Photothermal Agarose Microfabrication Technology for Collective Cell Migration Analysis.

Mitsuru Sentoku,Hiromichi Hashimoto,Masaharu Endo,Kento Iida,Kenji Yasuda

doi:10.3390/mi12091015

Abstract

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.

Highlights

As the thin agarose layer coated on the cultivation dish prevents the adhesion of cells on the dish, the vascular endothelial cells can attach only to the exposed region of the tissue culture dish for proliferation
The cells propagated from the reservoir remained inside the etched microchannels and no cells exceeded the etched structures even though a covering lid was not placed on the agarose microstructures
We have demonstrated another advantage of agarose microfabrication, which is the flexible stepwise microfabrication even during cell cultivation, for regulation of initial starting timing of cell propagation after the cells were confluent in the reservoir

Summary

Introduction

Microetching and microprinting are two major complementary approaches to form the confined spatial arrangement of cells The former is based on the semiconductor etching technology with photolithography or three-dimensional polymer structures [3,4]. Stepwise activation of stamped thermoresponsive molecules for direct control of neurite elongation in pre-designed patterns during cultivation was recently reported [7]. In both technologies, the designs are pre-determined by the photomasks or stamps and renders difficult the preparation of changes in complex structures and additional micropatterning after the cells are seeded onto the microengineered environment

Methods

Results

Discussion

Conclusion