bule-kineticore attachments as well as sufficient tension in microtubules themselves [17], the satisfaction of which results ultimately in cytokinesis and mitotic exit [18]. The cell division axis is also dependent on the orientation of ECM near the dividing cell and this effect requires an intact actin cytoskeleton [19]. This link between the ECM orientation, cytoskeleton, and condensed DNA is further supported by the co-localization of cytoskeletal binding proteins and the spindle apparatus during mitosis [20]. Centrosome number and polarity has been shown to depend not only on an intact cytoskeleton [21], but also on the phosphorylation state of focal adhesion kinase (FAK), further implicating a delicate force balance during mitosis [22]. We propose that aberrant mitotic outcomes, possibly due to altered cytoskeletal mechanics during mitosis, can be directed by an altered mechanical microenvironment. To explore this hypothesis, we have developed a novel microfluidic platform to confine a population of proliferating HeLa cells. This novel culture platform allows for both alterations in the geometry of the microenvironment, specifically the space in which the cell is allowed to grow and divide, as well as the elasticity of the substrate on which the cell is dividing. Figure 1 illustrates the device, with an example of a mitotic cell directly interacting with the elastic substrate (Fig. 1a). By using a microfluidic device to compress the cells, we minimize cell death due to lack of nutrients, as media is constantly perfused through the compression chamber. The device also allows for facile imaging of cells, as they are in a single focal plane. Results and Discussion Cells in confined 3D cultures exhibit drastic changes in size, shape, and symmetry of daughter cells when compared to unconfined 2D cultures. The height confinement also readily allowed visualization of condensed chromosomes at the mitotic plane. Within a 600 mm2 field-of-view cells are observed under time lapse bright-field microscopy for mitosis events (Video S1). In the unconfined device control, with posts in the up position (Fig. 2a), cells attain a spherical geometry during mitosis and complete the mitosis process within 140 minutes. However, in confined conditions, tri-daughter cytokinesis (Fig. 2b & 2c, Video S2 & S3), daughter cells with drastically different sizes (Fig. 2c, Video S3), tetra-daughter cytokinesis (Fig. 2d, Video S4), and mitosis resulting in cell death (Fig. 2e, Video S5) are commonly observed events that increase in frequency with increasing compression (decreasing Dy) and increasing stiffness (E). Since multi-polar divisions have previously been classified as divisions containing multiple centrosomes (N.2) during mitosis, to avoid confusion we refer to multi-daughter divisions as observable division events resulting in more than 2 daughter cells (including observations of tri-daughter through penta-daughter cytokinesis). Multi-daughter divisions induced by confinement lead to viable daughter cells with increased chances of chromosomal abnormal- ities. Interestingly, in figure 2b sequential tri-daughter divisions in consecutive cell cycles are observed for the high confinement, low stiffness condition (Dy = 3 mm, E = 130 kPa). In this case and others (Fig. 2d) we note that cells dividing in this manner will sometimes ‘‘re-fuse’’ after division before the next cell cycle. Further investigation is warranted to determine whether this behavior is indicative of a new class of checkpoint control programs that acts post-cytokinesis. Even considering potential corrective measures (re-fusion) that cells may employ, tri-daughter divisions (Fig. 2b & 2c), divisions resulting in unevenly sized daughter cells (Fig. 2c) and tetra-daughter divisions (Fig. 2d) are Aneuploidy in Confined Microenvironments likely to result in increased susceptibility to abnormal chromosome segregation. As the HeLa cell line is a 3N+ karyotype, the amount of assembly of mitotic spindles from each centrosome is increased by at least 50% when compared to diploid cells. With extra centrosomes (N.2) and their associated mitotic spindles, meta- phase and anaphase events are highly complex with respect to chromosome segregation to the poles even for bi-polar divisions. Even higher rates of missegregation is expected when cells divide in a multi-daughter fashion [5]. The degree of mechanical confinement affects the daughter cell size ratio in normal and multi-daughter cell divisions. Figure 3 illustrates the highly altered mitosis division shape and asymmetry under varied conditions of confinement. Upon the completion of telophase, cells in control conditions are , 20 mm in width and highly uniform (Fig. 3a), whereas in the extreme case of high confinement the daughter cells span between 40–80 mm in width and are highly asymmetric (Fig. 3d). For intermediate conditions, both increased stiffness and confinement act to increase division asymmetry, as observed in the average traces (Fig. 3b, c). Quantification of daughter cell traces (Fig. 3e) demonstrates that the difference in area between daughter cells increases for all cases compared to control, where statistical significance (p,0.001) is observed between both low confinement high stiffness and high confinement low stiffness compared to the control. Interestingly, a marked difference in type of multi-daughter division was observed when maintaining confinement, and changing only the substrate stiffness (Fig. 3a–d). Under low compression (Dy = 7 mm) when substrate stiffness is 130 KPa, multi-daughter divisions result primarily in three progeny (90%, Fig. 3b), however upon increasing substrate stiffness to 1 MPa the multi-daughter division mode shifts primarily to four progeny (85%, Fig. 3c). Assuming cell volume remains similar, the smaller cross-sectional cell area observed for softer 130 KPa substrates indicates that cells are able to apply significant force to deform the PDMS substrate and adopt a more rounded form. Note that a similar force is presumably applied by the cell to the stiffer 1 MPa substrate but this leads to less deformation. In both cases the force applied by the cell, and therefore the equal and opposite force applied by the substrate to the cell are similar, but the cell shape differs. Therefore, cell shape during mitosis may be a dominant factor in directing multi-daughter divisions (in which a more spherical shape is achievable for the softer substrates). A tendency towards tri- or tetra- daughter divisions may be due to spatial limits of chromosome assembly at the metaphase plane(s) in a confined shape, which is dependent on substrate stiffness. Alternatively, the location of mechanical loads may direct the geometry of these multi-daughter divisions, perhaps acting through cortical cues sensitive to environmental force magnitudes [23]. Along with possible effects of the uneven size of daughter cells and multi-daughter divisions on chromosomal segregation, alter- ation in cell shape with confinement may also pose difficulties in critical mitotic processes such as spindle assembly, signaling, and targeting processes during check point regulation and may be responsible for the increase in abnormal mitosis behavior. For example, signaling relying on either diffusion or active transport may proceed slower or be more error prone over the longer distances in an enlarged discoid shaped cell compared to a tight spherical cell. Additionally, different cytoskeletal filaments have a characteristic persistence length over which they are effective (i.e. able to transmit force between protein complexes) [24]. It is reasonable to postulate that by dramatically changing the shape of the cell from spherical to discoid, the lengths of force transmission (e.g. along microtubules from centrosome to kinetochore or from PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e38986