d-mannose-sensitive pilus of Acinetobacter baumannii is linked to biofilm formation and adherence onto respiratory tract epithelial cells.

BACKGROUND/PURPOSE: Acinetobacter baumannii is an important nosocomial pathogen. To better understand the role of CsuA/BABCDE pilus of A. baumannii in virulence, bacterial biofilm formation, adherence and carbohydrate-mediated inhibition were conducted. METHODS: CsuA/BABCDE pilus-producing (abbreviated Csu pilus) operon of A. baumannii ATCC17978 was cloned for analysis of biofilm formation on an abiotic plastic plate, bacterial adherence to respiratory epithelial human A549 cells and carbohydrate-mediated inhibition. The carbohydrates used for inhibition of biofilm formation and adherence to A549 cells included monosaccharides, pyranosides, and mannose-polymers. RESULTS: The Csu pilus of A. baumannii ATCC17978 was cloned and expressed into a non-pilus-producing Escherichia coli JM109, and was knocked out as well. The recombinant Csu (rCsu) pilus on E. coli JM109/rCsu pilus-producing clone observed by both electro-microscopy and atomic force microscopy showed abundant, while Csu-knockout A. baumannii ATCC17978 mutant appeared less or no pilus production. The E. coli JM109/rCsu pilus-producing clone significantly increased biofilm formation and adherence to A549 cells; however, the Csu-knockout mutant dramatically lost biofilm-making ability but, in contrast, increased adherence. Moreover, both of biofilm formation and adherence could be significantly inhibited by d-mannose and methyl-α-d-mannopyranoside in Csu pilus-producing E. coli JM109, whereas in A. baumannii ATCC17978, high concentration of carbohydrates was required for the inhibition, suggesting that Csu pilus is sensitive to d-mannose. CONCLUSION: This is the first study confirming that Csu pilus of A. baumannii belongs to mannose-sensitive type 1 pilus family and contributes to biofilm formation and bacterial adherence to human epithelial cells.

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.

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.

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.

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.

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 microm/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.

Stepped changes of monovalent ligand-binding force during ligand-induced clustering of integrin alphaIIB beta3.

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 alphaIIb beta3 (CHO alphaIIb beta3), to the bead carrier coated with the snake venom rhodostomin that served as an activated ligand for integrin alphaIIb beta3. A progressive increase of total binding force over time was noticed when the bead interacted with the CHO alphaIIb beta3 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.