Jongil Ju

Spheroid-based three-dimensional liver-on-a-chip to investigate hepatocyte–hepatic stellate cell interactions and flow effects

We have developed a three-dimensional (3D) liver-on-a-chip to investigate the interaction of hepatocytes and hepatic stellate cells (HSCs) in which primary 3D hepatocyte spheroids and HSCs are co-cultured without direct cell-cell contact. Here, we show that the 3D liver chip offers substantial advantages for the formation and harvesting of spheroids. The most important feature of this liver chip is that it enables continuous flow of medium to the cells through osmotic pumping, and thus requires only minimal handling and no external power source. We also demonstrate that flow assists the formation and long-term maintenance of spheroids. Additionally, we quantitatively and qualitatively investigated the paracrine effects of HSCs, demonstrating that HSCs assist in the maintenance of hepatocyte spheroids and play an important role in the formation of tight cell-cell contacts, thereby improving liver-specific function. Spheroids derived from co-cultures exhibited improved albumin and urea secretion rates compared to mono-cultured spheroids after 9 days. Immunostaining for cytochrome P450 revealed that the enzymatic activity of spheroids co-cultured for 8 days was greater than that of mono-cultured spheroids. These results indicate that this system has the potential for further development as a unique model for studying cellular interactions or as a tool that can be incorporated into other models aimed at creating hepatic structure and prolonging hepatocyte function in culture.

Cell encapsulation via microtechnologies

The encapsulation of living cells in a variety of soft polymers or hydrogels is important, particularly, for the rehabilitation of functional tissues capable of repairing or replacing damaged organs. Cellular encapsulation segregates cells from the surrounding tissue to protect the implanted cell from the recipients immune system after transplantation. Diverse hydrogel membranes have been popularly used as encapsulating materials and permit the diffusion of gas, nutrients, wastes and therapeutic products smoothly. This review describes a variety of methods that have been developed to achieve cellular encapsulation using microscale platform. Microtechnologies have been adopted to precisely control the encapsulated cell number, size and shape of a cell-laden polymer structure. We provide a brief overview of recent microtechnology-based cell encapsulation methods, with a detailed description of the relevant processes. Finally, we discuss the current challenges and future directions likely to be taken by cell microencapsulation approaches toward tissue engineering and cell therapy applications.

High-throughput microfluidics: improved sample treatment and washing over standard wells

Fluid flow in microchannels is used to treat or wash samples and can be incorporated into high-throughput applications such as drug screening, which currently use standard microtiter wells for performing assays. This paper provides theoretical and experimental data comparing microchannels and standard wells on the metrics of sample washing and experimental error in treatment concentrations. It is shown numerically and experimentally that microchannel concentration can be approximated with an inverse linear relationship to input volume. The experimentally supported mathematical approximation and error propagation methods are used to compare the accuracy and precision of treatments in microchannels vs. standard wells. Mathematical results suggest microchannels can provide 10 or more times the treatment precision of standard wells for volume ratios typical of high-throughput screening. Passive-pumping and diffusion are utilized to improve microchannel accuracy and precision even further in a treat-wait-treat method. The advantages of microchannels outlined here can have large-scale effects on cost and accuracy in screening applications.

Large-Scale, Ultrapliable, and Free-Standing Nanomembranes

The creation and characterization of large-area ultrathin highly pliable free-standing PDMS membranes and their application to the study of cellular epithelia is described. The ultra-thin membranes permitted the straight forward calculation of cell monolayer moduli, derived from measured stress-strain curves. These measurements allowed the unprecedented detection of cellular-level injury in the epithelia caused by the rupture of cell-cell tight junctions in response to stretching.

Optimizing human embryonic stem cells differentiation efficiency by screening size-tunable homogenous embryoid bodies.

Human embryonic stem cells (hESCs) are generally induced to differentiate by forming spherical structures termed embryoid bodies (EBs) in the presence of soluble growth factors. hEBs are generated by suspending small clumps of hESC colonies; however, the resulting hEBs are heterogeneous because this method lacks the ability to control the number of cells in individual EBs. This heterogeneity affects factors that influence differentiation such as cell-cell contact and the diffusion of soluble factors, and consequently, the differentiation capacity of each EB varies. Here, we fabricated size-tunable concave microwells to control the physical environment, thereby regulating the size of EBs formed from single hESCs. Defined numbers of single hESCs were forced to aggregate and generate uniformly sized EBs with high fidelity, and the size of the EBs was controlled using concave microwells of different diameters. Differentiation patterns in H9- and CHA15-hESCs were affected by EB size in both the absence and presence of growth factors. By screening EB size in the presence of various BMP4 concentrations, a two-fold increase in endothelial cell differentiation was achieved. Because each hESC line has unique characteristics, the findings of this study demonstrate that concave microwells could be used to screen different EB sizes and growth factor concentrations to optimize differentiation for each hESC line.

An electrofusion chip with a cell delivery system driven by surface tension

We have fabricated an electric cell fusion chip with an embedded cell delivery function driven by surface tension and evaluated its performance with several types of plant cells. The chip consists of a polydimethylsiloxane-based microchannel with a fusion chamber and gold‐titanium (Au‐Ti) electrodes. The velocity profiles of the microfluid in the channel and fusion chamber were calculated to predict cell movement, and the electric field distribution between the electrodes was also calculated in order to determine the appropriate electrode shape. The range of the fluid velocity in the fusion chamber is 20‐50 μ ms −1 and the measured speed of the cells is approximately 45 μ ms −1 , which is sufficiently slow for the motion of the cells in the fusion chamber to be monitored and controlled. We measured the variation of the pearl chain ratio with frequency for five kinds of plant cells, and determined that the optimal frequency for pearl chain formation is 1.5 MHz. The electrofusion of cells was successfully carried out under ac field (amplitude: 0.4‐0.5 kV cm −1 , frequency: 1.5 MHz) and dc pulse (amplitude: 1.0 kV cm −1 , duration: 20 ms) conditions. M This article features online multimedia enhancements S This article has associated online supplementary data files (Some figures in this article are in colour only in the electronic version)

Backward flow in a surface tension driven micropump

A surface tension driven micropump harnessing the pressure difference generated by drops of different curvature radii proves to be a simple and attractive passive method to drive fluid flow in microdevices. Here we observed the appearance of backward flow when the initial sizes of the droplets at the inlet and outlet ports are similar. To explain this phenomenon several hypotheses have been investigated. Consideration of the inertia of the fluid in the channel revealed that it alone is insufficient to explain the observed backward flow. We discovered that rotational flow inside the outlet droplet could be a source of inertia, explaining the generation of the backward flow. In addition, we have experimentally determined that the ratio of the volumes of the initial outlet drop and inlet drop correlates with the occurrence of the backward flow.

One-stop microfiber spinning and fabrication of a fibrous cell-encapsulated scaffold on a single microfluidic platform.

This paper provides a method for microscale fiber spinning and the in situ construction of a 3D fibrous scaffold on a single microfluidic platform. This platform was also used to fabricate a variety of fibrous scaffolds with diverse compositions without the use of complicated devices. We explored the potential utility of the fibrous scaffolds for tissue engineering applications by constructing a fibrous scaffold encapsulating primary hepatocytes. The cells in scaffold were cultured over seven days and maintained higher viability comparing with 3D alginate non-fibrous block. The main advantage of this platform is that the fibrous structure used to form a scaffold can be generated without damaging the mechanically weak alginate fibers or encapsulated cells because all procedures are performed in a single platform without the intervention of the operator. In addition, the proposed fibrous scaffold permitted high diffusion capability of molecules, which enabled better viability of encapsulated cells than non-fibrous scaffold even in massive cell culture.

Tobacco protoplast culture in a polydimethylsiloxane-based microfluidic channel

Summary.Several advances have been made in the use of microfluidic devices for insect and mammalian cell cultures, but no reports of their use for plant cell cultures have been published. We, therefore, conducted a plant cell culture in a microfluidic device using polydimethylsiloxane. Nicotiana tabacum protoplasts were cultured in a variously shaped polydimethylsiloxane channel containing Nitsch medium supplemented with 0.5 g of NLN-13 vitamin mixture, 2.0 mg of α-naphthaleneacetic acid, and 0.5 mg of 6-benzyladenine per liter and 9% mannitol. Protoplasts in the polydimethylsiloxane channel showed cell division and microcolony formation within 4 weeks. The use of a microfluidic channel is a novel technique in the field of plant cell culture. The results of this study will encourage the utilization of polydimethylsiloxane-based microfluidic devices in plant cell engineering and cell analysis.

Passive micromixer using by convection and surface tension effects with air-liquid interface

This article describes a passive micromixer that utilizes an air-liquid interface and surface tension effects to enhance fluid mixing via convection and Marangoni effects. Performance of the microfluidic component is tested within a passive-pumping-based device that consists of three microchannels connected in succession using passive micro-mixers. Mixing was quantified at 5 key points along the length of the device using microscope images of patterned streams of Alexa 488 fluorescent-dyed water and pure DI water flowing through the device. The passive micro-mixer mixed fluid 15–20 times more effectively than diffusion between laminar flow streams alone and is a novel micro-mixer embodiment that provides an additional strategy for removing external components from microscale devices for simpler, autonomous operation.