Abstract
Limited space for cell division, perhaps similar to the compressed microenvironment of a growing tumor, has been shown to induce phenotypic and karyotypic changes to a cell during mitosis. To expand understanding of this missegregation of chromosomes in aberrant multi-daughter or asymmetric cell divisions, we present a simple technique for subjecting mammalian cells to adjustable levels of confinement which allows subsequent interrogation of intracellular molecular components using high resolution confocal imaging. PDMS micropatterned confinement structures of subcellular height with neighboring taller media reservoir channels were secured on top of confluent cells with a custom compression well-plate system. The system improved ease of use over previous devices since confined cells could be initially grown on glass coverslips in a 12-well plate, and subsequently be imaged by high resolution confocal imaging, or during compression by live cell imaging. Live cell imaging showed a significant increase in abnormal divisions of confined cells across three different cell lines (HeLa, A375, and A549). Immunofluoresecence stains revealed a significant increase in cell diameter and chromosome area of confined cells, but no significant increase in centrosome-centromere distance upon division when compared to unconfined cells. The developed system could open up studies more broadly on confinement effects on mitotic processes, and increase the throughput of such studies.
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Acknowledgments
The authors would like to thank Yeganeh Amini, Coleman Murray, Dr. Matthew Schibler, and Nikhil Kalluri for their assistance in this work. Confocal laser scanning microscopy was performed at the CNSI Advanced Light Microscopy/Spectroscopy Shared Resource Facility at UCLA, supported with funding from NIH-NCRR shared resources grant (CJX1-443835-WS-29646) and NSF Major Research Instrumentation grant (CHE-0722519).
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The manuscript does not contain clinical studies or patient data. The authors declare that they have no conflict of interest.
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Kittur, H., Weaver, W. & Di Carlo, D. Well-plate mechanical confinement platform for studies of mechanical mutagenesis. Biomed Microdevices 16, 439–447 (2014). https://doi.org/10.1007/s10544-014-9846-4
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DOI: https://doi.org/10.1007/s10544-014-9846-4