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‘Optical Shaking’ of Red Blood Cells: A Strategy to Measure Cell-Fluid Coupling with Optical Tweezers
134a
Sunday, February 28, 2016
676-Pos Board B456 Dynamic Monitoring of Cell Mechanical Properties using Profile Microindentation Lionel Guillou1, Avin Babataheri1, Pierre-Henri Puech2, Abdul Barakat1, Julien Husson1. 1 Ecole Polytechnique, Palaiseau, France, 2Aix Marseille University, Marseille, France. We have developed a simple and relatively inexpensive system to visualize adherent cells in profile while measuring their mechanical properties using microindentation. The setup allows simultaneous control of cell microenvironment by introducing a micropipette for the delivery of soluble factors or other cell types. We validate this technique against atomic force microscopy measurements and, as a proof of concept, measure the viscoelastic properties of vascular endothelial cells in terms of an apparent stiffness and a dimensionless parameter that describes stress relaxation. Furthermore, we use this technique to monitor the time evolution of these mechanical properties as the cells’ actin is depolymerized using cytochalasin-D. 677-Pos Board B457 Dissecting the Role and Regulation of Mechanical Force at the T-APC Synapse using Atomic Force Microscopy Kenneth H. Hu1, Manish J. Butte2. 1 Biophysics, Stanford University, Stanford, CA, USA, 2Pediatrics, Stanford University, Stanford, CA, USA. T cell activation through formation of an immune synapse (IS) with an antigen presenting cell (APC) is a key step in the adaptive immune response. How the unique evolution and structure of the IS contributes to T cell activation is still being dissected. Here we hypothesize that the dynamic cytoskeletal changes that occur in the T cell generate mechanical forces that promote T cell activation either directly through the TCR or through formation of close contact regions to allow for TCR engagement through a dense glycocalyx. In order to monitor the T cell’s mechanical response to activation, we used an Atomic Force Microscopy (AFM) setup, with a peptide-MHC coated cantilever acting as a surrogate APC. Conversely, we also probed the role of forces during T cell activation by applying force with our setup. We found that T cells exhibit a characteristic profile of force, pushing and pulling on the cantilver. This force profile exhibits temporal correlation with the onset of calcium influx following TCR stimulation. Furthermore, inhibition of actin dynamics with Latrunculin A dramatically reduces calcium flux and force generation. Calcium influx can be partially rescued by application of external oscillatory forces through the cantilever. We find a characteristic timescale for integration of this signal suggesting some relaxation mechanism. To understand the role of the glycocalyx in T cell signaling, we coated the APC membrane with synthetic mucins of varying lengths. We found that T cell activation was reduced in the presence of large mucins, suggesting that the glycocalyx presents a barrier to TCR engagement. Our data shows the T cell acts mechanically on the APC during activation. Future work will need to be done to understand how this force generation is regulated. 678-Pos Board B458 ‘Optical Shaking’ of Red Blood Cells: A Strategy to Measure Cell-Fluid Coupling with Optical Tweezers Carla Zensen1,2, Isis E. Fernandez Buelvas2,3, Oliver Eickelberg2,3, Theobald Lohmu¨ller1,2, Jochen Feldmann1,2. 1 Department of Physics and Center of Nanoscience (CeNS), LudwigMaximilians-Universita¨t, Munich, Germany, 2Nanosystems Initiative Munich (NIM), Munich, Germany, 3Comprehensive Pneumology Center, Insititute of Lung Biology and Disease, Ludwig-Maximilians-Universita¨t and Helmholtz Zentrum, Munich, Germany. We introduce a strategy to use holographic optical tweezers for measuring the fluidic coupling of cells that are ‘shaken’ with a laser beam array. The flow generated by this periodic optical forcing is measured with a ‘detector’ micro particle that is trapped by a second, independent laser in the cell vicinity. A signal processing analysis of both the tracked cell and the trace of the detector bead provides detailed information about the cells reaction on a mechanical stimulation and how this is transferred through the fluid. Measurements of red blood cells exposed to different hypotonic media showed that different cellular edematous states result in a specific fluidic pattern in the flow observed around these cells. We motivate that the signal processing approach used here can be used to disentangle different mechano-biological features in the Fourier space by seeing them as independent analog filters. In a variation of the cell shaking technique, a frequency sweep was applied on lung cancer cells, providing a dynamical mechanical analysis on the single cell level with a fluidic readout. Our results illustrate a way how cell-fluid coupling or cell-cell-
interactions through the fluid can be characterized and modelled. Presumably, these play a comparable role as cell-substrate interactions. 679-Pos Board B459 Regulation of Cytoskeleton Contractility and Osteogenesis of Human Mesenchymal Stem Cells using Acoustic Tweezing Cytometry (ATC) Xufeng Xue1, Xiaowei Hong2, Jianping Fu1,2, Cheri Deng2. 1 Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, USA, 2Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA. Human mesenchymal stem cells (hMSCs) have great potential for cellular therapeutics for treating degenerative bone diseases such as osteoporosis. However, in vitro cell culture conditions for hMSC osteogenesis still remain suboptimal. In this study, we used an ultrasound-based technique, acoustic tweezing cytometry (ATC), to apply dynamic subcellular mechanical forces to enhance in vitro hMSC osteogenesis. In previous work, we have shown that single ATC application for 10-20 sec could lead to a sustained increase of the total cytoskeleton contractility of hMSCs for 30 min. As the contractile force of hMSCs has been implicated as a functional predictor of their long-term osteogenesis, we studied the effect of ATC stimulation on hMSC contractility for 24 hr. To identify the optimal experimental conditions to promote hMSC osteogenesis, we explored the effects of different ATC doses, as well as cell shape and substrate rigidity on cytoskeleton contractility of hMSCs. Substrate rigidity was modulated by varying the height of an elastomeric micropost array. We used micro-contact printing to print different adhesive islands to modulate cell shape. Moreover, we characterized the effect of ATC to regulate osteogenic differentiation of hMSCs. We plan to study how the RhoA/ROCK/myosin signaling axis is involved in intracellular force transduction by ATC in hMSCs in the future. In summary, our results showed that ATC was capable of modulating cytoskeleton contractility and enhancing osteogenesis of hMSCs in vitro for celltherapeutics applications for treating bone diseases. 680-Pos Board B460 Probing the Dose-Dependent Effect of Migration Stimulating Factor-Like Drug on Fibroblast Migration using Optical Tweezers Tung-Ju Tsai. Taipei Medical University, Taipei, Taiwan. Human dermal fibroblast migration is one of the most important step in wound healing. Normal and impaired wound healing has long been recognized to be coordinated by the soluble regulatory molecules and the insoluble extracellular matrix. Biological growth factors tested as wound care therapies include proteins and analogues that may function as wound healing cytokines, including epidermal growth factor (EGF), fibroblast growth factor (FGF), and plateletderived growth factor (PDGF). However, all the above growth factors are applied at relatively high concentrations over extended periods of time. Recently, migration stimulating factor (MSF) has been reported to stimulate the migration of fibroblasts, epithelial and endothelial cells; in addition, MSF is expected to induce cell motility at a low dose concentration. We propose an experimental approach to establish a dose dependent relationship with human dermal fibroblast cell at the single cell level. Our idea is that using polystyrene beads coated with different concentrations of migration stimulating factor-like small molecule MM-IGD-Az-2 as a point source of a chemoattractant to locally stimulate human dermal fibroblast cells CCD-996SK, where the cell response is mediated by the binding of the MM-IGD-Az-2 to integrin avb3 receptor. We then apply high-resolution optical tweezers system to conduct spatial and temporal regulation of cell locomotion at the single-cell level, where optically trapped bead is coated with the chemoattractant MM-IGDAz-2. We anticipate the proposed approach based on nano-biomedical technologies, together with the platform at single-cell level could be applied to build a methodology for wound healing behavior at the cellular level. 681-Pos Board B461 External Regulation of EGFR-Mediated Cell Locomotion using Optical Tweezers Hsin-Jui Wu. Taipei Medical University, Taipei City, Taiwan. Chemotaxis of cancer cells in the surrounding microenvironment is an essential component of tumour dissemination during progression and metastasis. Chemotaxis is the result of separate steps, including chemosensing, polarization and locomotion. A rigorous understanding of the mechanism of cancer cell chemotaxis will help us develop novel concepts and strategies for cancer therapy. Recently, concepts, principles and methods from the physical sciences have been applied to the cancer biology. In this study, we will present an innovative approach on chemotaxis assay and provide a bottom-up approach to
Sunday, February 28, 2016
676-Pos Board B456 Dynamic Monitoring of Cell Mechanical Properties using Profile Microindentation Lionel Guillou1, Avin Babataheri1, Pierre-Henri Puech2, Abdul Barakat1, Julien Husson1. 1 Ecole Polytechnique, Palaiseau, France, 2Aix Marseille University, Marseille, France. We have developed a simple and relatively inexpensive system to visualize adherent cells in profile while measuring their mechanical properties using microindentation. The setup allows simultaneous control of cell microenvironment by introducing a micropipette for the delivery of soluble factors or other cell types. We validate this technique against atomic force microscopy measurements and, as a proof of concept, measure the viscoelastic properties of vascular endothelial cells in terms of an apparent stiffness and a dimensionless parameter that describes stress relaxation. Furthermore, we use this technique to monitor the time evolution of these mechanical properties as the cells’ actin is depolymerized using cytochalasin-D. 677-Pos Board B457 Dissecting the Role and Regulation of Mechanical Force at the T-APC Synapse using Atomic Force Microscopy Kenneth H. Hu1, Manish J. Butte2. 1 Biophysics, Stanford University, Stanford, CA, USA, 2Pediatrics, Stanford University, Stanford, CA, USA. T cell activation through formation of an immune synapse (IS) with an antigen presenting cell (APC) is a key step in the adaptive immune response. How the unique evolution and structure of the IS contributes to T cell activation is still being dissected. Here we hypothesize that the dynamic cytoskeletal changes that occur in the T cell generate mechanical forces that promote T cell activation either directly through the TCR or through formation of close contact regions to allow for TCR engagement through a dense glycocalyx. In order to monitor the T cell’s mechanical response to activation, we used an Atomic Force Microscopy (AFM) setup, with a peptide-MHC coated cantilever acting as a surrogate APC. Conversely, we also probed the role of forces during T cell activation by applying force with our setup. We found that T cells exhibit a characteristic profile of force, pushing and pulling on the cantilver. This force profile exhibits temporal correlation with the onset of calcium influx following TCR stimulation. Furthermore, inhibition of actin dynamics with Latrunculin A dramatically reduces calcium flux and force generation. Calcium influx can be partially rescued by application of external oscillatory forces through the cantilever. We find a characteristic timescale for integration of this signal suggesting some relaxation mechanism. To understand the role of the glycocalyx in T cell signaling, we coated the APC membrane with synthetic mucins of varying lengths. We found that T cell activation was reduced in the presence of large mucins, suggesting that the glycocalyx presents a barrier to TCR engagement. Our data shows the T cell acts mechanically on the APC during activation. Future work will need to be done to understand how this force generation is regulated. 678-Pos Board B458 ‘Optical Shaking’ of Red Blood Cells: A Strategy to Measure Cell-Fluid Coupling with Optical Tweezers Carla Zensen1,2, Isis E. Fernandez Buelvas2,3, Oliver Eickelberg2,3, Theobald Lohmu¨ller1,2, Jochen Feldmann1,2. 1 Department of Physics and Center of Nanoscience (CeNS), LudwigMaximilians-Universita¨t, Munich, Germany, 2Nanosystems Initiative Munich (NIM), Munich, Germany, 3Comprehensive Pneumology Center, Insititute of Lung Biology and Disease, Ludwig-Maximilians-Universita¨t and Helmholtz Zentrum, Munich, Germany. We introduce a strategy to use holographic optical tweezers for measuring the fluidic coupling of cells that are ‘shaken’ with a laser beam array. The flow generated by this periodic optical forcing is measured with a ‘detector’ micro particle that is trapped by a second, independent laser in the cell vicinity. A signal processing analysis of both the tracked cell and the trace of the detector bead provides detailed information about the cells reaction on a mechanical stimulation and how this is transferred through the fluid. Measurements of red blood cells exposed to different hypotonic media showed that different cellular edematous states result in a specific fluidic pattern in the flow observed around these cells. We motivate that the signal processing approach used here can be used to disentangle different mechano-biological features in the Fourier space by seeing them as independent analog filters. In a variation of the cell shaking technique, a frequency sweep was applied on lung cancer cells, providing a dynamical mechanical analysis on the single cell level with a fluidic readout. Our results illustrate a way how cell-fluid coupling or cell-cell-
interactions through the fluid can be characterized and modelled. Presumably, these play a comparable role as cell-substrate interactions. 679-Pos Board B459 Regulation of Cytoskeleton Contractility and Osteogenesis of Human Mesenchymal Stem Cells using Acoustic Tweezing Cytometry (ATC) Xufeng Xue1, Xiaowei Hong2, Jianping Fu1,2, Cheri Deng2. 1 Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, USA, 2Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA. Human mesenchymal stem cells (hMSCs) have great potential for cellular therapeutics for treating degenerative bone diseases such as osteoporosis. However, in vitro cell culture conditions for hMSC osteogenesis still remain suboptimal. In this study, we used an ultrasound-based technique, acoustic tweezing cytometry (ATC), to apply dynamic subcellular mechanical forces to enhance in vitro hMSC osteogenesis. In previous work, we have shown that single ATC application for 10-20 sec could lead to a sustained increase of the total cytoskeleton contractility of hMSCs for 30 min. As the contractile force of hMSCs has been implicated as a functional predictor of their long-term osteogenesis, we studied the effect of ATC stimulation on hMSC contractility for 24 hr. To identify the optimal experimental conditions to promote hMSC osteogenesis, we explored the effects of different ATC doses, as well as cell shape and substrate rigidity on cytoskeleton contractility of hMSCs. Substrate rigidity was modulated by varying the height of an elastomeric micropost array. We used micro-contact printing to print different adhesive islands to modulate cell shape. Moreover, we characterized the effect of ATC to regulate osteogenic differentiation of hMSCs. We plan to study how the RhoA/ROCK/myosin signaling axis is involved in intracellular force transduction by ATC in hMSCs in the future. In summary, our results showed that ATC was capable of modulating cytoskeleton contractility and enhancing osteogenesis of hMSCs in vitro for celltherapeutics applications for treating bone diseases. 680-Pos Board B460 Probing the Dose-Dependent Effect of Migration Stimulating Factor-Like Drug on Fibroblast Migration using Optical Tweezers Tung-Ju Tsai. Taipei Medical University, Taipei, Taiwan. Human dermal fibroblast migration is one of the most important step in wound healing. Normal and impaired wound healing has long been recognized to be coordinated by the soluble regulatory molecules and the insoluble extracellular matrix. Biological growth factors tested as wound care therapies include proteins and analogues that may function as wound healing cytokines, including epidermal growth factor (EGF), fibroblast growth factor (FGF), and plateletderived growth factor (PDGF). However, all the above growth factors are applied at relatively high concentrations over extended periods of time. Recently, migration stimulating factor (MSF) has been reported to stimulate the migration of fibroblasts, epithelial and endothelial cells; in addition, MSF is expected to induce cell motility at a low dose concentration. We propose an experimental approach to establish a dose dependent relationship with human dermal fibroblast cell at the single cell level. Our idea is that using polystyrene beads coated with different concentrations of migration stimulating factor-like small molecule MM-IGD-Az-2 as a point source of a chemoattractant to locally stimulate human dermal fibroblast cells CCD-996SK, where the cell response is mediated by the binding of the MM-IGD-Az-2 to integrin avb3 receptor. We then apply high-resolution optical tweezers system to conduct spatial and temporal regulation of cell locomotion at the single-cell level, where optically trapped bead is coated with the chemoattractant MM-IGDAz-2. We anticipate the proposed approach based on nano-biomedical technologies, together with the platform at single-cell level could be applied to build a methodology for wound healing behavior at the cellular level. 681-Pos Board B461 External Regulation of EGFR-Mediated Cell Locomotion using Optical Tweezers Hsin-Jui Wu. Taipei Medical University, Taipei City, Taiwan. Chemotaxis of cancer cells in the surrounding microenvironment is an essential component of tumour dissemination during progression and metastasis. Chemotaxis is the result of separate steps, including chemosensing, polarization and locomotion. A rigorous understanding of the mechanism of cancer cell chemotaxis will help us develop novel concepts and strategies for cancer therapy. Recently, concepts, principles and methods from the physical sciences have been applied to the cancer biology. In this study, we will present an innovative approach on chemotaxis assay and provide a bottom-up approach to