Photothermal Agarose Microfabrication Technology for Collective Cell Migration Analysis

Agarose photothermal microfabrication technology is one of the micropatterning techniques that has the advantage of simple and flexible real-time fabrication even during the cultivation of cells. To examine the ability and limitation of the agarose microstructures, we investigated the collective epithelial cell migration behavior in two-dimensional agarose confined structures. Agarose microchannels from 10 to 211 micrometer width were fabricated with a spot heating of a focused 1480 nm wavelength infrared laser to the thin agarose layer coated on the cultivation dish after the cells occupied the reservoir. The collective cell migration velocity maintained constant regardless of their extension distance, whereas the width dependency of those velocities was maximized around 30 micrometer width and decreased both in the narrower and wider microchannels. The single-cell tracking revealed that the decrease of velocity in the narrower width was caused by the apparent increase of aspect ratio of cell shape (up to 8.9). In contrast, the decrease in the wider channels was mainly caused by the increase of the random walk-like behavior of component cells. The results confirmed the advantages of this method: (1) flexible fabrication without any pre-designing, (2) modification even during cultivation, and (3) the cells were confined in the agarose geometry.

Stepwise neuronal network pattern formation in agarose gel during cultivation using non-destructive microneedle photothermal microfabrication

Conventional neuronal network pattern formation techniques cannot control the arrangement of axons and dendrites because network structures must be fixed before neurite differentiation. To overcome this limitation, we developed a non-destructive stepwise microfabrication technique that can be used to alter microchannels within agarose to guide neurites during elongation. Micropatterns were formed in thin agarose layer coating of a cultivation dish using the tip of a 0.7 $$\upmu \mathrm{m}$$ μ m -diameter platinum-coated glass microneedle heated by a focused 1064-nm wavelength infrared laser, which has no absorbance of water. As the size of the heat source was 0.7 $$\upmu \mathrm{m}$$ μ m , which is smaller than the laser wavelength, the temperature fell to 45 $$^\circ \hbox {C}$$ ∘ C within a distance of 7.0 $$\upmu \mathrm{m}$$ μ m from the edge of the etched agarose microchannel. We exploited the fast temperature decay property to guide cell-to-cell connection during neuronal network cultivation. The first neurite of a hippocampal cell from a microchamber was guided to a microchannel leading to the target neuron with stepwise etching of the micrometer resolution microchannel in the agarose layer, and the elongated neurites were not damaged by the heat of etching. The results indicate the potential of this new technique for fully direction-controlled on-chip neuronal network studies.

In vitro studies on the behaviour of second-stage juveniles of Heterodera schachtii (Nematoda: Heteroderidae) in response to host plant root exudates

The behaviour of Heterodera schachtii second-stage juveniles in response to mustard (Sinapis alba) rooxudates was observed and analysed under aseptic conditions in a standardized bioassay. Aggregation of juveniles on an agarose layer occurred within less than 30 min in the area where root exudates had been applied and persisted for several hours. Analysis of time-lapse video recordings showed that the aggregation did not result from a directed orientation of the juvenile towards the root exudate. This was supported by an orientation assay using single juveniles. Aggregated juveniles showed pre-infection exploratory behaviour, including stylet thrusting and head-end bending, while staying at rest for several minutes.

Motility of Splenic Cells from Normal and Fibrosarcoma-Bearing Mice in Agarose Assay

When nonadherent splenic cells from normal and tumor-bearing (mouse fibrosarcoma, MFS) Swiss mice were added to wells made in agarose layers in plastic petri dishes, subpopulations of cells from tumor-bearing mice were seen to migrate out of the wells, whereas those from normal mice did not. The proportion of migratory cells among the lower density (< 1.057 to < 1.069 g/ml) cells was larger than that of higher density (<1.069 to < 1.087 g/ml) cells. When the plastic surface underneath the agarose layer was covered with a monolayer of MFS cells, the splenic cells from normal mice also migrated out of the wells. About 20 % dead MFS cells were observed in the zone of migration when the migratory cells were from normal mice, and about 30 % when the migratory cells were from tumor bearing mice. Apart from revealing the differences between the migratory behavior of splenic cells, the present work also suggests a novel application of agarose methodology in the study of interaction of cytotoxic cells with malignant cells.