Aneuploidy in Confined Microenvironments Figure 1. Microfluidic cell confinement device. (a) Device schematic with inset of confinement assay. The posts are 20680 mm spaced equally 40 mm apart. (b) In the unconfined state, the posts are raised from the glass substrate (top), upon applying pressure the confinement chamber is compressed such that the posts are in contact with the glass substrate. (c) Seeding cells in the confinement chamber (left), when the posts are lowered the cell is confined to 3 or 7 mm height and is squeezed out from the post area (middle). Cells spread and attach in the confined volume (right). doi:10.1371/journal.pone.0038986.g001 actin-based cortical cues to centrosomal microtubules [5,23]) become anisotropic in varying directions of division, which may result in the underlying multi-polar and asymmetric divisions. Similarly, nonmuscle myosin II (NMM-II) distribution and contractility, necessary to create a uniform cleavage furrow during cytokinesis, may be affected by the non-spherical shape and anisotropic mechanical stress in these confined conditions. Fundamentally, these results suggest a role for adoption of a spherical shape during mitosis in maintaining bipolar division events. Mechanical confinement leads to statistically significant differ- ences in the frequency of mitotic abnormalities. A summary of the abnormalities observed per cell cycle is shown in figure 4a. Here we define the total abnormalities per cell cycle as the combined frequency of multi-daughter divisions, divisions resulting in unevenly sized daughter cells, divisions resulting in cell death, and completed mitosis in greater than 140 minutes. Increasing both the geometric confinement of cells and substrate stiffness, leads to increasing frequencies of mitotic abnormalities (Fig. 4a). It should be noted that in the E = 1 MPa high confinement case no successful cell divisions were observed, but observable mitosis events (prophase, metaphase) were observed. Increased frequencies of multi-daughter divisions are observed in highly confined environments. A summary of multi-daughter divisions per cell cycle is shown in figure 4b. In the unconfined control condition less than 1% of cell divisions generate more than two daughter cells. In contrast, under low confinement (Dy = 7 mm) slight increases in the frequency of multi-daughter divisions are observed, and in high confinement cases (Dy = 3 mm) a drastic increase to 50% of all divisions are observed to lead to PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e38986