Droplet formation of biological non-Newtonian fluid in T-junction generators. I. Experimental investigation.

The extension of microfluidics to many bioassay applications requires the ability to work with non-Newtonian fluids. One case in point is the use of microfluidics with blood having different hematocrit levels. This work is the first part of a two-part study and presents the formation dynamics of blood droplets in a T-junction generator under the squeezing regime. In this regime, droplet formation with Newtonian fluids depends on T-junction geometry; however, we found that in the presence of the non-Newtonian fluid such as red blood cells, the formation depends on not only to the channel geometry, but also the flow rate ratio of fluids, and the viscosity of the phases. In addition, we analyzed the impact of the red blood cell concentration on the formation cycle. In this study, we presented the experimental data of the blood droplet evolution through the analysis of videos that are captured by a high-speed camera. During this analysis, we tracked several parameters such as droplet volume, spacing between droplets, droplet generation frequency, flow conditions, and geometrical designs of the T junction. Our analysis revealed that, unlike other non-Newtonian fluids, where the fourth stage exists (stretching stage), the formation cycle consists of only three stages: lag, filling, and necking stages. Because of the detailed analysis of each stage, a mathematical model can be generated to predict the final volume of the blood droplet and can be utilized as a guide in the operation of the microfluidic device for biochemical assay applications; this is the focus of the second part of this study [Phys. Rev. E 105, 025106 (2022)10.1103/PhysRevE.105.025106].

Droplet formation of biological non-Newtonian fluid in T-junction generators. II. Model for final droplet volume prediction.

This work represents the second part of a two-part series on the dynamics of droplet formation in a T-junction generator under the squeezing regime when using solutions of red blood cells as the dispersed phase. Solutions containing red blood cells are non-Newtonian; however, these solutions do not behave in the same way as other non-Newtonian fluids currently described in the literature. Hence, available models do not capture nor predict important features useful for the design of T-junction microfluidic systems, including droplet volume. The formation of a red blood cell-containing droplet consists of three stages: a lag stage, a filling stage, and a necking stage, with the lag stage only observed in narrow dispersed phase channel setups. Unlike other shear-thinning fluids, thread elongation into the main channel at the end of the necking stage is not observed for red blood cell solutions. In this work, a model that predicts the final droplet volume of a red blood cell containing droplets in T-junction generators is presented. The model combines a detailed analysis of the geometrical shape of the droplet during the formation process, with force and Laplace pressure balances to obtain the penetration depth (b_{fill}^{*}) and the critical neck thickness (2r_{pinch}^{*}) of the droplet. The performance of the model was validated by comparing the operational parameters (droplet volume, the spacing between the droplet, and the generation frequency) with the experimental data across a range of the dimensionless parameters (flow rate ratios, continuous phase viscosities, and channel geometries).

Rose petal topography mimicked poly(dimethylsiloxane) substrates for enhanced corneal endothelial cell behavior.

Low proliferation capacity of corneal endothelial cells (CECs) and worldwide limitations in transplantable donor tissues reveal the critical need of a robust approach for in vitro CEC growth. However, preservation of CEC-specific phenotype with increased proliferation has been a great challenge. Here we offer a biomimetic cell substrate design, by optimizing mechanical, topographical and biochemical characteristics of materials with CEC microenvironment. We showed the surprising similarity between topographical features of white rose petals and corneal endothelium due to hexagonal cell shapes and physiologically relevant cell density (≈ 2000 cells/mm2). Polydimethylsiloxane (PDMS) substrates with replica of white rose petal topography and cornea-friendly Young's modulus (211.85 ± 74.9 kPa) were functionalized with two of the important corneal extracellular matrix (ECM) components, collagen IV (COL 4) and hyaluronic acid (HA). White rose petal patterned and COL 4 modified PDMS with optimized stiffness provided enhanced bovine CEC response with higher density monolayers and increased phenotypic marker expression. This biomimetic approach demonstrates a successful platform to improve in vitro cell substrate properties of PDMS for corneal applications, suggesting an alternative environment for CEC-based therapies, drug toxicity investigations, microfluidics and organ-on-chip applications.

Bioinspired hydrogel surfaces to augment corneal endothelial cell monolayer formation.

Corneal endothelial cells (CECs) have limited proliferation ability leading to corneal endothelium (CE) dysfunction and eventually vision loss when cell number decreases below a critical level. Although transplantation is the main treatment method, donor shortage problem is a major bottleneck. The transplantation of in vitro developed endothelial cells with desirable density is a promising idea. Designing cell substrates that mimic the native CE microenvironment is a substantial step to achieve this goal. In the presented study, we prepared polyacrylamide (PA) cell substrates that have a microfabricated topography inspired by the dimensions of CECs. Hydrogel surfaces were prepared via two different designs with small and large patterns. Small patterned hydrogels have physiologically relevant hexagon densities (∼2000 hexagons/mm2 ), whereas large patterned hydrogels have sparsely populated hexagons (∼400 hexagons/mm2 ). These substrates have similar elastic modulus of native Descemet's membrane (DM; ∼50 kPa) and were modified with Collagen IV (Col IV) to have biochemical content similar to native DM. The behavior of bovine corneal endothelial cells on these substrates was investigated and results show that cell proliferation on small patterned substrates was significantly (p = 0.0004) higher than the large patterned substrates. Small patterned substrates enabled a more densely populated cell monolayer compared to other groups (p = 0.001 vs. flat and p < 0.0001 vs. large patterned substrates). These results suggest that generating bioinspired surface topographies augments the formation of CE monolayers with the desired cell density, addressing the in vitro development of CE layers.

Real-Time In Vivo Control of Neural Membrane Potential by Electro-Ionic Modulation.

Theoretically, by controlling neural membrane potential (Vm) in vivo, motion, sensation, and behavior can be controlled. Until now, there was no available technique that can increase or decrease ion concentration in vivo in real time to change neural membrane potential. We introduce a method that we coin electro-ionic modulation (EIM), wherein ionic concentration around a nerve can be controlled in real time and in vivo. We used an interface to regulate the Ca2+ ion concentration around the sciatic nerve of a frog and thus achieved stimulation and blocking with higher resolution and lower current compared with electrical stimulation. As EIM achieves higher controllability of Vm, it has potential to replace conventional methods used for the treatment of neurological disorders and may bring a new perspective to neuromodulation techniques.

Impedimetric detection and lumped element modelling of a hemagglutination assay in microdroplets.

Droplet-based microfluidic systems offer tremendous benefits for high throughput biochemical assays. Despite the wide use of electrical detection for microfluidic systems, application of impedimetric sensing for droplet systems is very limited. This is mainly due to the insulating oil-based continuous phase used for most aqueous samples of interest. We present modelling and experimental verification of impedimetric detection of hemagglutination in microdroplets. We have detected agglutinated red blood cells in microdroplets and screened whole blood samples for multiple antibody sera using conventional microelectrodes. We were able to form antibody and whole blood microdroplets in PDMS microchannels without any tedious chemical surface treatment. Following the injection of a blood sample into antibody droplets, we have detected the agglutination-positive and negative droplets in an automated manner. In order to understand the characteristics of impedimetric detection inside microdroplets, we have developed the lumped electrical circuit equivalent of an impedimetric droplet content detection system. The empirical lumped element values are in accordance with similar models developed for single phase electrical impedance spectroscopy systems. The presented approach is of interest for label-free, quantitative analysis of droplets. In addition, the standard electronic equipment used for detection allows miniaturized detection circuitries that can be integrated with a fluidic system for a quantitative microdroplet-based hemagglutination assay that is conventionally performed in well plates.