1 Scientific RepoRts | 6:19550 | DOI: 10.1038/srep19550 www.nature.com/scientificreports An approach to quantifying 3D responses of cells to extreme strain Yuhui Li 1,2,* , Guoyou Huang 1,2,* , Moxiao Li 2 , Lin Wang 1,2,3 , elliot L. elson 2,3,5 , tian Jian Lu 2 , Guy M. Genin 1,2,4,5 & Feng Xu 1,2 The tissues of hollow organs can routinely stretch up to 2.5 times their length. Although signifcant pathology can arise if relatively large stretches are sustained, the responses of cells are not known at these levels of sustained strain. A key challenge is presenting cells with a realistic and well-defned three-dimensional (3D) culture environment that can sustain such strains. Here, we describe an in vitro system called microscale, magnetically-actuated synthetic tissues (micro-MASTs) to quantify these responses for cells within a 3D hydrogel matrix. Cellular strain-threshold and saturation behaviors were observed in hydrogel matrix, including strain-dependent proliferation, spreading, polarization, and diferentiation, and matrix adhesion retained at strains sufcient for apoptosis. More broadly, the system shows promise for defning and controlling the efects of mechanical environment upon a broad range of cells. In vivo tissues commonly experience large deformation in both physiological and pathological conditions. For instance, the solid tissues of hollow organs (e.g., bladder) can routinely stretch up to 2.5 times their length, or a nominal strain of 150% 1 . Te alveolar can expand to 1.5 times their volume in normal mice and sustainably keep at 3 times in emphysematous mice 2 . Although signifcant pathology can arise if relatively large stretches are sustained, the responses of cells (e.g., fbroblasts) are not known at these levels of sustained strain 3–5 . Te responses of cells to sustained stretches in this upper range has not yet been established defnitively for cells in three-dimensional (3D) culture, in part due to challenges with presenting cells a realistic and well-defned culture environment that can sustain such strains 6,7 . Te need for efective 3D culture systems is particularly pressing because the well-established responses to mechanical stimuli of cells in two-dimensional (2D) culture appear to difer substantially from those of cells in 3D 8–11 . Even the toolbox of transmembrane molecules such as integrins that cells use to connect with their environment might be diferent in 3D environments 8 . Further, established tools for mechanobiology in 2D do not extend easily to 3D mechanobiology 12–15 . Observations acquired using tools such as micropost arrays 16 , 2D traction microscopy 17 , and stretchable substrata 18 are difcult to relate to 3D due to fundamental diferences in the morphologies of cells in 2D and 3D 19–21 . A number of systems have been developed to probe cell mechanobiology in 3D tissue constructs, but these cannot accommodate the high strains of interest. Although the strains experienced by cells in a tissue stretched 150% can be lower than 150% due to structural features such as crimping 22 and due to the details of load-sharing between cells and extracellular matrix (ECM) 23,24 , existing systems can sustain stretches that are only a small fraction of this, typically on the order of 30%. One of the earliest such systems is slab-like 10 or ring-like tissue constructs 25 consisting of cells seeded in a collagen tissue construct. Tese have provided much insight into issues of tissue remodeling that involve large deformations of the tissue constructs themselves, but only small strains at the level of cells 26–29 . Like these specimens, tissue constructs synthesized between fexible posts 30–32 and tissue constructs adhered to fexible substrates 33 cannot attain the high strain levels of interest. Another attractive 3D system is based on fbrous protein hydrogels, including collagen, gelatin, elastin and fbrin, which exhibit sim- ilar structural and mechanical properties with the native ECM 34,35 . A variety of fbrous hydrogel systems have been developed to help understand how ECM mechanics regulate cell behaviors 36,37 . For instance, alignment of vascular-derived cells was successfully engineered in collagen hydrogels with 10% cyclic strain 33 . Diferentiation 1 the Key Laboratory of Biomedical information engineering of Ministry of education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China. 2 Bioinspired Engineering and Biomechanics Center, Xi’an Jiaotong University, Xi’an 710049, China. 3 Department of Biochemistry and Molecular Biophysics, Saint Louis, Missouri 63110, USA. 4 Department of neurological Surgery, Washington University School of Medicine, Saint Louis, Missouri 63110, USA. 5 Department of Mechanical engineering and Materials Science, Washington University, Saint Louis, Missouri 63130, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to G.M.G. (email: genin@wustl.edu) or F.X. (email: fengxu@mail.xjtu.edu.cn) received: 26 June 2015 Accepted: 18 November 2015 Published: 18 February 2016 opeN