Long Hsu

Liver-cell patterning Lab Chip: mimicking the morphology of liver lobule tissue

A lobule-mimetic cell-patterning technique for on-chip reconstruction of centimetre-scale liver tissue of heterogeneous hepatic and endothelial cells via an enhanced field-induced dielectrophoresis (DEP) trap is demonstrated and reported. By mimicking the basic morphology of liver tissue, the classic hepatic lobule, the lobule-mimetic-stellate-electrodes array was designed for cell patterning. Through DEP manipulation, well-defined and enhanced spatial electric field gradients were created for in-parallel manipulation of massive individual cells. With this liver-cell patterning labchip design, the original randomly distributed hepatic and endothelial cells inside the microfluidic chamber can be manipulated separately and aligned into the desired pattern that mimicks the morphology of liver lobule tissue. Experimental results showed that both hepatic and endothelial cells were orderly guided, snared, and aligned along the field-induced orientation to form the lobule-mimetic pattern. About 95% cell viability of hepatic and endothelial cells was also observed after cell-patterning demonstration via a fluorescent assay technique. The liver function of CYP450-1A1 enzyme activity showed an 80% enhancement for our engineered liver tissue (HepG2+HUVECs) compared to the non-patterned pure HepG2 for two-day culturing.

Novel microchip for in situ TEM imaging of living organisms and bio-reactions in aqueous conditions

A novel and disposable microchip (K-kit) with SiO(2) nano-membranes was developed and used as a specimen kit for in situ imaging of living organisms in an aqueous condition using transmission electron microscopy (TEM) without equipment modification. This K-kit enabled the successful TEM observation of living Escherichia coli cells and the tellurite reduction process in Klebsiella pneumoniae. The K. pneumoniae and Saccharomyces cerevisiae can stay alive in K-kit after continuous TEM imaging for up to 14 s and 42 s, respectively. Besides, different tellurite reduction profiles in cells grown in aerobic and anaerobic environments can be clearly revealed. These results demonstrate that the K-kit developed in this paper can be useful for observing living organisms and monitoring biological processes in situ.

Dynamic manipulation and patterning of microparticles and cells by using TiOPc-based optoelectronic dielectrophoresis

We develop light-driven optoelectronic tweezers based on the organic photoconductive material titanium oxide phthalocyanine. These tweezers function based on negative dielectrophoresis (nDEP). The dynamic manipulation of a single microparticle and cell patterning are demonstrated by using this light-driven optoelectronic DEP chip. The adaptive light patterns that drive the optoelectronic DEP onchip are designed by using Flash software to approach appropriate dynamic manipulation. This is also the first reported demonstration, to the best of our knowledge, for successfully patterning such delicate cells from human hepatocellular liver carcinoma cell line HepG2 by using any optoelectronic tweezers.

A microfluidic chip with a U-shaped microstructure array for multicellular spheroid formation, culturing and analysis

Multicellular spheroids (MCS), formed by self-assembly of single cells, are commonly used as a three-dimensional cell culture model to bridge the gap between in vitro monolayer culture and in vivo tissues. However, current methods for MCS generation and analysis still suffer drawbacks such as being labor-intensive and of poor controllability, and are not suitable for high-throughput applications. This study demonstrates a novel microfluidic chip to facilitate MCS formation, culturing and analysis. The chip contains an array of U-shaped microstructures fabricated by photopolymerizing the poly(ethylene glycol) diacrylate hydrogel through defining the ultraviolet light exposure pattern with a photomask. The geometry of the U-shaped microstructures allowed trapping cells into the pocket through the actions of fluid flow and the force of gravity. The hydrogel is non-adherent for cells, promoting the formation of MCS. Its permselective property also facilitates exchange of nutrients and waste for MCS, while providing protection of MCS from shearing stress during the medium perfusion. Heterotypic MCS can be formed easily by manipulating the cell trapping steps. Subsequent drug susceptibility analysis and long-term culture could also be achieved within the same chip. This MCS formation and culture platform can be used as a micro-scale bioreactor and applied in many cell biology and drug testing studies.

Optical trapping of a spherically symmetric sphere in the ray-optics regime: a model for optical tweezers upon cells

Since their invention in 1986, optical tweezers have become a popular manipulation and force measurement tool in cellular and molecular biology. However, until recently there has not been a sophisticated model for optical tweezers on trapping cells in the ray-optics regime. We present a model for optical tweezers to calculate the optical force upon a spherically symmetric multilayer sphere representing a common biological cell. A numerical simulation of this model shows that not only is the magnitude of the optical force upon a Chinese hamster ovary cell significantly three times smaller than that upon a polystyrene bead of the same size, but the distribution of the optical force upon a cell is also much different from that upon a uniform particle, and there is a 30% difference in the optical trapping stiffness of these two cases. Furthermore, under a small variant condition for the refractive indices of any adjacent layers of the sphere, this model provides a simple approximation to calculate the optical force and the stiffness of an optical tweezers system.

Structural and Mechanical Properties of Klebsiella pneumoniae Type 3 Fimbriae

This study investigated the structural and mechanical properties of Klebsiella pneumoniae type 3 fimbriae, which constitute a known virulence factor for the bacterium. Transmission electron microscopy and optical tweezers were used to understand the ability of the bacterium to survive flushes. An individual K. pneumoniae type 3 fimbria exhibited a helix-like structure with a pitch of 4.1 nm and a three-phase force-extension curve. The fimbria was first nonlinearly stretched with increasing force. Then, it started to uncoil and extended several micrometers at a fixed force of 66 ± 4 pN (n = 22). Finally, the extension of the fimbria shifted to the third phase, with a characteristic force of 102 ± 9 pN (n = 14) at the inflection point. Compared with the P fimbriae and type 1 fimbriae of uropathogenic Escherichia coli, K. pneumoniae type 3 fimbriae have a larger pitch in the helix-like structure and stronger uncoiling and characteristic forces.

Cell patterning via diffraction-induced optoelectronic dielectrophoresis force on an organic photoconductive chip

A laser diffraction-induced dielectrophoresis (DEP) phenomenon for the patterning and manipulation of individual HepG2 cells and polystyrene beads via positive/negative DEP forces is reported in this paper. The optoelectronic substrate was fabricated using an organic photoconductive material, TiOPc, via a spin-coating process on an indium tin oxide glass surface. A piece of square aperture array grid grating was utilized to transform the collimating He-Ne laser beam into the multi-spot diffraction pattern which forms the virtual electrodes as the TiOPc-coating surface was illuminated by the multi-spot diffraction light pattern. HepG2 cells were trapped at the spot centers and polystyrene beads were trapped within the dim region of the illuminated image. The simulation results of light-induced electric field and a Fresnel diffraction image illustrated the distribution of trapped microparticles. The HepG2 morphology change, adhesion, and growth during a 5-day culture period demonstrated the cell viability through our manipulation. The power density inducing DEP phenomena, the characteristics of the thin TiOPc coating layer, the operating ac voltage/frequency, the sandwiched medium, the temperature rise due to the ac electric fields and the illuminating patterns are discussed in this paper. This concept of utilizing laser diffraction images to generate virtual electrodes on our TiOPc-based optoelectronic DEP chip extends the applications of optoelectronic dielectrophoretic manipulation.

Light-driven manipulation of picobubbles on a titanium oxide phthalocyanine-based optoelectronic chip

Microbubbles have a variety of applications in science and biological technology. Here, we demonstrate the manipulation of the picoliter gas bubble (picobubble) based on the optoelectronic-mechanism. The organic photoconductive material, titanium oxide phthalocyanine (TiOPc), was developed to make the light-sensitive substrate of this optoelectronic chip. The virtual electrodes are formed by projecting the dynamic light pattern onto TiOPc layer for generating the desired nonuniform electric field. The picobubble suspended in silicone oil can be manipulated with the velocity of 40–50 μm/s. The driving force up to 160 pico-Newtons could be generated for manipulating a gas bubble of 300 picoliters.

Stepped changes of monovalent ligand-binding force during ligand-induced clustering of integrin αIIBβ3

Recent evidence demonstrated that conformational changes of the integrin during receptor activation affected its binding to extracellular matrix; however, experimental assessment of ligand-receptor binding following the initial molecular interaction has rarely been carried out at a single-molecule resolution. In the present study, laser tweezers were used to measure the binding force exerted by a live Chinese hamster ovary cell that expressed integrin αIIbβ3 (CHO αIIbβ3), to the bead carrier coated with the snake venom rhodostomin that served as an activated ligand for integrin αIIbβ3. A progressive increase of total binding force over time was noticed when the bead interacted with the CHO αIIbβ3 cell; such an increase was due mainly to the recruitment of more integrin molecules to the bead-cell interface. When the binding strength exerted by a single ligand-receptor pair was derived from the “polyvalent” measurements, surprisingly, a stepped decrease of the “monovalent binding force” was noted (from 4.15 to 2.54 piconewtons (pN)); such decrease appeared to occur during the ligand-induced integrin clustering process. On the other hand, the mutant rhodostomin defective in clustering integrins exhibited only one (1.81 pN) unit binding strength.

Effects of Garlic Oil on the Migration of Neutrophil-Like Cell Studied by Using a Chemotactic Gradient Labchip

We have designed and fabricated a novel chemotactic gradient Labchip for studying cell migration quantitatively. Owing to the great potential of garlic and its preparations in developing antiinflammatory drugs, the aim of the present study is to investigate the effect of garlic oil on the locomotion of a neutrophil-like cell by measuring the dynamic features of cell migration including migration direction, average migration speed, chemotactic index (CI), and motility index (MI) with the newly designed Labchip. We found that garlic oil treatment lowered the values of CI and MI and reduced the average speed of cell migration from 13 to 8 μm/min. The results indicate that garlic oil is a potential inhibitor for neutrophil-like cell migration and chemotactic responsiveness. By comparing with the effects of nocodazole and cytochalasin B, we also suggest that the antiinflammatory activity exhibited by garlic oil was mainly through inhibiting the assembly-disassembly processes of the cytoskeleton.