| To precisely control the position of multiple cell types in a culture for the study of cell-cell contact interactions, we have developed a laser micropatterning technique. The technique employs the optical forces of a weakly focused laser beam. In the beam's focal region, the optical force draws microparticles, such as cells, into the center of the beam, propels them along the beam axis, and guides them onto a target surface. Specific patterns are created through computer-controlled micromanipulation of the substrate relative to the laser.; In this thesis, we systematically vary the controllable laser parameters, namely, wavelength, intensity, and exposure time of the laser on single cells to clearly establish laser parameters that allow negligible cell damage with significant cellular position control. To accomplish this goal, embryonic day 7 (E7) chick forebrains neurons were cultured with control and test cells selected one hour after seeding to allow attachment, at which time test cells were subjected to the laser. Cells were imaged at 4, 12, 24, and 36 hours after laser exposure to evaluate viability and functionality by using the Live/Dead Viability Cytotoxicity Kit (Molecular Probes L3224) and measuring neurite outgrowth, respectively. We have modified the COMET (single-cell gel electrophoresis) assay to quantify DNA damage on individual patterned cells and, thus, more extensively explore possible cell damage. For all parameters tested, our results show that there is no significant effect of laser exposure on cell viability or neurite outgrowth. |
