Abstract
Laser cell patterning is a distinctly effective technique for creating cell arrangements in a culture that replicates in vivo tissue structure for studying contact-mediated cell-cell interactions. Conventional laser-based single-cell-manipulation techniques are limited by their inability to pattern irregularly shaped cells, such as rod-shaped cardiomyocytes. We report the use of a spatial light modulator loaded with a computer-generated phase map to shape a single laser source into multiple laser-guidance beams distributed around the outer contour of an irregularly shaped cell to achieve accurate cell patterning. In addition to describing the principle and practice of the system design, we present what is to our knowledge the first achievement of patterning large, irregularly (rod) shaped adult rat cardiomyocytes in an end-to-end connected alignment to replicate the in vivo heart muscle cell connection without the use of substrate surface modifications, which can interfere with in vivo-like cell-cell and cell-extracellular matrix interactions. Our research demonstrates that two-stage multiple-beam laser guidance is effective: 1) A dual-beam configuration horizontally translates a cell from the outlet of the microfluidic cell delivery channel to a position above the cell deposition site and 2) A quad-beam configuration rapidly propels the cell axially through the suspension medium to the culture substrate with the vertical movement of the cell patterning chamber. Our study reveals that 90% of the patterned cells maintained end-to-end connection 30 minutes after patterning, and mechanical junctions could be reinstalled between laser connected cells after overnight incubation. This demonstrates that multiple-beam laser patterning is an outstanding tool for in vitro studying contact-mediated cell-cell interactions among irregularly shaped cells.
Highlights
With conventional cell-culture techniques, the spatial control of single cells necessary to recreate the cell-cell contact arrangement found in native tissue is difficult
In addition to describing the principle and practice of the system design, we present what is to our knowledge the first achievement of patterning large, irregularly shaped adult rat cardiomyocytes in an end-to-end connected alignment to replicate the in vivo heart muscle cell connection without use of substrate surface modifications, which can interfere with in vivo-like cell-cell and cell-extracellular matrix interactions
For cell guidance, used only a single laser beam. It could move large irregular cells, controlling cell orientation prior to patterning on a culture substrate was difficult due to the nonuniform hydrodynamic and optical forces cells experience during laser patterning: Viscous drag around the irregularly shaped cell body affects its 3D orientation during optical guidance in the laser patterning medium during transit from the cell injection biochip to the point where the cell is pushed to the culture substrate
Summary
With conventional cell-culture techniques, the spatial control of single cells necessary to recreate the cell-cell contact arrangement found in native tissue is difficult. A low NA microscope objective generates a weakly focused laser beam to simultaneously localize a particle to the beam axis and propel it in the beam-propagation direction With these principles, we developed a laser cellmicropatterning system in which the laser beam is focused in a transitional state between generating an optical trap and optical guidance [9]. We developed a laser cellmicropatterning system in which the laser beam is focused in a transitional state between generating an optical trap and optical guidance [9] Using this system, a single biological cell can be optically moved within a typical cell culture dish (e.g., 30 mm) and patterned into a predesignated cell culture microniche with very high spatial and temporal resolution [10,11,12]. This and the other currently available laser-based single-cellmanipulation techniques are limited by their inability to pattern irregularly shaped cells, such as rod-shaped cardiomyocytes
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