Kyoung Je Cha

Effect of Replicated Polymeric Substrate with Lotus Surface Structure on Adipose-Derived Stem Cell Behaviors

We fabricated polystyrene substrates with lotus leaf surface structure (LLSS) and investigated cell behaviors, including attachment, morphology, proliferation, and differentiation of adipose-derived stem cells (ASCs) on them. Compared to the flat substrate, the LLSS substrate induced higher cell attachment rate, but did not significantly change the cell proliferation rate. In addition, ASCs on the LLSS substrate exhibited relatively narrower spreading morphology and less organized cytoskeleton, there by resulting in smaller sizes of cells than those on the flat substrate. According to histochemical staining and RT-PCR analysis, the LLSS substrate induced higher adipogenic differentiation of ASCs than the flat substrate, while chondrogenic and osteogenic differentiation were decreased.

A portable pressure pump for microfluidic lab-on-a-chip systems using a porous polydimethylsiloxane (PDMS) sponge

In this paper, we propose a novel portable and disposable pressure pump using a porous polydimethylsiloxane (PDMS) sponge and demonstrate its application to a microfluidic lab-on-a-chip. The porous PDMS sponge was simply fabricated by a sugar leaching technique based on capillary suction of pre-cured PDMS into lumps of sugar, thereby enabling us to achieve the porous PDMS sponge composed of interconnected micropores. To indicate the characteristics of the porous PDMS sponge and pump, we measured the average porosities of them whose values were 0.64 and 0.34, respectively. A stress–strain relationship of the fabricated portable pressure pump represented a linear behavior in the compressive strain range of 0 to 20%. Within this range, a pumping volume of the pressure pump could be linearly controlled by the compressed strain. Finally, the fabricated porous PDMS pump was successfully demonstrated as a portable pressure pump for a disposable microfluidic lab-on-a-chip for efficient detection of agglutination. The proposed portable pressure pump can be potentially applicable to various disposable microfluidic lab-on-a-chip systems.

Enhanced osteogenic fate and function of MC3T3-E1 cells on nanoengineered polystyrene surfaces with nanopillar and nanopore arrays

During in vitro culture, cell fate and function, including cell adhesion, morphology, proliferation and differentiation, are affected by surface characteristics, such as geometry, wettability, hardness, chemistry and charge. This study replicated two different types of nanoengineered polystyrene surfaces (NPS) containing nanopillar (NPS-Pi) or nanopore (NPS-Po) arrays by hot embossing and investigated their topographical effects on cell behavior using osteoblast-like MC3T3-E1 cells. To mass-replicate NPS, rigid metal nano-stamps were manufactured by nickel electroforming onto two different nano-templates: (1) a nanopore-arrayed anodic aluminum oxide nano-template using two-step electrochemical oxidation and (2) a nanopillar-arrayed polymer using hot embossing process. The physical and mechanical properties of the NPS, including geometry, wettability, hardness and elastic modulus, were evaluated with the help of field emission-scanning electron microscopy, a contact angle meter, and a nanoindenter. The nanotopography maintained the bulk property, while drastically changing the surface properties. In vitro the NPS had significant effects on MC3T3-E1 cell morphology, attachment, proliferation and osteogenic differentiation compared to a flat substrate due to the altered physical and mechanical surface properties of the nanoengineered surface. Interestingly, the NPS-Po was more effective at enhancing cell proliferation and osteogenesis differentiation. One potential explanation for these results may be that the subcellular binding sites induced by the nanostructures changed the cell morphology and promoted contractile cytoskeletons, thereby enhancing osteogenic differentiation. This, which allows for the cost-effective replication of NPS and the control of cell behavior, has various applications with respect to biomedical and cell surface interaction studies, in addition to enhanced osteogenic cell fate and function.

Mass-producible nano-featured polystyrene surfaces for regulating the differentiation of human adipose-derived stem cells.

In this study, we report an efficient and cost-effective method of fabricating polystyrene (PS) nano-featured substrates containing nanopore (NPo) and nanopillar (NPi) arrays based on hot embossing using nickel nano-stamps. We investigate the behavior of adipose-derived stem cells (ASCs), including adhesion, morphology, proliferation and differentiation, on the replicated PS surfaces. Compared to a flat substrate, NPo- and NPi-featured substrates do not alter the morphology of stem cells. However, both NPo- and NPi-featured substrates induce different integrin expression and lower formation of focal adhesion complexes. In addition, ASCs on the NPo-featured substrate exhibit greater adipogenic differentiation, while the NPi-featured substrate induces higher osteogenic differentiation.

Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells.

Nano Petri dishes: a new polystyrene platform for studying cell-nanoengineered surface interactions

In this study, we fabricated and fully characterized a new type of polystyrene (PS) cell-culture platform containing nanoengineered surfaces (NES), referred to as a nano Petri dish, which can be used at the transition stage of basic cell–NES interaction studies for clinical applications. Nano-injection molding in this study was used for the mass production of the nano Petri dish having nanopore arrays. The effects of processing parameters of the injection molding on the replication quality of the nanopore arrays were quantitatively evaluated by means of design of experiments based on the Taguchi method. This allowed efficient and reliable cell culture studies by providing large numbers of the same dishes, in addition to removing the fixation step of the NES plates inside the cell-culture container. Physical, chemical and mechanical properties of the NES, as well as cell behavior including attachment and proliferation of human osteosarcoma MG-63 cells on the NES, were then characterized, with and without the oxygen plasma surface treatment.

Micropattern array with gradient size (µPAGS) plastic surfaces fabricated by PDMS (polydimethylsiloxane) mold-based hot embossing technique for investigation of cell–surface interaction

Recently, it was found that the variations of physical environment significantly affect cell behaviors including cell proliferation, migration and differentiation. Through a plastic surface with controlled mechanical properties such as stiffness, one can change the orientation and migration of cells in a particular direction, thereby determining cell behaviors. In this study, we demonstrate a polydimethylsiloxane (PDMS) mold-based hot embossing technique for rapid, simple and low-cost replication of polystyrene (PS) surfaces having micropatterns. The PDMS mold was fabricated by UV-photolithography followed by PDMS casting; the elastomeric properties of PDMS enabled us to obtain conformal contact of the PDMS mold to a PS surface and to create high transcription quality of micropatterns on the PS surface. Two different types of circular micropillar and microwell arrays were successfully replicated on the PS surfaces based on the suggested technique. The micropatterns were designed to have various diameters (2-150 µm), spacings (2-160 µm) and heights (1.4, 2.4, 8.2 and 14.9 µm), so as to generate the gradient of physical properties on the surface. Experimental parametric studies indicated that (1) the embossing temperature became a critical processing parameter as the aspect ratio of micropattern increased and (2) the PDMS mold-based hot embossing could successfully replicate micropatterns, even having an aspect ratio of 2.7 for micropattern diameter of 6 µm, with an optimal processing condition (embossing pressure and temperature of 0.4 MPa and 130 °C, respectively) in this study. We carried out cell experiments with adipose-derived stem cells on the replicated PS surface with the height of 1.4 µm to investigate cellular behaviors in response to the micropattern array with gradient size. Cellular experiment results showed that the micropillar-arrayed surface improved cell proliferation as compared with the microwell-arrayed surface. We could also estimate the ranges of pattern sizes having the desired effects on the cellular behaviors.

Nanofabrication of Nickel Stamp for Replication of Polymer Multilevel Nanolens Arrays via Modified Two-Step Anodization Process

In the present study, we propose a novel and low-cost nanofabrication technique for a nickel stamp for mass replication of polymer multilevel nanolens (MLNL) arrays. The present nanofabrication technique consists of a modified two-step anodization process and successive electroforming processes. As the first step in the fabrication of the nickel stamp, an aluminum template with multilevel nanodimple (MLND) arrays on an aluminum substrate was precisely fabricated by a modified two-step anodization, in which the processing condition of the second anodization step was changed. A nickel stamp with MLND arrays for the replication of a polymer substrate with MLNL arrays was achieved by electroforming onto the aluminum template with MLND arrays (a nickel template with MLNL arrays) followed by a second electroforming onto the obtained nickel template with MLNL arrays. The fabricated nickel stamp with MLND arrays was successfully used to replicate the polymer substrates with MLNL arrays via a hot embossing process.

Fabrication of Polymer Micropyramid/Nanolens Combined Structures Using Microstructured Nanodimpled Aluminum Stamp

In this study, we present an efficient and low-cost fabrication technique of polymer micropyramid/nanolens combined structures (MPNL-CS). The present fabrication technique is based on hot embossing of polymer substrates using a microstructured nanodimpled aluminum (MNA) stamp. The MNA stamp for the replication of polymer MPNL-CS was realized by combining three fabrication steps. (1) A nickel stamp with micropyramid array was manufactured by electroforming onto an anisotropically wet-etched silicon substrate. (2) A reverse micropyramid cavity array was transcribed on an electropolished aluminum substrate by microindentation with the nickel stamp. (3) The MNA stamp was finally realized by forming nanodimple arrays onto the reverse micropyramid array on the aluminum substrate through an electrochemical oxidation (anodization) process. In the final step, we have optimized the electrochemical oxidation process to realize nanodimple arrays at the sharp edge reverse micropyramid without defects. The MPNL-CS were successfully replicated on polymer substrates via a hot embossing process with the fabricated MNA stamp through investigating the effect of embossing pressure on the transcription quality of MPNL-CS. The present fabrication technique can be widely applied to the realization of various types of three-dimensional micro/nano combined structures.

Cell density-dependent differential proliferation of neural stem cells on omnidirectional nanopore-arrayed surface

Recently, the importance of surface nanotopography in the determination of stem cell fate and behavior has been revealed. In the current study, we generated polystyrene cell-culture dishes with an omnidirectional nanopore arrayed surface (ONAS) (diameter: 200 nm, depth: 500 nm, center-to-center distance: 500 nm) and investigated the effects of nanotopography on rat neural stem cells (NSCs). NSCs cultured on ONAS proliferated better than those on the flat surface when cell density was low and showed less spontaneous differentiation during proliferation in the presence of mitogens. Interestingly, NSCs cultured on ONAS at clonal density demonstrated a propensity to generate neurospheres, whereas those on the flat surface migrated out, proliferated as individuals, and spread out to attach to the surface. However, the differential patterns of proliferation were cell density-dependent since the distinct phenomena were lost when cell density was increased. ONAS modulated cytoskeletal reorganization and inhibited formation of focal adhesion, which is generally observed in NSCs grown on flat surfaces. ONAS appeared to reinforce NSC-NSC interaction, restricted individual cell migration and prohibited NSC attachment to the nanopore surface. These data demonstrate that ONAS maintains NSCs as undifferentiated while retaining multipotency and is a better topography for culturing low density NSCs.