Microdevice-based mechanical compression on living cells

Compressive stress enables the investigation of a range of cellular processes in which forces play an important role, such as cell growth, differentiation, migration, and invasion. Such solid stress can be introduced externally to study cell response and to mechanically induce changes in cell morphology and behavior by static or dynamic compression. Microfluidics is a useful tool for this, allowing one to mimic in vivo microenvironments in on-chip culture systems where force application can be controlled spatially and temporally. Here, we review the mechanical compression applications on cells with a broad focus on studies using microtechnologies and microdevices to apply cell compression, in comparison to off-chip bulk systems. Due to their unique features, microfluidic systems developed to apply compressive forces on single cells, in 2D and 3D culture models, and compression in cancer microenvironments are emphasized. Research efforts in this field can help the development of mechanoceuticals in the future

Application of sequential cyclic compression on cancer cells in a flexible microdevice

Mechanical forces shape physiological structure and function within cell and tissue microenvironments, during which cells strive to restore their shape or develop an adaptive mechanism to maintain cell integrity depending on strength and type of the mechanical loading. While some cells are shown to experience permanent plastic deformation after a repetitive mechanical tensile loading and unloading, the impact of such repetitive compression on deformation of cells is yet to be understood. As such, the ability to apply cyclic compression is crucial for any experimental setup aimed at the study of mechanical compression taking place in cell and tissue microenvironments. Here, we demonstrate such cyclic compression using a microfluidic compression platform on live cell actin in SKOV-3 ovarian cancer cells. Live imaging of the actin cytoskeleton dynamics of the compressed cells was performed for varying pressures applied sequentially in ascending order during cell compression. Additionally, recovery of the compressed cells was investigated by capturing actin cytoskeleton and nuclei profiles of the cells at zero time and 24 h-recovery after compression in end point assays. This was performed for a range of mild pressures within the physiological range. Results showed that the phenotypical response of compressed cells during recovery after compression with 20.8 kPa differed observably from that for 15.6 kPa. This demonstrated the ability of the platform to aid in the capture of differences in cell behaviour as a result of being compressed at various pressures in physiologically relevant manner. Differences observed between compressed cells fixed at zero time or after 24 h-recovery suggest that SKOV-3 cells exhibit deformations at the time of the compression, a proposed mechanism cells use to prevent mechanical damage. Thus, biomechanical responses of SKOV-3 ovarian cancer cells to sequential cyclic compression and during recovery after compression could be revealed in a flexible microdevice. As demonstrated in this work, the observation of morphological, cytoskeletal and nuclear differences in compressed and non-compressed cells, with controlled micro-scale mechanical cell compression and recovery and using livecell imaging, fluorescent tagging and end point assays, can give insights into the mechanics of cancer cells

Flexible microdevices for characterization of the role of bionanomechanics in cancer.

Evidence continues to emerge that cancer is not only a disease of genetic mutations, but also of altered mechanobiological profiles of the cells and microenvironment. This mutation-independent element might be a key factor in promoting development and spread of cancer. However, the nature and level of mechanical forces influencing the interactions between the physical micro- and nano-environment and cancer cells have yet to be comprehensively quantified. Biomechanical forces regulate tumour microen- vironment by solid stress, matrix mechanics, interstitial pressure and flow. Of these, compressive stress by tumour growth and stromal tissue alters the cell deformation, and recapitulates the biophysical properties of cells to grow, differentiate, spread or invade. The role of such solid stress can be studied by applying compression to cells in a localized, flexible and controlled manner. To achieve the latter, this thesis introduces a novel polydimethylsiloxane (PDMS)- based lab-on-a-chip platform containing a flexible microactuator with attached micro- piston. The platform is composed of a control microchannel for introducing external force and a PDMS membrane with monolithically integrated micro-piston suspended in a bottom microchannel to transduce this force in form of mechanical compression to ovarian cancer cells cultured on the glass surface enclosing the bottom layer. Before use with cells, mechanical parameters of the device were characterized via micro-piston actuation to ensure repeatability of compression. The characterization of the micro- piston actuation was done using optical imaging methods and two different external pressure system types, adding flexibility based on laboratory requirements. Experimen- tal data obtained from the vertical displacement measurements was used to establish a mechanical model of the platform. In turn, this model enabled the actual pressures in- side the device to be predicted based on externally applied pressures. After mechanical characterization, loading with cells and the culture of cancer cells in the micro-piston device were optimized experimentally. Initial cell experiments indicated directional alignment of monolayers of SKOV-3 ovarian cancer cells to the micro-pistons as hang- ing structure in the microchannel. This was followed by use of the device to study compression at physiologically-relevant pressure levels and to demonstrate mechanical lysis of cells. Cell viability responses were investigated for sequentially applied profiles and varying pressures in ascending order. Results demonstrated the ability of the platform to simulate cyclic and varying compression profiles comparable to those cells are exposed to in ovarian cancer metastasis. Detailed time-lapse live cell imaging showed the temporal evolution of dynamic pressure control on cell compression and deformation with different pressure amounts, time lengths and cyclic modes. Applicability of the cyclic compression facilitated by the developed platform was further demonstrated by capturing both actin and nuclei deformations occurring in cyclically compressed cells. Actin stress fibers showed distinct deformation in samples with applied pressures in ascending order. While compressed cell nuclei changed in circularity at different pressures, areal and axial deformations were non-permanent at milder pressures, such as 12.5 kPa and 17.9 kPa, and more permanent at higher pressures such as 20.7 kPa, for 1 hour-long cyclic compressions. To better mimic the in vivo cellular microenvironment, hydrogel functionalization of cell-culture chambers on the platform, as well as culture of the cells and application of cyclic compressions in these modified chambers were demonstrated. Additional modeling and experimental validation were performed to compare the actuation of micro-pistons on membranes with varying thicknesses. The sequential cyclic compression on the platform was then used to record dynamics of live cell actin, labelled with green fluorescent protein, in response to varying pressures applied in ascending order. Additionally, the recovery of the compressed cells was investigated using end point assays by capturing actin cytoskeleton and nuclei profiles of cells at zero time and at 24 h-recovery after compression in end point assays. This was performed for a range of mild pressures within the physiological range. Results showed that the phenotypical response of compressed cells during recovery after compression with 20.8 kPa differed observably from that for 15.6 kPa. This demonstrated the ability of the platform to aid in the capture of differences in cell behaviour as a result of being compressed at various pressures in physiologically relevant manner. Differences in actin signal and nuclei area and shape descriptors observed between compressed cells fixed at zero time or after 24 h-recovery suggest that SKOV-3 cells exhibit deformations at the time of the compression, a proposed mechanism cells use to prevent mechanical damage. Thus, biomechanical responses of SKOV-3 ovarian cancer cells to sequential cyclic compression and during recovery after compression could be revealed in a flexible microdevice. The observation of morphological, cytoskeletal and nuclear differences in compressed and non-compressed cells, with controlled micro-scale mechanical cell compression and recovery, can give insights into the mechanics of cancer cells. As demonstrated in this work, the platform introduced here can control the strength and duration of cyclic compression and enable the use of live-cell imaging, fluorescent tagging and end point assays to investigate cell response, thus providing a powerful new tool for the study of mechanobiological processes in cancer and cell biology

A microfluidic platform for applying localized and dynamically-controlled compression on cancer cells

In this work we report a microfluidic cell-culture platform with an integrated, actively-modulated actuator for the application of compressive forces on cancer cells. We show fabrication of the platform, mechanical characterization of the actuator and observed mechanotactic behavior of a monolayer of SKOV-3 ovarian cancer cells under static conditions. We further show compression and lysing of the cells, demonstrating suitability for mechanical stimulation with various pressures to study compressive forces in cancer microenvironments

A microfluidic platform for applying localized and dynamically-controlled compression on cancer cells

In this work we report a microfluidic cell-culture platform with an integrated, actively-modulated actuator for the application of compressive forces on cancer cells. We show fabrication of the platform, mechanical characterization of the actuator and observed mechanotactic behavior of a monolayer of SKOV-3 ovarian cancer cells under static conditions. We further show compression and lysing of the cells, demonstrating suitability for mechanical stimulation with various pressures to study compressive forces in cancer microenvironments

A flexible microdevice for mechanical cell stimulation and compression in microfluidic settings

Evidence continues to emerge that cancer is not only a disease of genetic mutations, but also of altered mechanobiological profiles of the cells and microenvironment. This mutation-independent element might be a key factor in promoting development and spread of cancer. Biomechanical forces regulate tumor microenvironment by solid stress, matrix mechanics, interstitial pressure and flow. Compressive stress by tumor growth and stromal tissue alters the cell deformation, and recapitulates the biophysical properties of cells to grow, differentiate, spread or invade. Such a solid stress can be introduced externally to change the cell response and to mechanically induce cell lysis by dynamic compression. In this work we report a microfluidic cell-culture platform with an integrated, actively-modulated actuator for the application of compressive forces on cancer cells. Our platform is composed of a control microchannel in a top layer for introducing external force and a polydimethylsiloxane (PDMS) membrane with monolithically integrated actuators. The integrated actuator, herein called micro-piston, was used to apply compression on SKOV-3 ovarian cancer cells in a dynamic and controlled manner by modulating applied gas pressure, localization, shape and size of the micro-piston. We report fabrication of the platform, characterization of the mechanical actuator experimentally and computationally, as well as cell loading and culture in the device. We further show use of the actuator to perform both, repeated dynamic cell compression at physiological pressure levels, and end-point mechanical cell lysis, demonstrating suitability for mechanical stimulation to study the role of compressive forces in cancer microenvironments. Finally, we extend cell compression applications in our device to investigating mechanobiologically-related protein and nuclei profile in cyclically compressed cells

SYNTHESIS AND REACTIONS OF SOME 1H-PYRAZOLE-3 CARBOXYLIC ACID CHLORIDE

The pyrazole-carboxylic acid chloride 2 was obtained from the reaction of 4-Benzoyl-1-(2,4-dinitrophenyl)-5-phenyl-1H-pyrazole-3-carboxylic acid I and thionyl chloride. 1H-Pyrazole-3 carboxylic acid chlorides 2 can easily be converted into corresponding 1H-pyrazole-3-carboxylic acid amide derivatives 4 and 1H-pyrazole-3-carboxamide derivatives 6 from the reaction with various aliphatic and aromatic amines. The structures of these new synthesized compounds were determined from the IR,(1)H and (13)C NMR spectroscopic data and elemental anaylsis

Breast cancer cells and macrophages in a paracrine-juxtacrine loop

Breast cancer cells (BCC) and macrophages are known to interact via epidermal growth factor (EGF) produced by macrophages and colony stimulating factor-1 (CSF-1) produced by BCC. Despite contradictory findings, this interaction is perceived as a paracrine loop. Further, the underlying mechanism of interaction remains unclear. Here, we investigated interactions of BCC with macrophages in 2D and 3D. While both BCC and macrophages showed invasion/chemotaxis to fetal bovine serum, only macrophages showed chemotaxis to BCC in custom designed 3D cell-on-a-chip devices. These results were in agreement with gradient simulation results and ELISA results showing that macrophage-derived-EGF was not secreted into macrophage-conditioned-medium. Live cell imaging of BCC in the presence and absence of iressa showed that macrophages but not macrophage-derived matrix modulated adhesion and motility of BCC in 2D. 3D co-culture experiments in collagen and matrigel showed that BCC changed their multicellular organization in the presence of macrophages. In custom designed 3D co-culture cell-on-a-chip devices, macrophages promoted and reduced migration of BCC in collagen and matrigel, respectively. Furthermore, adherent but not suspended BCC endocytosed EGFR when in contact with macrophages. Collectively, our data revealed that macrophages showed chemotaxis towards BCC whereas BCC required direct contact to interact with macrophage-derived-EGF. Therefore, we propose that the interaction between cancer cells and macrophages is a paracrine-juxtacrine loop of CSF-1 and EGF, respectively