Aneuploidy in Confined Microenvironments Figure 2. Abnormal cell divisions under mechanical confinement. (a) In an unconfined device control, mitosis occurs within 150 minutes. (b) A confinement condition (Dy = 3 mm, E = 130 kPa) showing sequential tri-daughter divisions with viable daughter cells. The first tri-daughter division is seen at 100 minutes, with cell fusion labeled by black arrows. The second tri-polar division is seen at 1550 minutes along with the viable 2nd daughter cell labeled in orange. (c) A confinement condition (Dy = 7 mm, E = 130 kPa) showing an asymmetric multi-daughter division of cytoplasm after mitosis (cell with arrow receives disproportionate cytoplasmic volume). (d) A confinement condition (Dy = 7 mm, E = 1 MPa) showing a tetra- daughter division with a cross geometry chromosome alignment (marked by black arrows at 0 minutes) and cell fusion at 60 minutes (labeled by black arrows). (e) A confinement condition (Dy = 7 mm, E = 1 MPa) showing a triangular chromosome arrangement (marked by black arrows) leading to cell death. All scale bars are 20 mm. doi:10.1371/journal.pone.0038986.g002 multi-daughter generation. Further, cells which have undergone 1 to 3 divisions remain viable and produce progeny of their own. Video S6 shows a cell undergoing a 1 to 3 division, and subsequently one of the original daughter cells undergoing a second 1 to 3 division. This high frequency of multi-daughter divisions under confinement is unexpected, given that it is 17 times higher than the rate of multi-polar division events reported in standard 2D culture by Ganem et al. [4], and suggests a fundamental relationship where confinement and elasticity of the environment can control the geometry of the cell division plane(s). Unevenly sized daughter cells, cell death rates, and mitosis duration increase with increasing confinement. Of the cells completing mitosis, the rate of unevenly sized (.15% difference) daughter cells produced per mitosis event significantly increased with both stiffness and confinement: up to 100% of all divisions under the highest confinement (Fig. 4c). In control conditions, Figure 3. Daughter cell asymmetry and multi-daughter division increases with confinement and stiffness. Overlaid cell traces for control and all experimental conditions are shown. (a) Cell traces are analyzed 5 minutes post-telophase. Average control divisions are shown. (b,c) Low confinement average traces show increased size and asymmetry in stiffer environments. Notably, multi-daughter division type (tri- vs. tetra- daughter) depends on microenvironment mechanical properties. (d) In high confinement states, the daughter cells are more spread out and highly asymmetric when compared to both low compression and control cases. (e) Area difference between daughter cells in each test case demonstrating asymmetry differences. Error bars are SEM. All scale bars are 20 mm. doi:10.1371/journal.pone.0038986.g003 PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e38986