Increased Asymmetric and Multi-Daughter Cell Division in Mechanically Confined Microenvironments Henry Tat Kwong Tse1., Westbrook McConnell Weaver1., Dino Di Carlo1,2* 1 Department of Bioengineering, University of California Los Angeles, Los Angeles, California, United States of America, 2 California NanoSystems Institute, Los Angeles, California, United States of America Abstract As the microenvironment of a cell changes, associated mechanical cues may lead to changes in biochemical signaling and inherently mechanical processes such as mitosis. Here we explore the effects of confined mechanical environments on cellular responses during mitosis. Previously, effects of mechanical confinement have been difficult to optically observe in three-dimensional and in vivo systems. To address this challenge, we present a novel microfluidic perfusion culture system that allows controllable variation in the level of confinement in a single axis allowing observation of cell growth and division at the single-cell level. The device is capable of creating precise confinement conditions in the vertical direction varying from high (3 mm) to low (7 mm) confinement while also varying the substrate stiffness (E = 130 kPa and 1 MPa). The Human cervical carcinoma (HeLa) model with a known 3N+ karyotype was used for this study. For this cell line, we observe that mechanically confined cell cycles resulted in stressed cell divisions: (i) delayed mitosis, (ii) multi- daughter mitosis events (from 3 up to 5 daughter cells), (iii) unevenly sized daughter cells, and (iv) induction of cell death. In the highest confined conditions, the frequency of divisions producing more than two progeny was increased an astounding 50-fold from unconfined environments, representing about one half of all successful mitotic events. Notably, the majority of daughter cells resulting from multipolar divisions were viable after cytokinesis and, perhaps suggesting another regulatory checkpoint in the cell cycle, were in some cases observed to re-fuse with neighboring cells post-cytokinesis. The higher instances of abnormal mitosis that we report in confined mechanically stiff spaces, may lead to increased rates of abnormal, viable, cells in the population. This work provides support to a hypothesis that environmental mechanical cues influences structural mechanisms of mitosis such as geometric orientation of the mitotic plane or planes. Citation: Tse HTK, Weaver WM, Di Carlo D (2012) Increased Asymmetric and Multi-Daughter Cell Division in Mechanically Confined Microenvironments. PLoS ONE 7(6): e38986. doi:10.1371/journal.pone.0038986 Editor: Wilbur Lam, Emory University/Georgia Insititute of Technology, United States of America Received March 28, 2012; Accepted May 17, 2012; Published June 25, 2012 Copyright: 2012 Tse et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: These authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dicarlo@seas.ucla.edu . These authors contributed equally to this work. Introduction An immense amount of past and current research is dedicated to understanding the control systems that govern the very complex network of chemical reactions that dictate cell biology. Of these cellular control systems, perhaps the most extensively studied and complex is the cell cycle regulatory system. Cell cycle regulation controls the progression of the life cycle of a cell, the growth of tissues, and is ultimately a significant contributor to the physiological homeostasis of complex multicellular organisms. However, recent research have also shown that non-conventional mitosis events contribute to natural genetic variation [1], as well as tumor progression[2–5]. Over a half century of research, sparked by Howard and Pelc’s observation that radio-labeled phosphorous incorporates differen- tially into cells not undergoing mitosis [6], has resulted in an increasingly complex understanding of cell cycle regulation. Regulation of the cycle depends on the constant production and degradation of proteins, and the activation or deactivation of the complexes responsible for targeting these proteins for degradation via ubiquitination. Environmental cues such as soluble factors have long been implicated in the cell cycle control system, however the past decade has given a new perspective on mechanical cues involved in cell biology. In order to gain a more complete understanding of cell biology and the cell cycle, considering both soluble and mechanical cues will be necessary. Mechanosensing is an important component of the physiology of the cell, as well as tissue homeostasis. Direct linkages between the extracellular matrix (ECM) and the intracellular environment allow external mechanical cues to alter the cellular state[7–9]. Conversely, these same linkages enable the cell to transmit forces extracellularly, altering the mechanical micro-environment itself [10]. Tipping this mechanical balance can result in cellular differentiation [11], morphology [12] and motility changes [13], as well as alterations in cell cycle control [14]. Mitosis is a highly regulated stage of the cell cycle, both biochemically and, more increasingly suspected, mechanically. The overall spherical shape that cells adopt during this phase and the internal organization of the cytoskeleton are directly implicat- ed in influencing the progression through mitosis [15]. The Spindle Assembly Checkpoint (SAC) has been identified as the major checkpoint responsible for ensuring correct chromosomal alignment during metaphase [16]. The SAC requires specific mechanical cues to proceed through mitosis, including microtu- PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e38986