Enhancing suspended cell transfection by inducing localized distribution of the membrane actin cortex before exposure to electromechanical stimulation.

OBJECTIVES: During physical transfection, an electrical field or mechanical force is used to induce cell transfection. We tested if the disruption of a dense actin layer underneath the membrane of a suspended cell enhances cell transfection. RESULTS: A bubble generator was used to electromechanically stimulate suspended cells. To clarify the influence of the actin layer (the actin cortex) on cell transfection efficiency, we used an actin polymerization inhibitor (cytochalasin D) to disrupt the actin cortex before electromechanical stimulation. Without cytochalasin D treatment, signals from the overall actin cortex decreased after electromechanical stimulation. With cytochalasin D treatment, there was localized F-actin aggregation under static conditions. After electromechanical stimulation, there was a partial loss (localized disruption), but no overall disruption, of the actin cortex. With the pretreatment with cytochalasin D, the transfection efficiency of plasmids (4.7, 8.3, or 11 kbp) into NIH/3T3 or UMR-106 cells increased significantly after exposure to electromechanical stimulation. CONCLUSIONS: Localized distribution of the actin cortex before exposure to electromechanical stimulation is crucial for inducing a partial loss of the cortex, which improves transfection efficiency and large plasmid delivery.

Degas-Driven Deterministic Lateral Displacement in Poly(dimethylsiloxane) Microfluidic Devices.

In this work, degas-driven microfluidic deterministic lateral displacement devices were fabricated from poly(dimethylsiloxane). Two device configurations were considered: one with a single input for the enrichment of particles and the other one with sheath inputs for the separation of particles based on their sizes. Using the single-input device, the characteristics of the degas-driven fluid through micropillars were investigated, and then selective enrichment of fluorescent polymer particles with diameters of around 13 µm mixed with similar 7 µm particles was demonstrated. Using the sheath-input device, the separation of 13 and 7 µm beads was achieved (the corresponding purities exceeded 92.62% and 99.98%, respectively). In addition, clusters composed of 7 µm beads (including doublets, triplets, and quadruplets) were fractionated based on their equivalent sizes. Finally, white blood cells could be separated from red blood cells at a relatively high capture efficiency (95.57%) and purity (86.97%).

Vessel specific imaging of glucose transfer with fluorescent glucose analogue in anesthetized mouse cortex.

The present study examined glucose transfer in the cellular scale of mouse brain microvasculature in vivo using two-photon microscopy and fluorescent glucose analogue (2-NBDG). The 2-NBDG was intravenously injected (0.04 mL/min) in the anesthetized Tie2-GFP mice in which the vascular endothelium expressed fluorescent protein. Time-lapse imaging was conducted on the cortical parenchyma, while the time-intensity change of the injected 2-NBDG was analysed in respective vascular compartments (artery, capillary, and vein). We observed that 2-NBDG signal increased monotonically in the vasculature during the period of the injection, and rapidly declined following its cessation. In tissue compartment, however, the signal intensity gradually increased even after cessation of the injection. Spatiotemporal analysis of the 2-NBDG intensity over the cross-sections of the vessels further showed distinct change of the 2-NBDG intensity across the vessel wall (endothelium), which may represents a regulation site of tissue glucose influx.

Tunable deterministic lateral displacement of particles flowing through thermo-responsive hydrogel micropillar arrays.

Deterministic lateral displacement (DLD) is a promising technology that allows for the continuous and the size-based separation of suspended particles at a high resolution through periodically arrayed micropillars. In conventional DLD, the critical diameter (Dc), which determines the migration mode of a particle of a particular size, is fixed by the device geometry. Here, we propose a novel DLD that uses the pillars of a thermo-responsive hydrogel, poly(N-isopropylacrylamide) (PNIPAM) to flexibly tune the Dc value. Upon heating and cooling, the PNIPAM pillars in the aqueous solution shrink and swell because of their hydrophobic-hydrophilic phase transitions as the temperature varies. Using the PNIPAM pillars confined in a poly(dimethylsiloxane) microchannel, we demonstrate continuous switching of particle (7-µm beads) trajectories (displacement or zigzag mode) by adjusting the Dc through temperature control of the device on a Peltier element. Further, we perform on/off operation of the particle separation (7-µm and 2-µm beads) by adjusting the Dc values.

Editorial for the Special Issue on Particles Separation in Microfluidic Devices, Volume II.

Particle separation in the nano- to microscale range is a significant step for biological, chemical, and medical analyses [...].

Viscosity-aided electromechanical poration of cells for transfecting molecules.

Cell poration technologies offer opportunities not only to understand the activities of biological molecules but also to investigate genetic manipulation possibilities. Unfortunately, transferring large molecules that can carry huge genomic information is challenging. Here, we demonstrate electromechanical poration using a core-shell-structured microbubble generator, consisting of a fine microelectrode covered with a dielectric material. By introducing a microcavity at its tip, we could concentrate the electrical field with the application of electric pulses and generate microbubbles for electromechanical stimulation of cells. Specifically, the technology enables transfection with molecules that are thousands of kDa even into osteoblasts and Chlamydomonas, which are generally considered to be difficult to inject. Notably, we found that the transfection efficiency can be enhanced by adjusting the viscosity of the cell suspension, which was presumably achieved by remodeling of the membrane cytoskeleton. The applicability of the approach to a variety of cell types opens up numerous emerging gene engineering applications.

Particle/cell separation using sheath-free deterministic lateral displacement arrays with inertially focused single straight input.

This paper proposes microfluidic particle separation by sheath-free deterministic lateral displacement (DLD) with inertial focusing in a single straight input channel. Unlike conventional DLD devices for size-based particle separation, in which sheath streams are used to focus the particles before the solution containing them reaches the DLD arrays, the proposed method uses inertial focusing to align the particles along the middle or the sidewalls of the straight rectangular input channel. The two-stage model of inertial focusing is applied to reduce the length of the side-focusing channel. The proposed method is demonstrated by using it to separate fluorescent polymer particles of diameters 13 and 7 µm, in the process of which the effect of the particle focusing regime on the separation performance is also investigated. Through middle focusing, the method is further used to separate MCF-7 cells (a model of circulating tumor cells (CTCs)) and blood cells, with ∼99.0% capture efficiency achieved.

Editorial for the Special Issue on Particles Separation in Microfluidic Devices.

The separation and sorting of micro- and nano-sized particles is an important step in chemical, biological, and medical analyses [...].