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Electroporation of dye-labeled bio-molecules into bacteria has proven to be a valuable route for single-molecule tracking in living cells. However, control over cell viability, electroporation efficiency, and environment conditions before, during, and after electroporation is difficult to achieve in bulk experiments. Here, a microfluidic platform is presented capable of single-cell electroporation with in situ microscopy and demonstrate delivery of DNA into bacteria. Via real time observation of the electroporation process, it is found that the effect of electrophoresis plays an important role when performing electroporation in a miniaturized platform and show that its undesired action can be balanced by using bipolar electrical pulses. It is suggested that a low temperature of the sample during electroporation is important for cell viability due to temperature-dependant viscoelastic properties of the cell membrane. It is further found that the presence of low conductive liquid between cells and the electrodes leads to a voltage divider effect that strongly influences the success of on-chip electroporation. Finally, it is concluded that electroporation is a highly stochastic process and envision that the microfluidic system presented here, capable of single-cell read-out, can be used for further fundamental studies to increase the understanding of the electroporation process in bacterial cells.

Culturing living cells in three-dimensional environments increases the biological relevance of laboratory experiments, but requires solutes to overcome a diffusion barrier to reach the centre of cellular constructs. We present a theoretical and numerical investigation that brings a mechanistic understanding of how microfluidic culture conditions, including chamber size, inlet fluid velocity and spatial confinement, affect solute distribution within three-dimensional cellular constructs. Contact with the chamber substrate reduces the maximally achievable construct radius by 15%. In practice, finite diffusion and convection kinetics in the microfluidic chamber further lower that limit. The benefits of external convection are greater if transport rates across diffusion-dominated areas are high. Those are omnipresent and include the diffusive boundary layer growing from the fluid-construct interface and regions near corners where fluid is recirculating. Such regions multiply the required convection to achieve a given solute penetration by up to 100, so chip designs ought to minimize them. Our results define conditions where complete solute transport into an avascular three-dimensional cell construct is achievable and applies to real chambers without needing to simulate their exact geometries.

Droplet-based microfluidics is a valuable tool in interdisciplinary research fields like cell biology and diagnostics. Newtonian fluids, like aqueous-based solutions, are commonly used for droplet generation. However, non-Newtonian fluids, e.g., hydrogels, are becoming increasingly popular as the dispersed phase. In this study, we investigate the dynamics of non-Newtonian ultra-low-gelling agarose droplet formation under different conditions to evaluate stability, with an aim to better understand the underlying physics of droplet formation. We varied the agarose gel concentration, temperature (40, 50, and 60 degrees C), and the flow rate ratio (phi) between the continuous and dispersed phase and observed droplet formation dynamics in the squeezing regime (capillary number, Ca-c < 0.015) in a T-junction under different flow conditions. We experimentally investigated the droplet size ( L (D) / w ) as a function of those four parameters and found that L- D / w depends strongly on phi, the agarose concentration, and temperature (which affects the viscosity ratio, lambda), but is only weakly dependent on Ca-c . We then confirmed our experimental findings with numerical simulations, which showed good agreement across all conditions. We numerically showed that the agarose droplet formation process consists of five stages, namely, filling, necking, pinching, threading, and breakup, where threading is an additional stage with a non-Newtonian dispersed phase. Finally, with numerical simulation, we concluded that threading length (l(thread )) is directly proportional to phi and has a complex relation with agarose concentration, and temperature.

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.