Sewoon Han

Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels

This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel-incorporating chambers between surface-accessible microchannels. By using this platform, well-defined biochemical and biophysical stimuli can be applied to multiple cell types interacting over distances of <1 mm, thereby replicating many aspects of the in vivo microenvironment. Capabilities exist for time-dependent manipulation of flow and concentration gradients as well as high-resolution real-time imaging for observing spatial-temporal single-cell behavior, cell-cell communication, cell-matrix interactions and cell population dynamics. These heterotypic cell type assays can be used to study cell survival, proliferation, migration, morphogenesis and differentiation under controlled conditions. Applications include the study of previously unexplored cellular interactions, and they have already provided new insights into how biochemical and biophysical factors regulate interactions between populations of different cell types. It takes 3 d to fabricate the system and experiments can run for up to several weeks.

In vitro 3D collective sprouting angiogenesis under orchestrated ANG-1 and VEGF gradients

Sprouting angiogenesis requires a coordinated guidance from a variety of angiogenic factors. Here, we have developed a unique hydrogel incorporating microfluidic platform which mimics the physiological microenvironment in 3D under a precisely orchestrated gradient of soluble angiogenic factors, VEGF and ANG-1. The system enables the quantified investigation in chemotactic response of endothelial cells during the collective angiogenic sprouting process. While the presence of a VEGF gradient alone was sufficient in inducing a greater number of tip cells, addition of ANG-1 to the VEGF gradient enhanced the number of tip cells that are attached to collectively migrated stalk cells. The chemotactic response of tip cells attracted by the VEGF gradient and the stabilizing role of ANG-1 were morphologically investigated, elucidating the 3D co-operative migration of tip and stalk cells as well as their structures. We found that ANG-1 enhanced the connection of the stalk cells with the tip cells, and then the direct connection regulated the morphogenesis and/or life cycle of stalk cells.

Sprouting angiogenesis under a chemical gradient regulated by interactions with an endothelial monolayer in a microfluidic platform.

Microfluidic cell culture assays are versatile tools for studying cell migration, particularly angiogenesis. Such assays can deliver precisely controlled linear gradients of chemical stimuli to cultured cells in a microfluidic channel, offering excellent optical resolution and in situ monitoring of cellular morphogenesis in response to a gradient. Microfluidic cell culture assays provide a chemical gradient subject to molecular diffusion, although cellular metabolism can perturb it. The actual gradient perturbed by cells has not been precisely described in the context of regulated cellular morphogenesis. We modeled the chemical gradient in a microfluidic channel by simulating the analyte(VEGF) distribution during cellular interactions. The results were experimentally verified by monitoring sprouting angiogenic response from a monolayer of human umbilical vein endothelial cells (hUVECs) into a type 1 collagen scaffold. The simulation provided a basis for understanding a real distribution of the analyte interrupted by cells in microfluidic device. The new protocol enables one to quantify the morphogenesis of hUVECs under a flat, less-steep, or steep gradient.

Three-dimensional extracellular matrix-mediated neural stem cell differentiation in a microfluidic device

Here, we report a unique method to quantify the effects of in vivo-like extracellular matrix (ECM) for guiding differentiation of neural stem cells (NSCs) in three-dimensional (3D) microenvironments using quantitative real-time polymerase chain reaction (qRT-PCR). We successfully monitored and quantified differentiation of NSCs in small volume ECMs and found that differentiation of NSCs, especially those differentiating towards neuronal and oligodendrocytic lineages, is significantly enhanced by 3D microenvironments reconstituted in the microfluidic channels.

Hydrodynamic effects on bacterial biofilm development in a microfluidic environment

In aquatic environments, microorganisms tend to form biofilms on surfaces to protect them from harsh conditions. The biofilms then accumulate into multilayered mat-like structures. In this study, we evaluated the effects of the hydrodynamic conditions on the ecology of biofilms produced by Pseudomonas aeruginosa (PA14). In microfluidic channels, we found that the development of biofilms was regulated by hydrodynamic conditions, but the developed biofilms also changed flow velocity by narrowing flow width. The coupled growing conditions were simplified by a new concept of consequent variables, and the dimensionless biofilm development (Ab/h(2) & Ab/w(cs)(2)) was successfully expressed by the Reynolds number (Re) and the dimension of the channel (r). At low Re, higher flow rates encouraged growth of biofilms, while higher flow rates with high Re suppressed growth of biofilms. These results provide a simple model as a theoretical basis for understanding development of biofilms in microfluidic channels.

A microfluidic array for quantitative analysis of human neural stem cell self-renewal and differentiation in three-dimensional hypoxic microenvironment

We report a microfluidic array for investigating and quantitatively analyzing human neural stem cell (hNSC) self-renewal and differentiation in an in vivo-like microenvironment. NSC niche conditions, including three-dimensional (3D) extracellular matrices and low oxygen tension, were effectively reconstituted in the microfluidic array in a combinatorial manner. The array device was fabricated to be detachable, rendering it compatible with quantitative real-time polymerase chain reaction for quantifying the effects of the biomimetic conditions on hNSC self-renewal and differentiation. We show that throughput of 3D cell culture and quantitative analysis can be increased. We also show that 3D hypoxic microenvironments maintain hNSC self-renewal capacity and direct neuronal commitment during hNSC differentiation.

Extracellular matrix heterogeneity regulates three-dimensional morphologies of breast adenocarcinoma cell invasion

Plasticity and reciprocity of breast cancer cells to various extracellular matrice (ECMs) are three-dimensionally analyzed in quantitative way in a novel and powerful microfluidic in vitro platform. This successfully demonstrates the metastatic potential of cancer cells and their effective strategies of ECM proteolytic remodeling and morphological change, while interacting with other cells and invading into heterogeneous ECMs.

Organic thin film transistors with ink-jet printed metal nanoparticle electrodes of a reduced channel length by laser ablation

The authors have demonstrated organic thin film transistors (OTFTs) based on the ink-jet printed electrodes in which a reduced channel length is accomplished by laser ablation. Laser ablation on the dried silver nanoparticle electrode formed by ink-jet printing effectively shortened the channel length down to 5μm, which is difficult to achieve by ink-jet printing alone. Reducing the channel length using this hybrid technique also allows them to observe the contact resistance effect in the OTFTs, which involves the printed silver nanoparticle electrode of a lower work function with respect to the ionization energy of the organic semiconductor.

Nanosecond laser ablation of silver nanoparticle film

Abstract. Nanosecond laser ablation of polyvinylpyrrolidone (PVP) protected silver nanoparticle (20 nm diameter) film is studied using a frequency doubled Nd:YAG nanosecond laser (532 nm wavelength, 6 ns full width half maximum pulse width). In the sintered silver nanoparticle film, absorbed light energy conducts well through the sintered porous structure, resulting in ablation craters of a porous dome shape or crown shape depending on the irradiation fluence due to the sudden vaporization of the PVP. In the unsintered silver nanoparticle film, the ablation crater with a clean edge profile is formed and many coalesced nanoparticles of 50 to 100 nm in size are observed inside the ablation crater. These results and an order of magnitude analysis indicate that the absorbed thermal energy is confined within the nanoparticles, causing melting of nanoparticles and their coalescence to larger agglomerates, which are removed following melting and subsequent partial vaporization.

Implantable microfluidic device for the formation of three-dimensional vasculature by human endothelial progenitor cells

Vasculogenesis is an important morphogenetic event for vascular tissue engineering and ischemic disease treatment. Stem and progenitor cells can contribute to vasculogenesis via endothelial differentiation and direct participation in blood vessel formation. In this study, we developed an implantable microfluidic device to facilitate formation of three-dimensional (3D) vascular structures by human endothelial progenitor cells (hEPCs). The microfluidic device was made of biodegradable poly(lactic-co-glycolic acid) (PLGA) using a microchannel patterned silicon wafer made by soft lithography. A collagen type I (Col I) hydrogel containing hEPCs filled the microfluidic channels to reconstitute a 3D microenvironment for facilitating vascular structure formation by hEPCs. The device seeded with hEPCs was implanted into the subcutaneous space of athymic mice and retrieved one and four weeks after implantation. Histology and immunohistochemistry revealed that hEPCs formed a 3D capillary network expressing endothelial cell-specific proteins in the channel of the PLGA microfluidic device. This result indicates that a 3D microscale extracellular matrix reconstituted in the microchannel can promote the endothelial differentiation of hEPCs and in turn hEPC-mediated vasculogenesis. The PLGA microfluidic device reported herein may be useful as an implantable tissue-engineering scaffold for vascularized tissue reconstruction and therapeutic angiogenesis.