liver microphysiological systems (MPS) via engineering of 3D stereolithography printed hydrogel chips with arrays of diffusion open synthetic vasculature channels at spacings approaching in vivo capillary distances. Chip designs are compatible with seeding of cell suspensions or preformed liver cell spheroids. Importantly, primary human hepatocytes (PHH) and hiPSC-derived hepatocyte-like cells remain viable, exhibit improved molecular phenotypes compared to isogenic monolayer and static spheroid cultures and form interconnected tissue structures over the course of multiple weeks in perfused culture. 3D optical oxygen mapping of embedded sensor beads shows that the liver MPS recapitulates oxygen gradients found in the acini, which translates into zone-specific acet-ami-no-phen toxicity patterns. Zonation, here naturally generated by high cell densities and associated oxygen and nutrient utilization along the flow path, is also documented by spatial proteomics showing increased concentration of periportal- versus perivenous-associated proteins at the inlet region and vice versa at the outlet region. The presented microperfused liver MPS provides a promising platform for the mesoscale culture of human liver cells at phenotypically relevant densities and oxygen exposures. STATEMENT OF SIGNIFICANCE: A full 3D tissue culture platform is presented, enabled by massively parallel arrays of high-resolution 3D printed microperfusion hydrogel channels that functionally mimics tissue vasculature. The platform supports long-term culture of liver models with dimensions of several millimeters at physiologically relevant cell densities, which is difficult to achieve with other methods. Human liver models are generated from seeded primary human hepatocytes (PHHs) cultured for two weeks, and from seeded spheroids of hiPSC-derived human liver-like cells cultured for two months. Both model types show improved functionality over state-of-the-art 3D spheroid suspensions cultured in parallel. The platform can generate physiologically relevant oxygen gradients driven by consumption rather than supply, which was validated by visualization of embedded oxygen-sensitive microbeads, which is exploited to demonstrate zonation-specific toxicity in PHH liver models.

Micropatterning of functional conductive polymers with multiple surface chemistries in register.

A versatile procedure is presented for fast and efficient micropatterning of multiple types of covalently bound surface chemistry in perfect register on and between conductive polymer microcircuits. The micropatterning principle is applied to several types of native and functionalized PEDOT (poly(3,4-ethylenedioxythiophene)) thin films. The method is based on contacting PEDOT-type thin films with a micropatterned agarose stamp containing an oxidant (aqueous hypochlorite) and applying a nonionic detergent. Where contacted, PEDOT not only loses its conductance but is entirely removed, thereby locally revealing the underlying substrate. Surface analysis showed that the substrate surface chemistry was fully exposed and not affected by the treatment. Click chemistry could thus be applied to selectively modify re-exposed alkyne and azide functional groups of functionalized polystyrene substrates. The versatility of the method is illustrated by micropatterning cell-binding RGD-functionalized PEDOT on low cell-binding PMOXA (poly(2-methyl-2-oxazoline)) to produce cell-capturing microelectrodes on a cell nonadhesive background in a few simple steps. The method should be applicable to a wide range of native and chemically functionalized conjugated polymer systems.

Directed self-organization of single DNA molecules in a nanoslit via embedded nanopit arrays.

We show that arrays of nanopit structures etched in a nanoslit can control the positioning and conformation of single DNA molecules in nanofluidic devices. By adjusting the spacing, organization and placement of the nanopits it is possible to immobilize DNA at predetermined regions of a device without additional chemical modification and achieve a high degree of control over local DNA conformation. DNA can be extended between two nanopits and in closely spaced arrays will self-assemble into "connect-the-dots" conformations consisting of locally pinned segments joined by fluctuating linkers. These results have broad implications for nanotechnology fields that require methods for the nanoscale positioning and manipulation of DNA.

Immobilization of Active Antibodies at Polymer Melt Surfaces during Injection Molding.

We demonstrate the transfer and immobilization of active antibodies from a low surface- energy mold surface to thermoplastic replica surfaces using injection molding, and we investigate the process at molecular scale. The transfer process is highly efficient, as verified by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) of the mold and replica surfaces. AFM analysis reveals partial nanometer-scale embedding of the protein into the polymer matrix as a possible mechanism of permanent immobilization. Replicas with rabbit anti-mouse IgG immobilized as capture antibody at the hot polymer melt surface during injection molding show similar affinity for their antigen (mouse IgG) in sandwich enzyme-linked immunosorbent assay (ELISA) as capture antibodies deposited by passive adsorption onto a bare thermoplastic replica. The transferred antibodies retain their functionality after incubation in serum-containing cell medium for >1 week. A mold coating time of 10 min prior to injection molding is sufficient for producing highly sensitive ELISA assays, thus enabling the short processing cycle times required for mass production of single-use biodevices relying on active immobilized antibodies.

Optical 4D oxygen mapping of microperfused tissue models with tunable in vivo-like 3D oxygen microenvironments.

Sufficient and controllable oxygen supply is essential for in vitro 3D cell and tissue culture at high cell densities, which calls for volumetric in situ oxygen analysis methods to quantitatively assess the oxygen distribution. This paper presents a general approach for accurate and precise non-contact 3D mapping of oxygen tension in high cell-density cultures via embedded commercially available oxygen microsensor beads read out by confocal phosphorescence lifetime microscopy (PLIM). Optimal acquisition conditions and data analysis procedures are established and implemented in a publicly available software package. The versatility of the established method is first demonstrated in model-assisted fluidic design of microperfused 3D printed hydrogel culture chips with the aim of full culture oxygenation, and subsequently for monitoring and maintenance of physiologically relevant spatial and temporal oxygen gradients in the 3D printed chips controlled by static or dynamic flow conditions during 3D culture.

Embedded 3D Printing in Self-Healing Annealable Composites for Precise Patterning of Functionally Mature Human Neural Constructs.

Human in vitro models of neural tissue with tunable microenvironment and defined spatial arrangement are needed to facilitate studies of brain development and disease. Towards this end, embedded printing inside granular gels holds great promise as it allows precise patterning of extremely soft tissue constructs. However, granular printing support formulations are restricted to only a handful of materials. Therefore, there has been a need for novel materials that take advantage of versatile biomimicry of bulk hydrogels while providing high-fidelity support for embedded printing akin to granular gels. To address this need, Authors present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self-Healing Annealable Particle-Extracellular matrix (SHAPE) composites. SHAPE composites consist of soft microgels immersed in viscous extracellular-matrix solution to enable precise and programmable patterning of human stem cells and consequent generation mature subtype-specific neurons that extend projections into the volume of the annealed support. The developed approach further allows multi-ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular-like channels. Due to its modularity and versatility, SHAPE biomanufacturing toolbox has potential to be used in applications beyond functional modeling of mechanically sensitive neural constructs.

Silk scaffolding drives self-assembly of functional and mature human brain organoids.

Human pluripotent stem cells (hPSCs) are intrinsically able to self-organize into cerebral organoids that mimic features of developing human brain tissue. These three-dimensional structures provide a unique opportunity to generate cytoarchitecture and cell-cell interactions reminiscent of human brain complexity in a dish. However, current in vitro brain organoid methodologies often result in intra-organoid variability, limiting their use in recapitulating later developmental stages as well as in disease modeling and drug discovery. In addition, cell stress and hypoxia resulting from long-term culture lead to incomplete maturation and cell death within the inner core. Here, we used a recombinant silk microfiber network as a scaffold to drive hPSCs to self-arrange into engineered cerebral organoids. Silk scaffolding promoted neuroectoderm formation and reduced heterogeneity of cellular organization within individual organoids. Bulk and single cell transcriptomics confirmed that silk cerebral organoids display more homogeneous and functionally mature neuronal properties than organoids grown in the absence of silk scaffold. Furthermore, oxygen sensing analysis showed that silk scaffolds create more favorable growth and differentiation conditions by facilitating the delivery of oxygen and nutrients. The silk scaffolding strategy appears to reduce intra-organoid variability and enhances self-organization into functionally mature human brain organoids.

Enhanced transduction of photonic crystal dye lasers for gas sensing via swelling polymer film.

We present the enhanced transduction of a photonic crystal dye laser for gas sensing via deposition of an additional swelling polymer film. Device operation involves swelling of the polymer film during exposure to specific gases, leading to a change in total effective refractive index. Experimental results show an enhancement of 16.09 dB in sensing ethanol vapor after deposition of a polystyrene film. We verify different responses of the polystyrene film when exposed to either ethanol vapor or increased humidity, indicating selectivity. The concept is generic and, in principle, straightforward in its application to other intracavity-based detection schemes to enable gas sensing.

Efficient large volume electroporation of dendritic cells through micrometer scale manipulation of flow in a disposable polymer chip.

We present a hybrid chip of polymer and stainless steel designed for high-throughput continuous electroporation of cells in suspension. The chip is constructed with two parallel stainless steel mesh electrodes oriented perpendicular to the liquid flow. The relatively high hydrodynamic resistance of the micrometer sized holes in the meshes compared to the main channel enforces an almost homogeneous flow velocity between the meshes. Thereby, very uniform electroporation of the cells can be accomplished. Successful electroporation of 20 million human dendritic cells with mRNA is demonstrated. The performance of the chip is similar to that of the traditional electroporation cuvette, but without an upper limit on the number of cells to be electroporated. The device is constructed with two female Luer parts and can easily be integrated with other microfluidic components. Furthermore it is fabricated from injection molded polymer parts and commercially available stainless steel mesh, making it suitable for inexpensive mass production.

Light-induced local heating for thermophoretic manipulation of DNA in polymer micro- and nanochannels.

We present a method for making polymer chips with a narrow-band near-infrared absorber layer that enables light-induced local heating of liquids inside fluidic micro- and nanochannels fabricated by thermal imprint in polymethyl methacrylate. We have characterized the resulting liquid temperature profiles in microchannels using the temperature dependent fluorescence of the complex [Ru(bpy)(3)](2+). We demonstrate thermophoretic manipulation of individual YOYO-1 stained T4 DNA molecules inside micro- and nanochannels.

Dual-Material 3D-Printed Intestinal Model Devices with Integrated Villi-like Scaffolds.

In vitro small intestinal models aim to mimic the in vivo intestinal function and structure, including the villi architecture of the native tissue. Accurate models in a scalable format are in great demand to advance, for example, the development of orally administered pharmaceutical products. Widely used planar intestinal cell monolayers for compound screening applications fail to recapitulate the three-dimensional (3D) microstructural characteristics of the intestinal villi arrays. This study employs stereolithographic 3D printing to manufacture biocompatible hydrogel-based scaffolds with villi-like micropillar arrays of tunable dimensions in poly(ethylene glycol) diacrylates (PEGDAs). The resulting 3D-printed microstructures are demonstrated to support a month-long culture and induce apicobasal polarization of Caco-2 epithelial cell layers along the villus axis, similar to the native intestinal microenvironment. Transport analysis requires confinement of compound transport to the epithelial cell layer within a compound diffusion-closed reservoir compartment. We meet this challenge by sequential printing of PEGDAs of different molecular weights into a monolithic device, where a diffusion-open villus-structured hydrogel bottom supports the cell culture and mass transport within the confines of a diffusion-closed solid wall. As a functional demonstrator of this scalable dual-material 3D micromanufacturing technology, we show that Caco-2 cells seeded in villi-wells form a tight epithelial barrier covering the villi-like micropillars and that compound-induced challenges to the barrier integrity can be monitored by standard high-throughput analysis tools (fluorescent tracer diffusion and transepithelial electrical resistance).

Complex surface concentration gradients by stenciled "electro click chemistry".

Complex one- or two-dimensional concentration gradients of alkynated molecules are produced on azidized conducting polymer substrates by stenciled "electro click chemistry". The latter describes the local electrochemical generation of catalytically active Cu(I) required to complete a "click reaction" between alkynes and azides at room temperature. A stencil on the counter electrode defines the shape and multiplicity of the gradient(s) on the conducting polymer substrate, while the specific reaction conditions control gradient steepness and the maximum concentration deposited. Biologically active ligands including cell binding peptides are patterned in gradients by this method without losing their biological function or the conductivity of the polymer.

Confinement spectroscopy: probing single DNA molecules with tapered nanochannels.

We demonstrate a confinement spectroscopy technique capable of probing small conformational changes of unanchored single DNA molecules in a manner analogous to force spectroscopy, in the regime corresponding to femtonewton forces. In contrast to force spectroscopy, various structural forms of DNA can easily be probed, as indicated by experiments on linear and circular DNA. The extension of circular DNA is found to scale according to the de Gennes exponent, unlike for linear DNA.

Leaky Optoelectrical Fiber for Optogenetic Stimulation and Electrochemical Detection of Dopamine Exocytosis from Human Dopaminergic Neurons.

In Parkinson's disease, the degeneration of dopaminergic neurons in substantia nigra leads to a decrease in the physiological levels of dopamine in striatum. The existing dopaminergic therapies effectively alleviate the symptoms, albeit they do not revert the disease progression and result in significant adverse effects. Transplanting dopaminergic neurons derived from stem cells could restore dopamine levels without additional motor complications. However, the transplanted cells disperse in vivo and it is not possible to stimulate them on demand to modulate dopamine release to prevent dyskinesia. In order to address these issues, this paper presents a multifunctional leaky optoelectrical fiber for potential neuromodulation and as a cell substrate for application in combined optogenetic stem cell therapy. Pyrolytic carbon coated optical fibers are laser ablated to pattern micro-optical windows to permit light leakage over a large area. The pyrolytic carbon acts as an excellent electrode for the electrochemical detection of dopamine. Human neural stem cells are genetically modified to express the light sensitive opsin channelrhodopsin-2 and are differentiated into dopaminergic neurons on the leaky optoelectrical fiber. Finally, light leaking from the micro-optical windows is used to stimulate the dopaminergic neurons resulting in the release of dopamine that is detected in real-time using chronoamperometry.