Mechanosensing and Shear Stress in Microfluidics

Microfluidic technology offers unprecedented spatiotemporal (space and time) control over the cellular microenvironment, driving innovation and discoveries in cell biology. This review, tailored for prospective and current users in the field of microfluidic cell culture, examines a fundamental physiological cue essential for replicating conditions inside living organisms within microfluidic systems: mechanical forces exerted on cells. Specifically, we will explore a mechanical force known as shear stress and its role in modulating cellular responses through a process known as mechanosensing. Further, we will explore the design considerations for shear stress control in microfluidic devices and discuss the diverse applications of this pioneering technology [1].

Advancing Science and Healthcare through Microfluidics 

Unraveling complex cellular interactions and cellular and tissue responses to physiological stimuli is vital to advancing science and improving healthcare. Traditional methods rely on in vivo (within a living organism) and in vitro (outside a living organism) testing, yet both methods have limitations. In vivo testing is limited by ethical concerns, cost, physiological variability, bias, and limited real-time analysis. In vitro testing, on the other hand, offers advantages because of the limited ethical concerns, decreased cost, and real-time analysis. Traditional in vitro studies, however, often lack the physiological cues necessary to translate results to whole organisms [2].

Bridging the Gap with Microfluidic Cell Culture

Microfluidic cell culture addresses challenges by controlling the microenvironment in the device, enabling the recreation of physiological conditions. This leverages the benefits of in vitro testing and bridges the gap between in vitro and in vivo research. Microfluidic technology can be adapted to culturing human cells and tissues within microchannels, where the device offers precise, spatiotemporal control over physiological conditions, such as nutrient supply, fluid flow, mechanical forces, and cell-cell interactions [2].

For a more in-depth overview of microfluidic cell culture, including beginner-friendly explanations, check out our “Cell Biology: Microfluidic Concepts and Methodologies” and “Microfluidic cell culture – a beginner-friendly review” resources.

Understanding Shear Stress: A Crucial Physiological Cue

Cells and tissues commonly experience various mechanical forces, including shear stress. But what exactly is shear stress? Shear stress is induced by fluid flow over a stationary phase, which creates a frictional force between the fluid layer and the solid. In vivo, shear stress is typically exerted on cells lining arteries and veins.

 In vivo, cells are not passive recipients of mechanical forces; mechanical forces function as fundamental physiological cues that induce cellular responses. Cells respond to mechanical forces using specialized sensors, known as mechanosensors, that can detect and respond to changes in mechanical forces exerted on them. Mechanosensors generally consist of proteins or structures outside of cells that perceive mechanical stimuli and convert them into chemical messages that elicit a cellular response. This entire process is known as mechanosensing (or mechanotransduction). In the case of shear stress, mechanosensing has been shown to promote cellular responses such as proliferation, migration, permeability, morphology, and gene expression. Thus, shear stress and mechanosensing are critical in physiological environments where fluids flow over cell and tissue surfaces [5]. 

Engineering Controlled Shear Stress in Microfluidic Devices

When designing a microfluidic cell culture setup, a user must consider several factors to incorporate shear stress into the microenvironment. For example, understanding the physiological environment being recapitulated is essential since it influences cell type and concentration [4, 6]. The design of the microfluidic device is also paramount. Microfluidic structural features that dictate the spatiotemporal magnitude of shear stress include microchannel dimensions, geometry, and methods for delivering and controlling the flow within the device [4].

1. Physiological Environment: The magnitude of shear forces exerted on cells depends on their locale and environment. For instance, the shear stress experienced by cells lining arteries is typically higher than that in veins (see Table 1). Understanding this variability is essential for designing devices that accurately recapitulate physiological states. This knowledge enables researchers to develop microfluidic devices that mimic conditions in arteries and veins and model diseased states where shear stress may be lower, higher, or variable [4].

2. Microchannel Dimensions and Geometry: The dimensions and geometry of microfluidic channels influence the magnitude of shear stress experienced by adhered cells and tissues within devices. Narrower channels or channels with higher aspect ratios tend to generate higher shear forces, while wider channels with lower aspect ratios produce lower shear stress. Additionally, introducing microstructures or coatings within microchannels can modulate shear stress distribution, enabling researchers to create spatially controlled shear force gradients in their experimental setup. Thus, precise control over device morphology is an essential tool for producing controlled microenvironments. (4)
3. Delivering and Controlling Flow: By precisely regulating the flow rate of fluids within microchannels of a device, the hydrodynamic flow across cells and tissues adhered to the walls of microfluidic channels creates shear stress. Higher shear stress results from higher flow rates, while lower shear stresses can be accomplished with lower flow rates. In microfluidic setups, flow rate can be generated by hydrostatic pressure, pressure generators, syringe pumps or liquid pumps such as peristaltic pumps. For more information on controlling the microfluidic flow rate, see our detailed review here.
   
   There are experimental and theoretical methods to measure shear stress in microfluidic systems that include:

• Particle image velocimetry (PIV) – PIV utilizes tracer particles that are imaged with microscopy to quantify flow velocities, which can be used to calculate shear stress via mathematical relationships. A complete understanding of mathematics and physics is not required with the Elveflow Shear Stress Calculator.

• Finite element analysis (FEA) – FEA is a computational technique that discretizes microchannel geometry and applies fluid flow boundary conditions to predict shear stress profiles. It is a complementary technique to experimental methods and beneficial to complex flow phenomena.
• Microfluidic shear stress sensors – Shear stress sensors are microfabricated and integrated into the microfluidic device. They directly measure shear stress through the deformation of cantilevers, membranes, etc. The deformation produces an electrical or mechanical signal, offering real-time shear stress monitoring and feed-back controlled experiments [4].

Diverse Microfluidic Platforms to Study Mechanosensing

 Monolayer Devices: Microfluidic devices can be designed to accommodate a monolayer of cells inside microchannels for studying the effects of mechanical forces. For example, researchers have applied monolayer setups to study physiological wound healing, creating what they referred to as a microfluidic wound-healing assay (Figure 3). In this study, fibroblasts were cultured as a monolayer within the system. A “wound” was created using precise spatial control afforded by microfluidics, where trypsin was applied to detach cells from the monolayer. Healing was defined as the regrowth of fibroblasts to cover the wound. The healing rates were quantified, and the influence of shear stress on the process was examined. The findings revealed a correlation between increased shear forces and faster healing times, suggesting mechanosensing plays a role in wound healing [8].

Three-dimensional (3D) Layer Devices: Microfluidic devices can also support 3D cell culture, often called “lab on a chip.” In one study, researchers designed a 3D microfluidic device by embedding cancer cells in a collagen-based hydrogel scaffold. This setup served as a model for tumors, where fluid flow could be modulated to mimic the higher interstitial fluid flow (higher shear forces) in the extracellular matrix (ECM) caused by cancer cells. The research demonstrated that shear stress promoted the directional migration of cancer cells in a simulated ECM environment [9]. 

In addition to monolayer and 3D layer devices, microfluidic cell culture setups can be hybridized to incorporate both 2D and 3D cells within the same device. Furthermore, microfluidic devices can also be designed to facilitate co-culture experiments. Co-culture experiments involve culturing two or more cell types together to study cell-cell interactions with physiological cues, either in 2D or 3D. Gradient- or oscillatory-generating devices can also be designed to mimic dynamic in vivo conditions [1].

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