Aneuploidy in Confined Microenvironments Figure 4. Quantitative effects of confinement on cell-cycle abnormalities. (a) Summary of experimental data in terms of abnormalities per cell cycle characterized by: multi-daughter divisions, asymmetric divisions, apoptotic divisions, and divisions lasting for more than 140 minutes. (b) Multi-daughter divisions per cell cycle character- ized as divisions producing more than two daughter cells. Error bars are SEP. (c) Asymmetric divisions are characterized by daughter cells having a difference greater than 15% in area. Error bars are SEP. (d) Cell deaths per cell cycle are characterized by cells that cease activity and contract prior to completion of mitosis. Error bars are SEP. (e) Comparing the mitosis duration of the high confinement (Dy = 3 mm, E = 130 kPa) condition to control and both low confinement conditions. Error bars are SEM. (f) Summary of divisions in each case completing division within 140 minutes. For high confinement E = 1 MPa, no data (ND) was available as no completed mitosis events was observed. Error bars are SEP. doi:10.1371/journal.pone.0038986.g004 mitosis resulting in cell death, possibly due to intact checkpoint failures, is observed in less than 1% of all division events. Cell death increases significantly to 4% and 8% at low confinement at E = 130 kPa and 1 MPa, respectively, and up to 70% under high confinement E = 130 kPa (Fig. 4d). The duration of mitosis shown in figure 4e & 4f suggests increasing confinement is sufficient to increase mitotic duration. This corroborates previous observations by Kwon et al. [21] and Maresca et al. [17] that mitotic processes are dependent on correct orientation of, and force balance in, the cytoskeleton in order to achieve the proper signaling state to proceed through mitotic checkpoints in a timely manner. A unique mitotic event resulting in five daughter cells under partial confinement was observed in which confinement was not complete such that the gap size was between 3–7 mm with E = 130 kPa (Fig. 5a). The cell in metaphase is 60 mm in diameter, 4 times wider than the 15 mm spherical cells undergoing mitosis during unconfined division. The metaphase lineup of chromo- somes is highly abnormal as visual inspection suggests (Fig. 5a, left image) with at least 4 centrosome pole regions. Yet during anaphase (middle image), chromosome segregation seems to move toward 5 poles, leading to 5 daughter cells. Although the quantification of chromosome content is not available for these cells, it is reasonable to postulate that the chromosomes were incorrectly and unevenly segregated in this division, as with the other multi-daughter divisions (specifically to three daughter cells). In the case of the three daughter divisions, one replication cycle (S- phase), cannot be properly split into three without an extra half S- phase. In the case of the penta-daughter division, the same logic remains, however the mitotic planes appear to be even more complex. Although these penta-daughter division events are rare (observed twice over 500 single-cell observations), our single-cell analysis platform enables the capture of these rare atypical biological processes. Conclusion To elucidate the effects of confined mechanical microenviron- ments on cell behavior and mitotic processes, the microfluidic platform described here successfully creates confined culture environments that may mimic aspects of in vivo tumor mechanical properties. The data presented shows high instances of abnormal multi-daughter division events induced simply by increasing the confinement of a cell (i.e. decreasing the available volume to divide in) and by increasing the substrate stiffness. Further, the frequencies at which these abnormalities occur is found at much higher rates than previously observed. The ability to induce these abnormal divisions to drastically higher rates (half of all divisions in some cases) suggests a fundamental interaction between the PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e38986