3D Cell Culture Method in Channel-Free Water-in-Oil Droplets.

A new channel-free water-in-oil (WO) droplet 3D cell culture method is proposed to address the challenges while maintaining the advantages of the conventional 3D cell culture methods. The proposed WO method can fundamentally solve the constraint of spheroids size, a common challenge in conventional 3D culture, by using droplet size controllability. The 3D cell culture performance of the WO method is verified by comparing it with the conventional 3D cell culture methods. A systematic investigation of the culture conditions of the WO method confirms the working range of cell concentration and droplet size, as well as the scalability of spheroid size. Adjusting droplet size and cell concentration enables rapid spheroid formation with large and high cell concentration droplets or fast spheroid growth with small and low cell concentration droplets, providing control over the spheroid size and growth rate according to the purpose. Furthermore, long-term culture is demonstrated for 1 month with the proposed method, showing the largest spheroid culture and demonstrating the possibility that this method can be used not only for spheroid formation but also for organoid studies. Finally, if a WO-based automated 3D cell culture system is developed, it will be a useful tool for organoid research.

Simultaneous Separating, Splitting, Collecting, and Dispensing by Droplet Pinch-Off for Droplet Cell Culture.

Simultaneous separating, splitting, collecting, and dispensing a cell suspension droplet has been demonstrated by aspiration and subsequent droplet pinch-off for use in microfluidic droplet cell culture systems. This method is applied to cell manipulations including aliquots and concentrations of microalgal and mammalian cell suspensions. Especially, medium exchange of spheroid droplets is successfully demonstrated by collecting more than 99% of all culture medium without damaging the spheroids, demonstrating its potential for a 3D cell culture system. Through dimensional analysis and systematic parametric studies, it is found that initial mother droplet size together with aspiration flow rate determines three droplet pinch-off regimes. By observing contact angle changes during aspiration, the difference in the large and the small droplet pinch-off can be quantitatively explained using force balance. It is found that the capillary number plays a significant role in droplet pinch-off, but the Bond number and the Ohnesorge number have minor effects. Since the dispensed droplet size is mainly determined by the capillary number, the dispensed droplet size can be controlled simply by adjusting the aspiration flow rate. It is hoped that this method can contribute to various fields using droplets, such as droplet cell culture and digital microfluidics, beyond the generation of small droplets.

Safe and efficient RNA and DNA introduction into cells using digital electroporation system.

We systematically compared the delivery and expression efficiencies according to cell types (plant and animal cells) and genetic materials (RNA and DNA) to deliver RNA using a digital electroporation system. Despite the significantly lower RNA delivery in Chlamydomoans reinhartii than DNA delivery due to RNA secondary structure and cell wall, the expression/delivery ratio of RNA was significantly higher than that of DNA (up to 90%), confirming the generally known fact that RNA is more favorable for expression than DNA. On the other hand, in K562 cells, the difference in RNA and DNA delivery efficiency was negligible. Therefore, structural differences between DNA and RNA affect delivery efficiency differently depending on the cell type. RNA delivery efficiency of K562 cells was high, but expression efficiency was much lower than that of microalgae. According to the proposed strategy, compatibility between K562 cells and the nucleic acids used in this study is presumed to be one of the reasons for this low expression efficiency. Gene regulation by delivering small interfering RNA (siRNA) was demonstrated in K562 cells, confirming the feasibility of the digital electroporation system for RNA interference (RNAi) research as a safe and efficient delivery system.

Microfluidics-Assisted Synthesis of Hierarchical Cu2 O Nanocrystal as C2 -Selective CO2 Reduction Electrocatalyst.

Copper-based catalysts have attracted enormous attention due to their high selectivity for C2+ products during the electrochemical reduction of CO2 (CO2 RR). In particular, grain boundaries on the catalysts contribute to the generation of various Cu coordination environments, which have been found essential for C-C coupling. However, smooth-surfaced Cu2 O nanocrystals generally lack the ability for the surface reorganization to form multiple grain boundaries and desired Cu undercoordination sites. Flow chemistry armed with the unparalleled ability to mix reaction mixture can achieve a very high concentration of unstable reaction intermediates, which in turn are used up rapidly to lead to kinetics-driven nanocrystal growth. Herein, the synthesis of a unique hierarchical structure of Cu2 O with numerous steps (h-Cu2 O ONS) via flow chemistry-assisted modulation of nanocrystal growth kinetics is reported. The surface of h-Cu2 O ONS underwent rapid surface reconstruction under CO2 RR conditions to exhibit multiple heterointerfaces between Cu2 O and Cu phases, setting the preferable condition to facilitate C-C bond formation. Notably, the h-Cu2 O ONS obtained the increased C2 H4 Faradaic efficiency from 31.9% to 43.5% during electrocatalysis concurrent with the morphological reorganization, showing the role of the stepped surface. Also, the h-Cu2 O ONS demonstrated a 3.8-fold higher ethylene production rate as compared to the Cu2 O nanocube.

Effect of Deformation on Droplet Contact Charge Electrophoresis.

The effect of deformation on the droplet contact charge electrophoresis (CCEP) was investigated for consistent droplet movement control. Through systematic experiments and numerical simulations, it has been found that overcharging by deformation is up to about 130% of the sphere and is mainly driven by the concentration of the electric field near the tip of the droplet rather than an increase in the surface area. Dimensional analysis revealed a consistent droplet CCEP motion with the electric capillary number range of 0.01-0.09. We also found that the dimensionless droplet charge follows a universal curve proportional to the electric capillary number, regardless of the droplet size, and the weak dependence on the droplet size shown in the experimental results is due to hydrodynamic effects, not electrostatic ones. Changes in droplet velocity distribution with droplet size and the electric capillary number were also investigated. Using the perfect conductor theory and Stokes law, we derived an analytical relationship between the droplet center velocity and the electric capillary number and analyzed the experimental results based on this relationship. This study implies that if proper hydrodynamic correction is applied, the droplet CCEP and its deformation effect can be explained by a perfect conductor theory.

Wall Effects on Hydrodynamic Drag and the Corresponding Accuracy of Charge Measurement in Droplet Contact Charge Electrophoresis.

Droplet size dependent wall effects on hydrodynamic drag and the corresponding droplet contact charge estimations were experimentally and theoretically investigated. The consistent reduction in the dimensionless droplet contact charges proportional to droplet size was reported and explained by the parallel and approaching wall effects on the drag coefficient. Extrapolation of the size dependent droplet charge data showed that the droplet charge follows the perfect conductor theory when the droplet radius approaches zero. The proposed model was applied to the drag calculation to estimate and compare dimensionless charges before and after consideration of the wall effects. The droplet free fall test concluded that the droplets in the current experimental setup follow Stokes' law. The theoretical velocity profile of the droplet approaching the wall perpendicularly is proposed considering the approaching wall effect on hydrodynamic drag and verified by comparison with the experiment. The droplet size dependent velocity profile shape change was also explained by this approaching wall effect. The shape of the asymmetric velocity profile along the direction of droplet movement was explained by the effect of the image charge through direct numerical calculation of the electric force. The direct calculation of the electric force also showed that the electric correction at the center of the cuvette is negligible; thus, it is sufficient to consider only hydrodynamic correction for accurate charge measurement in this experimental system. The present study will contribute to the accurate measurement of the droplet charges under contact charge electrophoresis. It also provides the basis for precise control of droplet movement in lab-on-a-chip devices.

Enhanced Controllability of Fries Rearrangements Using High-Resolution 3D-Printed Metal Microreactor with Circular Channel.

High-resolution 3D-printed stainless steel metal microreactors (3D-PMRs) with different cross-sectional geometry are fabricated to control ultrafast intramolecular rearrangement reactions in a comparative manner. The 3D-PMR with circular channel demonstrates the improved controllability in rapid Fries-type rearrangement reactions, because of the superior mixing efficiency to rectangular cross-section channels (250 µm × 125 µm) which is confirmed based on the computational flow dynamics simulation. Even in case of very rapid intramolecular rearrangement of sterically small acetyl group occurring in 333 µs of reaction time, the desired intermolecular reaction can outpace to the undesired intramolecular rearrangement using 3D-PMR to result in high conversion and yield.

Optimization of the droplet electroporation method for wild type Chlamydomonas reinhardtii transformation.

We performed the transformation of a wild type Chlamydomonas reinhardtii by optimizing previously developed droplet EP method. For more effective and faster optimization, we used DNA dying fluorescent molecule (Yo-Pro-1) for finding optimal EP conditions instead of using protein expression based evaluation method. By examining wider range of electrical parameter space together with the analysis of total current flow of EP process, we found optimal EP conditions. The obtained optimal EP conditions were verified by CFP transgene expression experiments. By applying the optimal EP conditions to the transformation of C. reinhardtii, we obtained transformants and analyzed them using PCR. Finally, implications and future work are discussed.

A continuous droplet electroporation system for high throughput processing.

A continuous droplet electroporation (EP) system capable of handling a billion cells has been proposed and demonstrated using a proof-of-concept prototype design. Numerical simulations were conducted to design the new system and to compare the continuous droplet EP system with the previous single droplet EP system. Through parametric studies on the applied voltage and flow rate, a much higher cyan fluorescent protein transgene expression efficiency (38.8 ± 8.9%) was accomplished over that of the previous single droplet EP system. A parallel continuous droplet EP system is also demonstrated by introducing additional electrode pairs into the continuous droplet EP system to achieve ultrahigh throughput. Finally, the significance and meaning of the present work and future development direction have been discussed.

Indirect fabrication of versatile 3D microfluidic device by a rotating plate combined 3D printing system.

In the past decade, 3D-printing technology has been applied in the field of microfluidics to fabricate microfluidic devices for wide-ranging areas of study including chemistry, biology, medicine, and others. However, these methods face several limitations such as insufficient resolution and long fabrication time. In this study, versatile microfluidic devices with different functions were indirectly fabricated by a rapid sacrificial template printing process using a photocurable fluoropolymer with chemical durability. The Pluronic® F127 hydrogel as a sacrificial template was rapidly patterned on substrates by a non-lithographic printing process using a computer-controlled 3D-printing system. Viscous fluoropolymer was cast on the non-deformable template that was consequently removed by applying heat and negative pressure after UV curing. The chemical-resistant and transparent microchannels were oblate-hemispherical on the cross section. They were tested by performing a heterogeneous catalytic reaction as well as a photochemical reaction. The microchannels with controlled heights were devised to induce convection for functioning as a micromixer with asymmetric flows. Moreover, upon printing the Pluronic® F127 on both sides of the PFPE (perfluoropolyether-urethane dimethacrylate) membrane substrate, the 3D hybrid microfluidic device was embedded with a permeable membrane between the lower and upper channels, which is useful for gas-liquid chemical processes.

Correction: Indirect fabrication of versatile 3D microfluidic device by a rotating plate combined 3D printing system.

[This corrects the article DOI: 10.1039/C8RA08465C.].

Electrostatic Origins of the Positive and Negative Charging Difference in the Contact Charge Electrophoresis of a Water Droplet.

The positive and negative charging difference in the contact charge electrophoresis of a water droplet suspended in oil was investigated to find out the origin of this charging difference. Through numerous experiments and numerical analysis, the charging difference has been found to be mainly originated from electrostatic sources. Two electrostatic sources were found in the present experimental setup, and by excluding those two sources the charging difference was successfully diminished. The present findings well explain previous experimental results and also provide design guidelines for consistent droplet movement control in contact charge electrophoresis-based digital microfluidic systems. Finally, further discussions on the obtained results, its implications, and future work are discussed.

Electrically Controllable Microparticle Synthesis and Digital Microfluidic Manipulation by Electric-Field-Induced Droplet Dispensing into Immiscible Fluids.

The dispensing of tiny droplets is a basic and crucial process in a myriad of applications, such as DNA/protein microarray, cell cultures, chemical synthesis of microparticles, and digital microfluidics. This work systematically demonstrates droplet dispensing into immiscible fluids through electric charge concentration (ECC) method. It exhibits three main modes (i.e., attaching, uniform, and bursting modes) as a function of flow rates, applied voltages, and gap distances between the nozzle and the oil surface. Through a conventional nozzle with diameter of a few millimeters, charged droplets with volumes ranging from a few µL to a few tens of nL can be uniformly dispensed into the oil chamber without reduction in nozzle size. Based on the features of the proposed method (e.g., formation of droplets with controllable polarity and amount of electric charge in water and oil system), a simple and straightforward method is developed for microparticle synthesis, including preparation of colloidosomes and fabrication of Janus microparticles with anisotropic internal structures. Finally, a combined system consisting of ECC-induced droplet dispensing and electrophoresis of charged droplet (ECD)-driven manipulation systems is constructed. This integrated platform will provide increased utility and flexibility in microfluidic applications because a charged droplet can be delivered toward the intended position by programmable electric control.

Submillisecond organic synthesis: Outpacing Fries rearrangement through microfluidic rapid mixing.

In chemical synthesis, rapid intramolecular rearrangements often foil attempts at site-selective bimolecular functionalization. We developed a microfluidic technique that outpaces the very rapid anionic Fries rearrangement to chemoselectively functionalize iodophenyl carbamates at the ortho position. Central to the technique is a chip microreactor of our design, which can deliver a reaction time in the submillisecond range even at cryogenic temperatures. The microreactor was applied to the synthesis of afesal, a bioactive molecule exhibiting anthelmintic activity, to demonstrate its potential for practical synthesis and production.

Multilayered film microreactors fabricated by a one-step thermal bonding technique with high reproducibility and their applications.

We report the versatile uses of multilayered polyimide (PI) film microreactors with various functions including pressure tolerance, three-dimensional mixing and multistep membrane emulsification. Such PI film microreactors were fabricated by a simple one-step thermal bonding technique with high reproducibility. Upon bonding at 300 °C for 1 hour, the thin and flexible film microdevices could withstand pressure up to 8.6 MPa and 16.3 MPa with PI adhesive film or fluoropolymer adhesive, respectively, due to differences in wettability. The hydrophilic and hydrophobic microchannel devices were used to generate monodisperse oil-in-water (O/W) and water-in-oil (W/O) droplets, and polymer micro/nanoparticles at a high generation frequency. A monolithic and chemical resistant film microreactor with a three-dimensional serpentine microchannel was used for the selective reduction of ester to aldehyde by efficient mixing and quenching in a flash chemistry manner, within a several 10(1) millisecond time scale. Furthermore, a novel multilayered film microreactor for organic-aqueous biphasic interfacial reactions was devised by embedding a membrane layer to induce chaotic mixing in both the interface and emulsified phase by flowing through multiple numbers of meshed structures along the hydrophobic channel. This simple and economic fabrication technique significantly facilitates mass production of multilayered film devices that could be useful as a platform for various microfluidic applications in chemistry and biology.

Digital Microfluidic Approach for Efficient Electroporation with High Productivity: Transgene Expression of Microalgae without Cell Wall Removal.

A unique digital microfluidic electroporation (EP) system successfully demonstrates higher transgene expression than that of conventional techniques, in addition to reliable productivity and feasible integrated processes. By systematic investigations into the effects of the droplet EP conditions for a wild-type microalgae, 1 order of magnitude higher transgene expression is accomplished without cell wall removal over the conventional bulk EP system. In addition, the newly proposed droplet EP method by a droplet contact charging phenomena shows a great potential for the integration of EP processes and on-chip cell culture providing easy controllability of each process. Finally, the implications of the accomplishments and future directions for development of the proposed technology are discussed.

Characterization of electrode alignment for optimal droplet charging and actuation in droplet-based microfluidic system.

The actuation method using electric force as a driving force is utilized widely in droplet-based microfluidic systems. In this work, the effects of charging electrode alignment on direct charging of a droplet on electrified electrodes and a subsequent electrophoretic control of the droplet are investigated. The charging characteristics of a droplet according to different electrode alignments are quantitatively examined through experiments and systematic numerical simulations with varying distances and angles between the two electrodes. The droplet charge acquired from the electrified electrode is directly proportional to the distance and barely affected by the angle between the two electrodes. This implies that the primary consideration of electrode alignment in microfluidic devices is the distance between electrodes and the insignificant effect of angle provides a great degree of freedom in designing such devices. Not only the droplet charge acquired from the electrode but also the force exerted on the droplet is analyzed. Finally, the implications and design guidance for microfluidic systems are discussed with an electrophoresis of a charged droplet method-based digital microfluidic device.

A monolithic and flexible fluoropolymer film microreactor for organic synthesis applications.

A photocurable and viscous fluoropolymer with chemical stability is a highly desirable material for fabrication of microchemical devices. Lack of a reliable fabrication method, however, limits actual applications for organic reactions. Herein, we report fabrication of a monolithic and flexible fluoropolymer film microreactor and its use as a new microfluidic platform. The fabrication involves facile soft lithography techniques that enable partial curing of thin laminates, which can be readily bonded by conformal contact without any external forces. We demonstrate fabrication of various functional channels (~300 µm thick) such as those embedded with either a herringbone micromixer pattern or a droplet generator. Organic reactions under strongly acidic and basic conditions can be carried out in this film microreactor even at elevated temperature with excellent reproducibility. In particular, the transparent film microreactor with good deformability could be wrapped around a light-emitting lamp for close contact with the light source for efficient photochemical reactions with visible light, which demonstrates easy integration with optical components for functional miniaturized systems.

Three-dimensional flash flow microreactor for scale-up production of monodisperse PEG-PLGA nanoparticles.

We present a pressure-tolerant 3D parallel polyimide (PI) film microreactor operating at up to ~160 bars with direct 3D flow focusing geometry for mass production of PEG-PLGA nanoparticles in a ~10(1) gram-scale (g h(-1)).

Chitosan-microreactor: a versatile approach for heterogeneous organic synthesis in microfluidics.

Microreactors have been proven to be efficient tools for a variety of homogeneous organic transformations due to their mixing efficiency, which results in very fast reactions, better heat and mass transfer, and simple scale-up. However, in heterogeneous catalytic reactions each catalyst needs an individual substrate as support. Herein, a versatile approach to immobilize metal catalysts on chitosan as a common substrate is presented. Chitosan, accommodating many metal catalysts, is grafted onto the microchannel surface as nanobrush. The versatility, catalytic efficiency, and stability/durability of the microreactor are demonstrated for a number of organic transformations involving various metal compounds as catalysts.