Mechanobiology and Microscale Devices - Neeraj, Robert Sterian, Eugene Cheong, Luke Boudreau
Every organism, be it an amoeba or a complex multicellular organism like human being, is composed of cells which are constantly in communication with the physical environment like extracellular matrix (ECM), fluid flow, or with adjacent cells. Cells sense their surroundings through biochemical signals like cytokines and growth factors or through mechanical stimuli like osmotic forces, shear stresses, ECM stiffness or geometry. Recently, scientists are beginning to understand that mechanical signals that are either intrinsic or extrinsic play a vital role in cell migration, morphology, proliferation, differentiation and homeostasis. Mechanotransduction is the process by which a mechanical stimulus elicits a biological response from a cell/tissue which changes the intrinsic properties of a cell or its microenvironment. This in turn modifies the mechanical properties around the cell and creates a feedback loop. Mechanobiology is the field of science which seeks to study the responsiveness and signaling of tissues and cells by altering physical and mechanical properties in order to elucidate the relationships observed in mechanotransduction and cell signaling. For many years, most of the experiments designed to understand the fundamental questions in cell and molecular biology have been performed in vivo with limited control or in vitro in a way that lacks many physiologically relevant parameters. As such microfluidics has become a staple for use in mechanobiology since the micro- and nanoscale aspects of these platforms can allow for a level of control often seen with in vitro experiments, while also providing the robustness of cell differentiation seen with in vivo experiments.
Types of mechanical stimulus
In order to develop more accurate experiments using mechanobiology, microfluidics will be used to answer mechanobiological questions by developing different microenvironments for cells and tissues to undergo different situations using mechanical devices. The goal in simulating these environments is to gain more knowledge on the interaction of cells and develop more realistic models for in vivo models. The development of realistic models will lead to more efficient methodologies on how to develop experiments with the end goal of achieving organ equilibrium and/or treatment of diseases. The capability of a 3D view of cellular compound interactions between a compound and a surface. For many examples, PDMS is used as a material for many devices. These interactions from different mechanical stimuli will also lead to more accurate observations rather than viewing cell at a two dimensional plane.
Shear stress in blood vessels
Endothelial shear stress is the friction of blood flow through an endothelial surface. If a blood vessel were to have a high endothelial shear stress, then there is very high friction leading to decrease of blood flow. Where as a blood vessel with a low endothelial shear stress has a low friction leading to an increase of blood flow. With the use of mechanobiology, scientists are able to study the effects of fluid flow by using a constricted pathway to emulate a blood vessel with shear stress. Some responses as a result of this observation are cell elongation, increased cell contractibility, and decreased permeability of blood vessels. Blood vessels are made of endothelial cells (ECs) lining the inner surface, smooth muscle cells and pericytes surrounding the endothelial cell layer and blood cells comprising of erythrocytes, leukocytes and monocytes that travel inside the blood vessel. These cells undergo various mechanical stimuli inside the body, among which the most important component is contributed by hemodynamic forces due to the flow of blood. The ECs experience shear stress on their apical surface generated by the blood flow which influences cell proliferation, morphology, migration, wound healing, permeability and gene expression.
To understand how shear stresses are induced in a blood vessel, let us consider the example of water flowing in a pipe. The flowing water applies a physical force on the walls of the pipe. This force can be resolved into two components: the force parallel to the pipe wall manifests as shear stress and the force perpendicular to the wall exerts a tensile stress. In blood vessels, the shear stress is a frictional force which is exerted by blood on the endothelial side of the vessel whereas the tensile stress is the cause of blood pressure which dilates the vessel wall rhythmically as the heart pumps making it pulsatile in nature. Previous studies involving parallel plate flow chamber models were used to demonstrate the effect of shear flow on ECs but nevertheless they are difficult to optimize and customize due to the requirement of large amounts of cells and reagents in addition to low experimental efficiency due to its inability to generate multiple shear stress profiles in a single apparatus. In order to eliminate the drawbacks of these macroscale systems, novel microfluidic platforms were used to develop various shear flow assays. Chau et al. have developed a device which allows up to ten different shear stresses spanning across 0.07 – 13 Pa to be induced on the ECs simultaneously. This was achieved by varying the length of the channel which in turn affected the resistance of the channel (R), volume flow rate (Q) and shear stress (τ).
Transendothelial and Interstitial flow in blood vessels
Besides shear flow, the ECs experience transendothelial flow which influences the force and the mass transport across the endothelial layer. These phenomena have been mimicked in vitro using various microfluidic platforms that use 3D collagen hydrogels to study the effect on sprouting angiogenesis. Song et al. have shown using a microfluidic tissue analog of angiogenic sprouting that fluid shear stress exerted during blood flow reduces EC sprouting in a nitric-oxide dependent manner and the interstitial flow witnessed in an extravasating plasma directs EC morphogenesis and sprout formation. This interstitial flow is defined as the motion of fluids through an extracellular matrix such as tissues. This type of flow is encouraged in order to help transport large proteins and induce physiological responses from interstitial cells. By implementing a pressure fluid gradient on a hydrogel, motion and alignment of cells can be mimicked as interstitial flow. According to figure 3 (right), the blue arrows represent a culture of cancer cells migrating at an upstream motion.
Strain, stretching, and stiffness in blood vessels
Due to the pulsatile nature of blood flow in the vessels, the ECs, smooth muscle cells (SMCs) and mesenchymal stem cells (MSCs) are exposed to cyclic substrate strain. Various microfluidic assays were developed to study this effect in MSCs and SMCs. Zhou et al. have developed a device to simulate and study the effect of substrate strains when applied on MSCs. The study has reported that cells aligned for strains greater than 10% and activated SMAD1/SMAD2 and Wnt/β-catenin pathways. The hMSCs were cultured on flexible membranes which inflates and deflates when pressure is applied and released respectively mimicking the arterial vessels. While strain is often seen in blood vessels, stretching and stiffness is more often observed within muscle cells. Stretching is a form of mechanical stimulation that engineers the physical lining of the membrane and modifies it. This technique is primarily used for promoting the process of myogenesis, the formation of muscular tissue. Stretching can also play a major role in the growth and functionality of muscle.  There are multiple methods for how a membrane can be stretched depending on using one more more axels. Studies have shown that muscle tissue under Embryonic Chick Cardiomyocyte and Neonatal Rat Cardiomyocyte culture have been studied for approximately a week in order observe the effects while under mechanical stimulation. The results presented evidence of the muscle tissue becoming more enlarged, improved functionality of contracting, and increased tensile strength., Similar to stretching, stiffness gradient has an influence in myogenesis. In addition, with an in vitro model, stiffness has the ability to control a range of cellular functionality such as motility, morphology, and response signaling.
Measurement of cellular traction force using microfluidic devices
The cellular traction force (CTF) is defined as the tangential force exerted by cells on the extracellular matrix (ECM) or any underlying substrate. CTFs play a vital role in various biological processes like wound healing, inflammation, cell migration, angiogenesis and metastasis. Generation of CTFs in non-muscle cells is accomplished using actin filament bundles, also called as stress fibers, and powered by the hydrolysis of ATP generates tension that contracts the cell body due to actomyosin cross bridges. This tension is then transmitted to the extracellular matrix (ECM) and this force is called cellular traction force. The secondary source of CTF is through actin polymerization which drives the forward protrusion of the leading edge of a migrating cell. These forces are transferred from actin fibers to the ECM through focal adhesions (FA) which consists of a complex assembly of ECM proteins, transmembrane receptors and signaling molecules like integrins, paxillin, vinculin, tensin, talin, kinases and phosphatases. Among the FA proteins, integrins play an important role in cell mechanotransduction by providing a physical connection between the ECM and actin cytoskeleton and this helps in sensing various mechanical cues around the cell microenvironment.
It is always important to study the cellular microenvironment to understand various biological processes as it gives a real picture of various biochemical, bioelectric and mechanical signaling that takes place. To replicate these cellular microenvironments and various other in vivo conditions in the lab, microfluidic device is an ideal candidate and provides a clear and robust insight into a variety of cellular aspects like cell migration, CTFs, cell differentiation and cell death. For example, a microfluidic device was developed to study the effect of chemotaxis on migration of a group of cells. A chemical gradient of hepatocyte growth factor (HGF) was induced, and the collective movement of cells cultured on PDMS substrates in micropatterned islands were analyzed and the data was interpreted in terms of monolayer traction and stress microscopy. The PDMS substrate is embedded with fluorescent nanoparticles to track the motion of cells and coated with collagen solution to facilitate cell attachment and proliferation. During the initial time points, all cases showed similar trends where the cells on the edge of the island showed strong inward traction while cells within the island had fluctuations in the traction distribution. After the application of HGF gradient for 10 h, the degree of expansion of cell island varied but the traction force distributions largely remained the same. However, the monolayer stress maps showed a trend where the stresses were high (200 Pa) when the HGF concentration was low as compared to 100 Pa when the HGF concentration was high.
Traction forces play a vital role in angiogenesis where the coordinated cell movement and ECM remodeling is carried out. The cells migrate in response to complex mechanical and chemical signals around their microenvironment. Mechanical signals like fluid shear stress and ECM stiffness help in blood vessel sprouting and formation. The response of the endothelial cells is communicated in the form of forces exerted by cells on the substrate to remodel them to form appropriate network of blood vessels and capillaries. This forms a feedback loop where mechanosensing of cells through focal adhesions gives them a sense of the mechanical environment around them and in turn they produce traction forces to remodel the matrix to form new blood vessels or repair and maintain existing ones. Microfluidic devices also provide an alternative, easy and efficient way to compute viscoelastic parameters of cells, binding strengths of biomolecules inside and on the plasma membrane and mechanics of dorsal side of the cell. Atomic force microscopy and optical trap provides a way to compute the molecular binding strengths, TFM and micropost deflection method helps in quantifying CTFs and magnetic tweezers help in studying the viscoelastic parameters of the cell. As mentioned above, we can quantify cellular traction forces through the deflection of an array of compliant PDMS microposts produced by cells cultured on them. Raman et al. have fabricated a microfluidic device consisting of PDMS microposts of varying area. NIH 3T3 and human osteosarcoma cells were used to study the effect of myosin-II and confinement of cells in narrow channels. Inhibition of myosin-II using blebbistatin did not affect the traction force or migration speed of cells in confined narrow channels but in wide microchannels whereas activation of myosin-II using calyculin A did not affect the CTF in confined environments but increased them in wide microchannels. This study shows the importance of physical environment of cells in different biological processes like cancer progression and metastasis and how the advent of microfluidics played a pivotal role in unravelling these mechanisms that occur in vivo and are otherwise difficult to replicate in in vitro models.
Effect of electroporation on cell mechanics
Electroporation (EP) is a common clinical and lab technique where electric pulses are used to transiently permeabilize the cell membrane allowing for rapid delivery of nanoparticles, drugs and genetic material. Generally, cells maintain an electric potential difference between the inner and outer side of the cell membrane due to a complex system of regulation using ion pumps and ion channels in their plasma membrane and this is termed as resting transmembrane voltage (TMV). In eukaryotes, the resting TMV ranges between -40 and -70 mV, i.e. the electric potential inside the cell is lower than the outer one. When an external electric field is applied, it manifests as an additional induced TMV on the cell proportional to the external field besides the resting TMV. This induced voltage is experienced by the cell as long as the duration of the applied field. When the induced TMV exceeds a certain range, then the cell starts to undergo certain structural and molecular changes in its plasma membrane that are absent under normal conditions. One of the changes witnessed due to applied electric field is formation of nano pores on the plasma membrane termed as electropermeabilization which rapidly increases the membrane permeability.
Besides membrane permeabilization, EP induces change in the actin cytoskeleton and thus affects other mechanical properties of the cell which are essential for its function. The mechanical properties of a cell play a central role in a variety of biological functions such as cell aging, differentiation, migration, mechanotransduction and cell death. These properties can be exploited to serve as a reliable alternative to conventional biomarkers for disease diagnosis. EP can also be used to treat tumors where in the mechanical properties can be changed in order to induce cell death.,  Bai et al. have used a novel microfluidic approach to characterize the mechanical properties of a cell using dielectrophoresis (DEP) based cell stretching and actin cytoskeleton modeling method. Leukemia cells were treated with a chemotherapeutic drug – doxorubicin that induces apoptosis and altered the actin cytoskeleton. Post treatment, the cells were stretched using two different methods - the DEP setup and optical tweezers to compare the biophysical properties in each case. People have also studied the effect of EP on cell swelling using a novel high throughput microfluidic device. In this device, EP was performed on a large cell population but on a single cell at a time. CHO cells were allowed to travel through a narrow microchannel where electric field (DC) is greater than the rest of the channel that can cause EP due to change in the length and cross-sectional area for a constant time which can be adjusted based on the velocity of the cells in the fluid. Using the device, Wang et al. were able to capture images and study the changes in cellular morphology and swelling for up to 30 cells per second.
Pressure effects on cells
Every cell in the human body experiences some form of pressure. From the bone cells, which experience immense weight to keep our bodies upright, to the blood and aortic cells, which experience large amounts of hydrostatic pressure changes every time out heart beats, pressure is an important variable when studying cells and cell culture, though it is often overlooked. In the field of mechanobiology, researchers aim to study the effect of pressure on cells. Most of these studies involve the use of big and expensive bioreactors that cam modulate the pressure of the entire system to ensure that all the cells are experiencing the same pressure equally. Though there is a lot to find out from these studies, almost of these studies focus on a few aspects of the cells. These are the cell morphology, the rate of diffusion of substances such as growth media and oxygen and the growth rate of the cells. Studies that aim to use pressure as a controlled variable must be completed in large and expensive bioreactors that are able to modulate pressure of the system and ensure equal pressure distribution across the cell cultures. Though there is still much to learn about cell's interactions with pressure, some of the most well studied areas pertain to pressure effects on cellular morphology, mass transport cellular systems, and cellular growth rate. In regard to cellular morphology, cells will often attempt to compensate immense pressure by increasing pressure internally to counteract the force. Should the cell not compensate, the cell will collapse under the pressure and die. However, if cells are able to balance the pressure with its environment, cells will adopt an elongated morphology.
As immense pressure changes the shape of the cell, the cell's membrane and its components are also likely to be affected though researchers are likely to overlook this. For example, the protein channels within the fluid mosaic such as the aquaporin, which allow for the flow of water into and out of the cell, could become deformed and alter rates of diffusion. Alternatively, ion channels could be affected and a neural cell's ability to transmit signals or even synthesize ATP may be affected. Microfluidics may present a novel lens to study pressure on cells. While large bioreactors are extremely viable for pressurizing cell cultures over a large range of pressures, microfluidic devices may be able to bring a similar if not better systems using less resources and space. One example of this comes from Liu et al. where they created a microfluidic device that has a built-in electrofluidic pressure sensor to study endothelial cells under shear stress as well as hydrostatic presure. Ho et al. additionally created a microfluidic device that exerts a cyclic hydrostatic pressure on the cells. The system allows for the study of cyclic compression on adherent cells, mimicking the environment experienced by cells in blood vessels.
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