Mechanical Properties of the Tumor Stromal Microenvironment Probed In Vitro and Ex Vivo by In Situ-Calibrated Optical Trap-Based Active Microrheology

Jack R. Staunton, Wilfred Vieira, King Leung Fung, Ross Lake, Alexus Devine, Kandice Tanner

One of the hallmarks of the malignant transformation of epithelial tissue is the modulation of stromal components of the microenvironment. In particular, aberrant extracellular matrix (ECM) remodeling and stiffening enhances tumor growth and survival and promotes metastasis. Type I collagen is one of the major ECM components. It serves as a scaffold protein in the stroma contributing to the tissue’s mechanical properties, imparting tensile strength and rigidity to tissues such as those of the skin, tendons, and lungs. Here we investigate the effects of intrinsic spatial heterogeneities due to fibrillar architecture, pore size and ligand density on the microscale and bulk mechanical properties of the ECM. Type I collagen hydrogels with topologies tuned by polymerization temperature and concentration to mimic physico-chemical properties of a normal tissue and tumor microenvironment were measured by in situ-calibrated Active Microrheology by Optical Trapping revealing significantly different microscale complex shear moduli at Hz-kHz frequencies and two orders of magnitude of strain amplitude that we compared to data from bulk rheology measurements. Access to higher frequencies enabled observation of transitions from elastic to viscous behavior that occur at ~200–2750 Hz, which largely was dependent on tissue architecture well outside the dynamic range of instrument acquisition possible with SAOS bulk rheology. We determined that mouse melanoma tumors and human breast tumors displayed complex moduli ~5–1000 Pa, increasing with frequency and displaying a nonlinear stress–strain response. Thus, we show the feasibility of a mechanical biopsy in efforts to provide a diagnostic tool to aid in the design of therapeutics complementary to those based on standard histopathology.

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Red Blood Cell Aging During Storage, Studied Using Optical Tweezers Experiment

Justyna Czerwinska, Stefan Michael Wolf, Hanieh Mohammadi, Sylvia Jeney

This paper presents experimental and numerical studies of erythrocyte stretching, with a focus on the aging of red blood cells in an in vitro environment during storage. The experimental studies were performed using optical tweezers. The laser beam was used to pull and stretch a cell sedimented on a flat surface. A force calibration was obtained via a comparison of the experimental data with results from finite element simulations of the cell stretching. The experiments were performed using blood samples from blood bank donations made by three donors. The experiments were performed over 21 days of storage, and the estimate erythrocyte membrane shear modulus during this period increased from 2.5 to 13 μN/m.

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Non-Processive Force Generation by Mammalian Axonemal Dynein In Situ on Doublet Microtubules

David P. Lorch, Kathleen A. Lesich, Charles B. Lindemann, Alan J. Hunt
We utilize optical tweezers to examine displacements produced by small numbers of dynein motors located in situ on doublet microtubules from disintegrated mammalian sperm axonemes. In contrast with cytoplasmic dynein, we find that axonemal dynein is not processive, and the duration of individual force-generating interactions with a microtubule are longer than predicted from the velocity of movements generated by large ensembles of motors. These findings suggest that tension is required for rapid release of dynein following a power stroke and may explain how axonemal dynein is adapted to work in arrays within an axoneme, where cyclical bending patterns require motors to function over a range of sliding velocities.

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Cardiogenic Regulation of Stem-Cell Electrical Properties in a Laser-Patterned Biochip

Zhen Ma, Qiuying Liu, Honghai Liu, Huaxiao Yang, Julie X. Yun, Meifeng Xu, Carol A. Eisenberg, Thomas K. Borg, Roger Markwald and Bruce Z. Gao

Normal cardiomyocytes are highly dependent on the functional expression of ion channels to form action potentials and electrical coupling with other cells. To fully determine the scientific and therapeutic potential of stem cells for cardiovascular-disease treatment, it is necessary to assess comprehensively the regulation of stem-cell electrical properties during stem cell-cardiomyocyte interaction. It has been reported in the literature that contact with native cardiomyocytes induced and regulated stem-cell cardiogenic differentiation. However, in conventional cell-culture models, the importance of cell–cell contact for stem-cell functional coupling with cardiomyocytes has not been elucidated due to insufficient control of the cell-contact mode of individual cells. Using microfabrication and laser-guided cell micropatterning techniques, we created two biochips with contact-promotive and -preventive microenvironments to systematically study the effect of contact on cardiogenic regulation of stem-cell electrical properties. In contact-promotive biochips, connexin 43 expression was upregulated and relocated to the junction area between one stem cell and one cardiomyocyte. Only stem cells in contact with cardiomyocytes were induced by adjacent cardiomyocytes to acquire electrophysiological properties for action-potential formation similar to that of a cardiomyocyte.

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Advances in Experiments and Modeling in Micro- and Nano-Biomechanics: A Mini Review

Mian Long, Masaaki Sato, Chwee Teck Lim, Jianhua Wu, Taiji Adachiand Yasuhiro Inoue

Recent advances in micro- and nano-technologies and high-end computing have enabled the development of new experimental and modeling approaches to study biomechanics at the micro- and nano-scales that were previously not possible. These new cutting-edge approaches are contributing toward our understanding in emerging areas such as mechanobiology and mechanochemistry. Another important potential contribution lies in translational medicine, since biomechanical studies at the cellular and molecular levels have direct relevance in areas such disease diagnosis, nano-medicine and drug delivery. Thus, the developed experimental and modeling approaches are critical in elucidating important mechanistic insights in both basic sciences and clinical treatment. While it is hard to cover all the recent advances in this mini-review, we focus on several important approaches. For experimental techniques, we review the assays involving shear flow, cellular imaging, microbead, microcontact printing, and micropillars at the micro-scale, and micropipette aspiration, optical tweezers, parallel flow chamber, and atomic force microscopy at the nano-scale. In modeling and simulations, we outline the theoretical modeling for actin dynamics in migrating cell and actin-based cell motility in cellular mechanics, as well as the receptor–ligand binding in cell adhesion and the application of free, steered, and flow molecular dynamics simulations in molecular biomechanics. Relevant scientific issues and applications are also discussed.

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Manipulation of Suspended Single Cells by Microfluidics and Optical Tweezers

Nathalie Nève, Sean S. Kohles, Shelley R. Winn and Derek C. Tretheway

Chondrocytes and osteoblasts experience multiple stresses in vivo. The optimum mechanical conditions for cell health are not fully understood. This paper describes the optical and microfluidic mechanical manipulation of single suspended cells enabled by the μPIVOT, an integrated micron resolution particle image velocimeter (μPIV) and dual optical tweezers instrument (OT). In this study, we examine the viability and trap stiffness of cartilage cells, identify the maximum fluid-induced stresses possible in uniform and extensional flows, and compare the deformation characteristics of bone and muscle cells. These results indicate cell photodamage of chondrocytes is negligible for at least 20 min for laser powers below 30 mW, a dead cell presents less resistance to internal organelle rearrangement and deforms globally more than a viable cell, the maximum fluid-induced shear stresses are limited to ~15 mPa for uniform flows but may exceed 1 Pa for extensional flows, and osteoblasts show no deformation for shear stresses up to 250 mPa while myoblasts are more easily deformed and exhibit a modulated response to increasing stress. This suggests that global and/or local stresses can be applied to single cells without physical contact. Coupled with microfluidic sensors, these manipulations may provide unique methods to explore single cell biomechanics.

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Validation, In-Depth Analysis, and Modification of the Micropipette Aspiration Technique

Yong Chen, Baoyu Liu, Gang Xu and Jin-Yu Shao

The micropipette aspiration technique (MAT) has been successfully applied to many studies in cell adhesion such as leukocyte–endothelium interactions. However, this technique has never been validated experimentally and it has been only employed to impose constant forces. In this study, we validated the force measurement of the MAT with the optical trap and analyzed two technical issues of the MAT, force-transducer offset and cell-micropipette gap, with finite element simulation. We also modified the MAT so that increasing or decreasing forces can be applied. With the modified MAT, we studied tether extraction from endothelial cells by pulling single tethers at increasing velocities and constant force loading rates. Before the onset of tether extraction, an apparently linear surface protrusion of a few hundred nanometers was observed, which is likely related to membrane receptors pulling on the underlying cytoskeleton. The strength of the modified MAT lies in its capability and consistency to apply a wide range of force loading rates from several piconewtons per second up to thousands of piconewtons per second. With this modification, the MAT becomes more versatile in the study of single molecule and single cell biophysics.

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