Aneuploidy in Confined Microenvironments Figure 5. A penta-daughter cell division may lead to aneuploid daughter cells. As a qualitative example, this cell is in a partially confined state between 3–7 mm. Extreme confinement conditions observed here can lead to aberrant mitotic orientation resulting in highly asymmetric divisions. These microscopic images show the time lapse of a cell undergoing a single 1R5 successful mitosis. Scale bar: 20 mm. doi:10.1371/journal.pone.0038986.g005 extracellular mechanical environment and the overall orientation and the multi-polarity of the cellular division axis. The ability of this highly confined microenvironment to induce aberrant divisions, specifically multi-daughter divisions, is an intriguing finding in itself. Previously, work has been done to elucidate the biomolecular players involved in multi-centrosome divisions by Ganem et al. [4], however here we contribute by supporting the hypothesis that multi-daughter divisions may be due, at least in part, to abnormal mechanical interactions between the cell and the immediate environment. The observations made and metrics analyzed here are purely phenotypic. While chromosome quantification is not presented here, these abnormal phenotypic divisions strongly suggest aberrations in chromosome segregation, and should be investigat- ed molecularly. These results provide strong initial evidence for the dependence of the mitotic geometry and polarity on the extracellular mechanical environment, and warrants dedicated, in- depth molecular characterization of the phenomena. Materials and Methods Microdevice Design To simulate aspects of a compliant confinement microenviron- ment we designed a perfusable microfluidic device with a compressible assay chamber (4 mm66 mm) (Fig. 1). This setup allows for ease of observation while simulating a pseudo-3D environment. The device’s low fluidic resistance allows uniform high density cell seeding and low shear stress during perfusion. The device operates under continuous perfusion with a total chamber volume exchange every two minutes to maintain the cells during assays. The array of 20 mm680 mm posts spaced 40 mm apart within the chamber provides control of the confinement height, structural support to resist sagging, and a pseudo-3D confined environment. In contrast to 3D culture in hydrogel systems our system provides precise control of the confinement volume, active uniform delivery of nutrients to each cell, and a single imaging plane. Microfluidic Chip Fabrication The master molds for the mechanical micro-confinement culture device were fabricated using negative photoresist (SU-5, Microchem Corporation). The base layer was spun on at 950 RPM to yield a 15 mm channel height, and the post heights (Dy) were 7 mm and 3 mm, corresponding to 1750 RPM and 3300 RPM, respectively. Poly(dimethylsiloxane) (PDMS) was prepared with varying (v/v) ratios of crosslinker to base (1:10 for E = 1 MPa and 1:20 for E = 130 kPa) [25]. The elastomer devices were cured at 65uC overnight, then cut from the mold, punched, and bonded to clean glass slides (Fisher Scientific) after treatment of both the glass and PDMS with Oxygen Plasma (0.5 Torr, 35 W) for 20 seconds. Cell Culture & Experimental Setup Human cervical carcinoma (HeLa) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, catalog CCL-2). Cells were maintained in high glucose Dulbecco’s modified Eagle’s medium (DMEM), 100 mg mL21 penicillin, 100 mg mL21 streptomycin, and 10% fetal bovine serum (Gibco). Cell synchronization was performed by a two day serum starve followed by a 2 mM double thymidine block [26]. Prior to the assay, the device was primed and flushed with ethanol at 20 mL min21 for 15 minutes, followed by a PBS flush at 20 mL min21 for 15 minutes. The device was then coated with bovine fibronectin (Invitrogen) 50 mg mL21 adsorbed nonspecifically for 90 minutes at room temperature. Cells were loaded as a suspension of 1 million cells mL21 to achieve a final in-device density of approximately 100 cells per 600 mm2 in the confinement assay area (Fig. 1a). Cells were then placed in a microscope incubator for 20 minutes to allow for attachment. For confinement assays, the device was compressed via a calibrated weight. To determine the proper mass to use for each confinement and stiffness case, a separate device, not containing cells, perfused with blue dye was used to determine when the posts came in contact with the glass substrate (i.e. the posts became optically clear). This process is graphically represented in figure 1b. The mass of calibration weights (80 g–100 g) were also confirmed by streak imaging of 2.2 mm beads which allowed tracking of compression progress (Fig. S1). Once the beads were no longer transiting under the posts, an effective calibration weight was arrived at. The heights of the devices were kept approximately constant by pouring an equal volume of PDMS:crosslinker each time a new device was molded from the master to achieve a bulk height of approximately 5 mm. Upon confinement, cells are subjected to a confinement force that is equal to the force exerted by the cell onto the substrate. This confinement force is dependent on the confinement height but not the substrate elasticity. The perfusion of supplemented DMEM media began at one half of a volume exchange of the chamber per minute (750 nL min21). Live-cell imaging was performed in a microscope incubator (temperature and CO2 controlled) with an inverted microscope (Nikon Eclipse TI). Images were captured with a Coolsnap HQ2 camera on the Nikon Advance Research software (Nikon) every five minutes with experiments lasting up to six days. PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38986