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Engineering vasculature networks in physiologically relevant hydrogelsrepresents a challenge in terms of both fabrication, due to the cell–bioinkinteractions, as well as the subsequent hydrogel-device interfacing. Here, anew cell-friendly fabrication strategy is presented to realize perfusablemulti-hydrogel vasculature models supporting co-culture integrated in amicrofluidic chip. The system comprises two different hydrogels to specificallysupport the growth and proliferation of two different cell types selected for thevessel model. First, the channels are printed in a gelatin-based ink bytwo-photon polymerization (2PP) inside the microfluidic device. Then, ahuman lung fibroblast-laden fibrin hydrogel is injected to surround the printednetwork. Finally, human endothelial cells are seeded inside the printedchannels. The printing parameters and fibrin composition are optimized toreduce hydrogel swelling and ensure a stable model that can be perfused withcell media. Fabricating the hydrogel structure in two steps ensures that nocells are exposed to cytotoxic fabrication processes, while still obtaining highfidelity printing. In this work, the possibility to guide the endothelial cellinvasion through the 3D printed scaffold and perfusion of the co-culturemodel for 10 days is successfully demonstrated on a custom-made perfusionsystem.

In healthy individuals, the intestinal epithelium forms a tight barrier to prevent gut bacteria from reaching blood circulation. To study the effect of probiotics, dietary compounds and drugs on gut barrier formation and disruption, human gut epithelial and bacterial cells can be cocultured in an in vitro model called the human microbial crosstalk (HuMiX) gut-on-a-chip system. Here, we present the design, fabrication and integration of thin-film electrodes into the HuMiX platform to measure transepithelial electrical resistance (TEER) as a direct readout on barrier tightness in real-time. As various aspects of the HuMiX platform have already been set in their design, such as multiple compressible layers, uneven surfaces and nontransparent materials, a novel fabrication method was developed whereby thin-film metal electrodes were first deposited on flexible substrates and sequentially integrated with the HuMiX system via a transfer-tape approach. Moreover, to measure localized TEER along the cell culture chamber, we integrated multiple electrodes that were connected to an impedance analyzer via a multiplexer. We further developed a dynamic normalization method because the active measurement area depends on the measured TEER levels. The fabrication process and system setup can be applicable to other barrier-on-chip systems. As a proof-of-concept, we measured the barrier formation of a cancerous Caco-2 cell line in real-time, which was mapped at four spatially separated positions along the HuMiX culture area.

The high mortality associated with certain cancers can be attributed to the invasive nature of the tumor cells. Yet, the complexity of studying invasion hinders our understanding of how the tumor spreads. This work presents a microengineered three-dimensional (3D) in vitro model for studying cancer cell invasion and interaction with endothelial cells. The model was generated by printing a biomimetic hydrogel scaffold directly on a chip using 2-photon polymerization that simulates the brain's extracellular matrix. The scaffold's geometry was specifically designed to facilitate the growth of a continuous layer of endothelial cells on one side, while also allowing for the introduction of tumor cells on the other side. This arrangement confines the cells spatially and enables in situ microscopy of the cancer cells as they invade the hydrogel scaffold and interact with the endothelial layer. We examined the impact of 3D printing parameters on the hydrogel's physical properties and used patient derived glioblastoma cells to study their effect on cell invasion. Notably, the tumor cells tended to infiltrate faster when an endothelial cell barrier was present. The potential for adjusting the hydrogel scaffold's properties, coupled with the capability for real-time observation of tumor-endothelial cell interactions, offers a platform for studying tumor invasion and tumor–endothelial cell interactions.

In disciplines like toxicology and pharmacology, oxygen (O-2) respiration is a universal metric for evaluating the effects of chemicals across various model systems, including mammalian and microalgal cells. However, for these cells the common practice is to segregate populations into control and exposure groups, which assumes direct equivalence in their responses and does not take into account heterogeneity among individual cells. This lack of resolution impedes our ability to precisely investigate differences among experimental groups with small or limited sample sizes. To overcome this barrier, we introduce SlipO(2)Chip, an innovative glass microfluidic platform for precisely quantifying single-cell O-2 respiration in the coordinated absence and presence of chemical solutes. SlipO(2)Chip comprises a wet-etched fused silica channel plate on the top and a dry-etched borosilicate microwell plate at the bottom. The microwells are coated with Pt(ii) meso-tetra(pentafluorophenyl)porphine (PtTFPP), an O-2 sensing optode material and an O-2-independent reference dye. A custom 3D-printed holder facilitates the controlled horizontal movement ('slipping') of the channel plate over the microwell plate, thereby establishing or disrupting the fluid path over microwells. Collectively, these design elements enable the immobilization of single-cells in microwells, their exposure to controlled fluid flows, the coordinated opening and closing of microwells and repeated measurements of single-cell O-2 respiration. Uniquely, by sequentially executing opening and closing it becomes possible to measure single-cell respiration prior to and after exposure to chemical solutes. In a proof-of-concept application, we utilized SlipO(2)Chip to measure the impact of increasing exposures of the marine bacterial signal 2-heptyl-4-quinolone (HHQ) on the dark respiration of the diatom Ditylum brightwellii at single-cell resolution. Results revealed a concentration-dependent decrease in per-cell O-2 dark respiration, with a maximum reduction of 40.2% observed at HHQ concentrations exceeding 35.5 mu M, and a half-maximal effective concentration (EC50) of 5.8 mu M, consistent with that obtained via conventional bulk respiration methods. The ability of SlipO(2)Chip to sequentially assess the effects of chemical substances on single-cell O-2 metabolism is advantageous for research where sample volumes are limited, such as clinical biopsies, studies involving rare microbial isolates, and toxicological studies aiming to address exposure effects while accounting for cell-to-cell variability.<br />

Droplet-based microfluidics involves the generation of discrete volumes in a continuous and immiscible phase. It is an attractive technique for biological, especially single-cell level, assays due to its ability to encapsulate particles and cells within individual reaction vessels at high throughput. Bulk-wave-acoustophoresis is a contactless and label free method to spatially localize particles. It has emerged as a suitable technique to concentrate and separate droplet contents on-chip. In traditional droplet microfluidics for biological applications fluorinated oils, e.g. Novec HFE-7500, are favored due to insolubility for most organic compounds, which prevents cross-contamination between droplets and their high gas permeability. However, a strong and uniform field cannot be formed inside aqueous droplets generated in HFE-7500 due to a mismatch in the acoustic properties of the two phases. Previously, the individual contributions of the acoustic properties, i.e. density, compressibility, and speed of sound, to the focusing quality were not fully investigated. We demonstrate in this work, that the speed of sound dominates the focusing performance. It is shown experimentally and numerically that matching the speed of sound between continuous and discrete phase is paramount to high quality acoustic focusing. To this end the acoustic focusing of polystyrene particles inside aqueous droplets immersed in different continuous phase fluids (hydrocarbon oil, fluorinated oil and silicone oil) is investigated. We demonstrate that a match in the speed of sound between the two phases results in higher droplet-internal particle focusing quality.

Droplet-based microfluidics involves the generation of discrete volumes of a dispersed phase (often water) in a continuous and immiscible phase (oil). Bulk acoustic wave acoustophoresis can be combined with droplet microfluidics for droplet handling, or handling of droplet content. Fluorinated oils, e.g. Novec HFE-7500, possess properties such as high permeability for gases and insolubility for most organic compounds, which makes them favorable for droplet-based applications. However, it has been observed that a deterioration in the average magnitude of the primary acoustic radiation force inside water droplets generated in HFE-7500 results in poor acoustophoretic focusing quality [2,3]. As a follow up to these studies, we compared the droplet internal acoustophoretic focusing performance inside aqueous droplets generated in a selection of oils that have previously been used in two-phase applications [4]. Based on these experiments, we conclude that a match in the speed of sound between the dispersed and continuous phase is paramount to high quality acoustic focusing. Here, we present simulation results of four selected cases, aqueous droplets immersed in linseed oil, light mineral oil, silicone oil 50 cSt and HFE-7500, and offer a theoretical explanation to why it is speed of sound that dominates acoustophoretic focusing performance.

The ability to manipulate particles or cells inside water-in-oil droplets is essential to various chemical, biological and biomedical assays performed using droplet microfluidics systems. Bulk acoustophoresis permits spatial localization of cells and particles in a label-free and non-contact manner by utilizing acoustic standing wave and is therefore a suitable particle manipulation method for integration with droplet microfluidics. Fluorinated oils, e.g., Novec HFE-7500, are most commonly used as the continuous phase in typical droplet applications as they are permeable to gases and insoluble for most organic compounds. Previous studies, however, have concluded that sufficient acoustic focusing capability cannot be achieved inside aqueous droplets generated in HFE-7500 due to a mismatch in the acoustic properties of the two phases used. The critical acoustic properties that underlie this difference in acoustic focusing capability were however not identified. This work presents a study where the suitability of a selection of oils, from three categories commonly used in droplet microfluidics (hydrocarbon oil, fluorinated oil and silicone oil), was examined for acoustic particle manipulation in water-in-oil droplets. We hypothesized that when performing acoustic focusing inside aqueous droplets confined in a microfluidic channel (i.e., plugs), due to the presence of a thin oil film between the channel wall and the droplet, the acoustic impedance between the two fluid phases must match in order to increase acoustic coupling efficiency between the two liquid phases. Through this experimental work, we demonstrate that hydrocarbon oils are most compatible with acoustic focusing in a two-phase system that generates channel-confined water-in-oil droplets due to matching acoustic properties with water. We also conclude that acoustic impedance matching between the two fluid phases is not critical for optimal droplet-internal particle manipulation. Our results suggest that a match in the speed of sound of the two phases may be more relevant in practice.

Since its introduction in the 1980s, 3D printing has advanced as a versatile and reliable tool with applications in different fields. Among the available 3D printing techniques, two-photon polymerization is regarded as one of the most promising technologies for microscale printing due to its ability to combine a high printing fidelity down to submicron scale with free-form structure design. Recently, the technology has been enhanced through the implementation of faster laser scanning strategies, as well as the development of new photoresists. This paves the way for a wide range of applications, which has resulted in an increasing number of available commercial systems. This work aims to provide an overview of the technology capability by comparing three commercial systems in a round-robin test. To cover a wide range of applications, six test structures with distinct features were designed, covering various aspects of interest, from single material objects with sub-micron feature sizes up to multi-material millimeter-sized objects. Application-specific structures were printed to evaluate surface roughness and the stitching capability of the printers. Moreover, the ability to generate free-hanging structures and complex surfaces required for cell scaffolds and microfluidic platform fabrication was quantitatively investigated. Finally, the influence of the numerical aperture of the fabrication objective on the printing quality was assessed. All three printers successfully fabricated samples comprising various three-dimensional features and achieved submicron resolution and feature sizes, demonstrating the versatility and precision of two-photon polymerization direct laser writing. Our study will facilitate the understanding of the technology maturity level, while highlighting specific aspects that characterize each of the investigated systems.