Joong Yull Park

Differentiation of Neural Progenitor Cells in a Microfluidic Chip‐Generated Cytokine Gradient

In early embryonic development, spatial gradients of diffusible signaling molecules play important roles in controlling differentiation of cell types or arrays in diverse tissues. Thus, the concentration of exogenous cytokines or growth factors at any given time is crucial to the formation of an enriched population of a desired cell type from primitive stem cells in vitro. Microfluidic technology has proven very useful in the creation of cell‐friendly microenvironments. Such techniques are, however, currently limited to a few cell types. Improved versatility is required if these systems are to become practically applicable to stem cells showing various plasticity ranges. Here, we built a microfluidic platform in which cells can be exposed to stable concentration gradients of various signaling molecules for more than a week with only minimal handling and no external power source. To maintain stability of the gradient concentration, the osmotic pumping performance was optimized by balancing the capillary action and hydraulic pressure in the inlet reagent reservoirs. We cultured an enriched population of neural progenitors derived from human embryonic stem cells in our microfluidic chamber for 8 days under continuous cytokine gradients (sonic hedgehog, fibroblast growth factor 8, and bone morphogenetic protein 4). Neural progenitors successfully differentiated into neurons, generating a complex neural network. The average numbers of both neuronal cell body clusters and neurite bundles were directly proportional to sonic hedgehog concentrations in the gradient chip. The system was shown to be useful for both basic and translational research, with straightforward mechanisms and operational schemes. STEM CELLS 2009;27:2646–2654

Biomimetic soft multifunctional miniature aquabots.

Engineered locomotive systems are widespread on the macro scale (e.g., planes, trains, automobiles), yet miniaturized mobile systems are largely limited to the research lab. In particular, integration of specific functions such as sensing or drug delivery into a small robot is a great challenge. The ability to miniaturize parts affecting motion, such as MEMS-based components and electroactive polymers, while widely demonstrated has not led to practical advances in miniature multifunctional mobile robots. Rather complex construction, limits in the use of diverse materials, and cost (in case of the MEMS-based bot) all hinder the production of these robots. The small (micro-/millimeter scale) system within aqueous environments is particularly challenging, yet there are important potential applications for robots in such environments. To date, engineers have explored soft materials for creating components at the micro-/millimeter scales; for example, actuators such as ionic polymer metal composites, conducting polymers, and carbon nanotube actuators, and engineered devices housing hydrogel valves, filters, and lenses, as well as other soft material actuators that mimic natural movement (earthworm, myriapod, etc.). Still, the creation of miniature multifunctional robots that operate in aqueous environments has been elusive. Nature provides numerous examples of small, multifunctional and autonomous ‘‘robots’’ in the insect and marine worlds after which we aimed to model miniature polymeric aquabots. Here we present mini (microto millimeter) soft aquabots that combine multiple functionalities to perform multifunctional operations in aqueous environments, effectively simulating their natural counterparts. Despite extensive research on macroscale robotics and miniature micro-electromechanical systems, relatively little attention has been paid to the creation of miniature soft robots with diverse shapes, actuation mechanisms, and integrated functionalities. As a fabrication method, we describe a technique that takes advantage of the small volume required by amicrofluidic chamber; this will be used for photopolymerizing multifunctional minibots that possess several ‘‘organs,’’ allowing for movement, sensing/signaling, and capture/transport/release. A diverse group of aquabots mimicking three kinds of living organisms – octopus, sperm, and myriapod, each representing a different mode of locomotion – has been developed. These aquabots have the following key features: 1) diverse shape and small size (sub-millimeter feature sizes); 2) a variety of functions (e.g., actuation, sensing) integrated in a single robot; and 3) rapid, scalable fabrication both in dimension and quantity. The aquabots were fabricated out of soft materials, due to the ease of polymerization, as well as the ability to tailor the material composition towards achieving certain functionality (e.g., responsiveness to pH, temperature, or electric/ magnetic field). Sequential in situ photopolymerization within a microfluidic device allowed for precise fabrication of different components (arms, legs, body; see detailed procedure for photopolymerization in Experimental section). The bodies were constructed using a relatively inert polyethylene glycol (PEG) material, whereas ionic electroactive polymers (electroactive hydrogels) were chosen from a large number of available soft materials to serve as the actuators, thus facilitating controlled movement. Depending on the application, some aquabots contained temperature-, pH-, or chemical-sensitive polymers in addition to their electroactive hydrogel and PEG components. The gel network of polymerized actuator material was anionic, causing positively charged surfactant molecules to bind to its surface; this surfactant binding leads to an osmotic pressure difference between the gel interior and the external solution, thus inducing the contraction and curvature of the gel strip. Four parameters control electroactive hydrogelbased actuators: 1)magnitude of the applied potential; 2) angle of the applied potential; and both 3) width and 4) length of the actuator. Figure 1A illustrates the behavior of a rectangular gel actuator under changing parameters (width of post and applied voltage; see Supporting Information Fig. S1 for more complete parameter characterization). In situ photopolymerization facilitates the control of the geometric parameters and is amenable to the use of laser or multiphoton polymerization methods for improved resolution (e.g., further reduce actuator width to achieve faster and larger motion). To date, diverse electroactive hydrogels have been broadly applied to creating polymeric microactuators (see the detailed behavior of electric-sensitive hydrogel in Supporting Information). Here, we demonstrate the use of electroactive hydrogel for fabricating octopus-, sperm-, and myriapod-like aquabots that retain respective characteristic movement (Fig. 2; movies are available for octopus-like aquabot and sperm-like aquabots in Supporting Information – Videos 1 and 2, respectively). The repeated bending test under aquatic conditions was carried out to investigate the durability of the electroactive hydrogel strips (see Supporting Information for a detailed durability test conditions). The operation of the communications

Shear stress induced by an interstitial level of slow flow increases the osteogenic differentiation of mesenchymal stem cells through TAZ activation.

Shear stress activates cellular signaling involved in cellular proliferation, differentiation, and migration. However, the mechanisms of mesenchymal stem cell (MSC) differentiation under interstitial flow are not fully understood. Here, we show the increased osteogenic differentiation of MSCs under exposure to constant, extremely low shear stress created by osmotic pressure-induced flow in a microfluidic chip. The interstitial level of shear stress in the proposed microfluidic system stimulated nuclear localization of TAZ (transcriptional coactivator with PDZ-binding motif), a transcriptional modulator of MSCs, activated TAZ target genes such as CTGF and Cyr61, and induced osteogenic differentiation. TAZ-depleted cells showed defects in shear stress-induced osteogenic differentiation. In shear stress induced cellular signaling, Rho signaling pathway was important forthe nuclear localization of TAZ. Taken together, these results suggest that TAZ is an important mediator of interstitial flow-driven shear stress signaling in osteoblast differentiation of MSCs.

Electrically-driven hydrogel actuators in microfluidic channels: fabrication, characterization, and biological application

The utility of electro-responsive smart materials has been limited by bubble generation (hydrolysis) during application of electrical fields and by biocompatibility issues. Here we describe the design of a device that overcomes these limitations by combining material properties, new design concepts, and microtechnology. 4-hydroxybutyl acrylate (4-HBA) was used as a backbone hydrogel material, and its actuating behavior, bending force, and elasticity were extensively characterized as a function of size and acrylic acid concentration. To prevent bubble generation, the system was designed such that the hydrogel actuator could be operated at low driving voltages (<1.2 V). A microfluidic channel with an integrated electroactive hydrogel actuator was developed for sorting particles. This device could be operated in cell culture media, and the sorting capabilities were initially assessed by sorting droplets in an oil droplet emulsion. Biocompatibility was subsequently tested by sorting mouse embryoid bodies (mEBs) according to size. The sorted and collected mEBs maintained pluripotency, and selected mEBs successfully differentiated into three germ layers: endoderm, mesoderm, and ectoderm. The electroactive hydrogel device, integrated into a microfluidic system, successfully demonstrated the practical application of smart materials for use in cell biology.

Simultaneous generation of chemical concentration and mechanical shear stress gradients using microfluidic osmotic flow comparable to interstitial flow.

Cells are very sensitive to various microenvironmental cues, including mechanical stress and chemical gradients. Therefore, physiologically relevant models of cells should consider how cells sense and respond to microenvironmental cues. This can be accomplished by using microfluidic systems, in which fluid physics can be realized at a nanoliter scale. Here we describe a simple and versatile method to study the generation of chemical concentration and mechanical shear stress gradients in a single microfluidic chip. Our system uses an osmotic pump that produces very slow (<a few microm/s) and controlled flow, allowing a wide and stable diffusion of specific chemical concentration. We also established a shear stress gradient passively via a circular channel in the interstitial level. For evaluation of the system, we used L929 mouse fibroblast cells and simultaneously exposed them to a mechanical stress gradient and a chemical nutrient gradient. The interstitial shear stress level clearly affected cell alignment, mobility velocity, and attachment. At the same time, cell proliferation reflected nutrient concentration level. Our system, which enables continuous and long-term culture of cells in a combined chemical and mechanical gradient, provides physiologically realistic conditions and will be applicable to studies of cancer metastasis and stem cell differentiation.

Regulating microenvironmental stimuli for stem cells and cancer cells using microsystems

Cells express hundreds of different types of receptors, which they use to continuously monitor their chemical and mechanical microenvironments. Stem cells and cancer cells are particularly sensitive to microenvironmental cues because their interactions have profound effects on stem cell potency and tumorigenesis, respectively. Unlike conventional tissue culture in wells and dishes, microtechnology with dimensions on the cellular scale can be combined with materials, chemicals, physiological flows, and other effectors to provide high levels of control in a format more flexible than macroscale in vitro or in vivo systems, revealing stimulation-specific responses of stem cells and cancer cells. Microtechnology-integrated biology enable the simultaneous control of multiple numbers of biological microenvironmental factors in a high-throughput manner. In this review we present representative examples of the use of microtechnology systems to regulate the mechanical, chemical, topological, adhesive, and other environments of individual stem cells and cancer cells. We then explore the possibilities for simultaneous multimodal control of combinations of these environmental factors.

Neurotoxic amyloid beta oligomeric assemblies recreated in microfluidic platform with interstitial level of slow flow

Alzheimers disease is accompanied by progressive, time-dependent changes of three moieties of amyloid beta. In vitro models therefore should provide same conditions for more physiologic studies. Here we observed changes in the number of fibrils over time and studied the correlation between amyloid beta moieties and neurotoxicity. Although the number of fibrils increased dramatically, the change in neurotoxicity with time was small, suggesting that fibrils make little contribution to neurotoxicity. To study the neurotoxicity of diffusible moieties by regulating microenvironments, we created a bio-mimetic microfluidic system generating spatial gradients of diffusible oligomeric assemblies and assessed their effects on cultured neurons. We found amyloid beta exposure produced an atrophy effect and observed neurite extension during the differentiation of neural progenitor cells increased when cells were cultured with continuous flow. The results demonstrate the potential neurotoxicity of oligomeric assemblies and establish a prospective microfluidic platform for studying the neurotoxicity of amyloid beta.

Ice-lithographic fabrication of concave microwells and a microfluidic network

We describe a novel method to produce concave microwells utilizing solid–liquid phase change. This method, named ‘ice-lithography’, does not require any lithographic processes and consists of a few simple steps that yield multiple concave microwells. We demonstrated that the shape and size of the microwells can be controlled by varying substrates and vapor-collection time. Patterned wells with sizes in the range of 10μm to several millimeters in diameter could be produced. Additionally, we fabricated a uniformly aligned concave microwell pattern and a microfluidic network. Ice-lithography has potential biological and biomedical applications in areas such as the fabrication of cell docking devices and microbioreactors as well as the formation of uniformly sized embryoid bodies.

Cell morphological response to low shear stress in a two-dimensional culture microsystem with magnitudes comparable to interstitial shear stress.

Slow interstitial flow can lead to large changes in cell morphology. Since conventional biological assays are adapted to two-dimensional culture protocols, there is a need to develop a microfluidic system that can generate physiological levels of interstitial flow. Here we developed a system that uses a passive osmotic pumping mechanism to generate sustained and steady interstitial flows for two-dimensional cultures. Two different cell types, fibroblasts and mesenchymal stem cells, were selected because they are generally exposed to interstitial flow. To quantify the cellular response to interstitial shear flow in terms of proliferation and alignment, 4 rates of flow were applied. We found that the proliferation rate of fibroblasts varied linearly with wall shear stress. In addition, alignment of fibroblast cells depended linearly on the magnitude of the shear stress, whereas mesenchymal stem cells were aligned regardless of the magnitude of shear stress. This suggested that mesenchymal stem cells are very sensitive to shear stresses, even at levels generated by interstitial flow. The results presented here emphasize the need to consider the mechanosensitivity and the natural role of different cell types when evaluating their responses to fluid flow.

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)