Fig 5 - uploaded by Jeroen Eyckmans
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Magnetic tweezer system. (a) shows a schematic and (b) shows a photograph of the unit mounted on a microscope. The long, tapered rod is an iron core that acts as a magnetic pole tip projecting into a P35 μTUG sample dish. The tweezer solenoid surrounds the core between the arms of the aluminum bracket on a 3-axis micromanipulator and is encased in an aluminum heat-sinking block. A second manipulator and coil assembly (unused in this application) is shown at left in (b) without its core. The image in (b) is reproduced from A. S. Liu, Ph.D. Thesis, the Johns Hopkins University (2015). Used by permission
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Cell interactions with the extracellular matrix (ECM) are critical to cell and tissue functions involving adhesion, communication, and differentiation. Three-dimensional (3D) in vitro culture systems are an important approach to mimic in vivo cell–matrix interactions for mechanobiology studies and tissue engineering applications. This chapter descr...
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... It employs a dual magnetic tweezer (Fig. 5) to ensure that only one cell is exposed to the magnetic field at a time, but if this is not a consideration, then alternative approaches may be employed to generate the needed field. Detailed information on the construction of a magnetic tweezer system is provided in several publications (Bose, Huang, Eyckmans, Chen, & Reich, 2018;Kramer, 2009;Lin, Kramer, Chen, & Reich, 2012;Zhao, Boudou, Wang, Chen, & Reich, 2014). ...
... Basic Protocol 3. Several experimental considerations must be addressed to acquire accurate data for the frequency dependence of the local cellular rheology with magnetic nanowires embedded in microposts and a magnetic tweezer system (Bose et al., 2018;Kramer, 2009;Lin et al., 2012;Zhao et al., 2014). To minimize evaporation and thermal fluctuations, we describe a customized sample chamber that allows the tips of the magnetic tweezer poles to be brought within 2 mm of the AMPAD array without contacting the culture media (Shi et al., 2019). ...
The dynamics of the cellular actomyosin cytoskeleton are crucial to many aspects of cellular function. Here, we describe techniques that employ active micropost array detectors (AMPADs) to measure cytoskeletal rheology and mechanical force fluctuations. The AMPADS are arrays of flexible poly(dimethylsiloxane) (PDMS) microposts with magnetic nanowires embedded in a subset of microposts to enable actuation of those posts via an externally applied magnetic field. Techniques are described to track the magnetic microposts’ motion with nanometer precision at up to 100 video frames per second to measure the local cellular rheology at well‐defined positions. Application of these high‐precision tracking techniques to the full array of microposts in contact with a cell also enables mapping of the cytoskeletal mechanical fluctuation dynamics with high spatial and temporal resolution. This article describes (1) the fabrication of magnetic micropost arrays, (2) measurement protocols for both local rheology and cytoskeletal force fluctuation mapping, and (3) special‐purpose software routines to reduce and analyze these data. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.
Basic Protocol 1 : Fabrication of magnetic micropost arrays
Basic Protocol 2 : Data acquisition for cellular force fluctuations on non‐magnetic micropost arrays
Basic Protocol 3 : Data acquisition for local cellular rheology measurements with magnetic microposts
Basic Protocol 4 : Data reduction: determining microposts’ motion
Basic Protocol 5 : Data analysis: determining local rheology from magnetic microposts
Basic Protocol 6 : Data analysis for force fluctuation measurements
Support Protocol 1 : Fabrication of magnetic Ni nanowires by electrodeposition
Support Protocol 2 : Configuring Streampix for magnetic rheology measurements
... Arrays of microfabricated tissue gauge ( µTUG) devices were fabricated as previously described [35,36]. Master versions of the µTUG arrays were created in SU-8 photoresist on silicon substrates by a multilayer microlithography technique. ...
... The ECM solution was then added to µTUG devices that were placed on ice packs (432014, Corning), and degassed. hiPSC-CMs, human umbilical vein endothelial cells (HUVECs) (CC-2935, Lonza) and human adult ventricular cardiac fibroblasts (FB) (6310, ScienCell) were counted and mixed at a ratio of 75:10:15 in ECM solution (seeding density 2.5 10 5 cells/cm 2 ), added to the µTUGs and spun down into the microwells at 225 g for 1.5 min, after which the excess ECM solution was removed [35]. hiPSC-CM solutions without fibroblasts did not form compact tissue structures, and a range of fibroblast seeding densities (2.5 -10 10 4 cells/cm 2 ) were tested to ensure compaction. ...
... Videos of CMTs undergoing spontaneous beating during culture were acquired by an EVOS FL Cell Imaging System at 20x magnification at 20 fps. The motions of the micropillars were measured from the videos by custom MATLAB scripts [35] or manual tracking using ImageJ. The CMTs' contraction forces were calculated using F = kδ where δ is the average deflection of the two micropillars and k = 0.25 μN/μm. ...
Recent advances in the understanding and use of pluripotent stem cells have produced major changes in approaches to the diagnosis and treatment of human disease. An obstacle to the use of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for regenerative medicine, disease modeling and drug discovery is their immature state relative to adult myocardium. We show the effects of a combination of biochemical factors, thyroid hormone, dexamethasone, and insulin-like growth factor-1 (TDI) on the maturation of hiPSC-CMs in 3D cardiac microtissues (CMTs) that recapitulate aspects of the native myocardium. Based on a comparison of the gene expression profiles and the structural, ultrastructural, and electrophysiological properties of hiPSC-CMs in monolayers and CMTs, and measurements of the mechanical and pharmacological properties of CMTs, we find that TDI treatment in a 3D tissue context yields a higher fidelity adult cardiac phenotype, including sarcoplasmic reticulum function and contractile properties consistent with promotion of the maturation of hiPSC derived cardiomyocytes.
... previously. 24,33,34 Poly(dimethylsiloxane) (PDMS) negative molds were made from the masters and the final μTUG devices were cast in PDMS from the molds. Six different μTUG well geometries with varying shapes, sizes, and pillar positions were used to control the microtissues' shape (Figures 1A−C and 2A−C). ...
... P24443) prior to microtissue seeding to prevent adhesion of cells to the walls of the μTUG wells. 34 2.2. Cell Culture and Microtissue Seeding. ...
... For the experiments, cells at passage 8−14 (HDF) and <17 (3T3s) were suspended in 2 mg/mL rat-tail collagen type I (BD Bioscience) at a cell density of 1 × 10 5 cells/mL (150−200 cells per microtissue) for HDFs and 5 × 10 5 cells/mL (750−1000 cells per microtissue) for 3T3s and seeded into the microwells following previously published protocols. 24,[26][27][28]30,34 The microtissues were allowed to self-assemble and compact in DMEM + 2% FBS (HDFs) or DMEM + 10% FBS (3T3s) for 72 h prior to the experiments. For the HDFs, it was necessary to use lower cell and FBS concentrations than for the 3T3s to compensate for the HDF's higher contractility and obtain stable microtissues. ...
The structure and stiffness of the extracellular matrix (ECM) in living tissues plays a significant role in facilitating cellular functions and maintaining tissue homeostasis. However, the wide variation and complexity in tissue composition across different tissue types make comparative study of the impact of matrix architecture and alignment on tissue mechanics difficult. Here we present a microtissue-based system capable of controlling the degree of ECM alignment in 3D self-assembled fibroblast-populated collagen matrix, anchored around multiple elastic micropillars. The pillars provide structural constraints, control matrix alignment, enable measurement of the microtissues’ contractile forces, and provide the ability to apply tensile strain using magnetic particles. Utilizing finite element models (FEM) to parameterize results of mechanical measurements, spatial variations in the microtissues’ Young’s moduli across different regions were shown to be correlated with the degree of ECM fiber alignment. The aligned regions were up to six times stiffer than the unaligned regions. The results were not affected by suppression of cellular contractile forces in matured microtissues. However, comparison to a distributed fiber anisotropic model shows that variations in fiber alignment alone cannot account for the variations in the observed moduli, indicating that fiber density and tissue geometry also play important roles in the microtissues’ properties. These results suggest a complex interplay between mechanical boundary constraints, ECM alignment, density, and mechanics, and offers an approach combining engineered microtissues and computational modeling to elucidate these relationships.
Cardiovascular diseases bear strong socioeconomic and ecological impact on the worldwide healthcare system. A large consumption of goods, use of polymer-based cardiovascular biomaterials, and long hospitalization times add up to an extensive carbon footprint on the environment often turning out to be ineffective at healing such cardiovascular diseases. On the other hand, cardiac cell toxicity is among the most severe but common side effect of drugs used to treat numerous diseases from COVID-19 to diabetes, often resulting in the withdrawal of such pharmaceuticals from the market. Currently, most patients that have suffered from cardiovascular disease will never fully recover. All of these factors further contribute to the extensive negative toll pharmaceutical, biotechnological, and biomedical companies have on the environment. Hence, there is a dire need to develop new environmentally-friendly strategies that on the one hand would promise cardiac tissue regeneration after damage and on the other hand would offer solutions for the fast screening of drugs to ensure that they do not cause cardiovascular toxicity. Importantly, both require one thing–a mature, functioning cardiac tissue that can be fabricated in a fast, reliable, and repeatable manner from environmentally friendly biomaterials in the lab. This is not an easy task to complete as numerous approaches have been undertaken, separately and combined, to achieve it. This review gathers such strategies and provides insights into which succeed or fail and what is needed for the field of environmentally-friendly cardiac tissue engineering to prosper.
