In vivo, most cell types exist surrounded by moving fluid that influences their phenotype and function. The flow created from moving fluids creates shear stress, which can influence cytokine production, polarization, transcription factor expression, organization, and morphology of many cell types (Tzima, E., 2005, Shi, X., 2016, Chen, C., 2014). However, despite these facts, many biological investigations are conducted in vitro. Despite the convenience of in vitro cell cultures for modeling physiological processes, in vitro cultures are critically limited by the static nature of the culture system. This limits the translation of in vitro results to in vivo systems. Therefore, the ability to accurately mimic in vivo environments using physiological shear flow is critical to understanding the true biological responses of cells. Here, we highlight several flow-dependent biological processes that cannot easily be studied in traditional cell culture dishes, as well as a simple solution to investigate these cells under shear flow.
Figure 1. Shear stress on the vascular wall
Atherosclerosis
Atherosclerosis can lead to restricted blood flow, causing a range of complications. The restriction in flow is due to a combination of factors, including reduced nitric oxide (NO) bioavailability, which impairs vessel dilation, and plaque accumulation, which reduces the size of the vessel, thereby limiting blood flow. Flow of different levels, high to low, can be utilized to simulate different parts of this disease to elucidate the mechanisms of atherosclerosis. For example, shear flow not only impacts endothelial cell alignment (Figure 2) but also influences signaling pathways and gene expression within endothelial cells. Furthermore, leukocyte adhesion to the endothelium and transmigration into the arterial intima, important steps in the development and progression of atherosclerosis, are impacted by shear flow. Therefore, accurate in vitro investigation of atherosclerosis mechanisms in a simulated in vivo environment can speed discovery and development of atherosclerosis treatments and preventions.
Figure 2. Impact of shear flow on endothelial cells
Blood Clotting
Platelets are critical for the return to homeostasis following physical wounding, as they bind to damaged blood vessels to prevent excessive bleeding. Additionally, platelets play a major role in multiple diseases, including sickle cell disease, atherosclerosis, and hemophilia. Dysregulated platelet function can lead to poor clotting and subsequent bleeding or to the formation of thrombi, which can occlude blood flow to tissue. Although aggregometry is typically used to study the mechanisms of platelet function, this method is low-throughput, requires relatively large amounts of blood, and perhaps most importantly, is not performed under flow conditions. These critical limitations reduce the relevance of many platelet investigations because platelets can be activated by high shear stress, such as injuries to capillaries or turbulent flow near atherosclerotic plaques. The ability to investigate and image platelet interactions and thrombus formation under physiological and pathological shear flow, aids the understanding of the mechanisms that contribute to platelet function.
Video 1. Thrombin-loaded injury-site-targeted lipid nanoparticles
Video 1. t-TLNPs (thrombin-loaded injury-site-targeted lipid nanoparticles) enhance fibrin generation under simulated vascular flow environment imaged in real-time using BioFlux microfluidic setup. Human plasma containing fluorescently labeled platelets and fibrinogen (by Calcein and AlexaFluor647, respectively) (Girish, A., 2022).
Cancer cells
Cancer cell chemotaxis and transmigration contribute to metastasis and other oncological processes in the vascular system. Examining migration over time under shear flow can help screen compounds that inhibit cancer cell invasion, leading to better treatment options. Furthermore, tumors are known to alter the microenvironments that surround them. This can promote immunotolerance, encourage tumor angiogenesis, and negatively impact nearby tissue. In vivo examination of these responses is difficult due to the heterogeneity of the cells in the tumor’s environment. Stepwise mechanistic examination of tumors and their impact on the surrounding microenvironment is much more feasible in a controlled modular system that simulates in vivo conditions.
Figure 3. Shear stress by vessel
CAR-T cells
Immunotherapies like CAR-T and TCR require interactions between multiple cell types and target cells and tissues. Real-time cell adhesion, transmigration, binding strength, and other aspects of T cell therapies can be assessed under physiological flow as compounds are introduced. In addition, this type of tightly controlled assay can enhance CAR-T therapy by ensuring that engineered cells have the desired functional properties before delivering treatment to patients.
Bacterial and Fungal Biofilms
Bacteria and fungi often form biofilms, which are complex communities of microbes bound by an extracellular matrix. Biofilms are beneficial to microbes for various reasons, including adhesion, nutrient cycling, exchange of genetic material, and division of metabolic labor amongst different subpopulations. However, the development of biofilms is often detrimental to humans, such as in the case of plaque accumulation on teeth and medical device infection. Although biofilms can be investigated in traditional cultures, the static nature of the culture can lead to nutrient depletion and metabolite accumulation, which does not reflect natural systems where biofilms modulate their response to environmental changes. Therefore, efficient development of antibiotic and antimicrobial drugs should be done under shear flow conditions.
Figure 4. Confocal Laser Scanning Microscopy of viable P338-adsorbed and unadsorbed biofilms
Conclusions
Accurate investigation of processes that are impacted by flow in the body requires similar flow in vitro to examine relevant responses. Instruments such as BioFlux can create shear stress at specific intensities that can be adjusted to mimic the physiological or pathological shear stress that cells of interest experience in vivo. A key component of the BioFlux system is the ability to provide real-time imaging, enabling a clearer understanding of the interactions between cells or the effects of compounds during drug discovery and development investigations. Adding shear stress to cells in the form of flow can provide many benefits to in vitro experiments that induce physiological responses similar to those that occur in vivo.
References
Lexi Land contributes to developing scientific workflows that are compatible with the CellRaft® AIR System. Her focus at Cell Microsystems involves stem cell research, 3D organoid work, as well as adherent and suspension cell culture. Lexi received a Bachelor of Science in Biological Sciences from North Carolina State University with a focus in molecular, cellular, and developmental biology.
In the ongoing battle against antibiotic-resistant bacteria, or “superbugs,” scientists are constantly seeking innovative tools to understand the mechanisms of resistance and develop effective treatments. One such groundbreaking technology is shear flow systems, which have emerged as game-changers in microbiological research. By simulating the dynamic conditions of bacteria’s natural environments, such as the human body or food processing, shear flow systems provide unique insights into bacterial behavior and resistance mechanisms that traditional static methods simply cannot achieve. In this blog post, we’ll explore the benefits of using shear flow systems to combat antibiotic resistance and develop next-generation therapies.
Antibiotic resistance is a complex phenomenon influenced by a variety of factors, including genetic mutations, horizontal gene transfer, and the formation of biofilms. Traditional laboratory methods often fail to capture the dynamic nature of these processes, leading to incomplete or misleading results. Shear flow systems address this limitation by providing a more realistic environment for bacterial growth and interaction. Here’s how:
1. Mimicking Real-World Conditions
In the human body, bacteria are rarely in a static state. They are constantly exposed to fluid flow, such as blood circulation or urine flow, which influences their behavior and resistance mechanisms. Shear flow systems can replicate these conditions, allowing researchers to study how bacteria respond to antibiotics under realistic physiological conditions. This is critical for understanding how resistance develops and persists in vivo. This benefit was recently highlighted in a webinar presented by Dr. Katharina Richter, a microbiology researcher from the University of Adelaide in Australia. Using a high-throughput BioFlux Shear Flow System, Dr. Richter and her team were able to test 3 different methods of superbug treatments under physiological conditions.
2. Studying Biofilm Formation and Resistance
Biofilms—structured communities of bacteria encased in a protective matrix—are a major contributor to antibiotic resistance. Biofilms are notoriously difficult to treat because they shield bacteria from antibiotics and the immune system. Shear flow systems enable researchers to study biofilm formation in real time, observing how bacteria adhere to surfaces, form microcolonies, and develop resistance under flow conditions. For example, using a BioFlux, Dr. Richter was able to leverage high-resolution imaging to obtain unprecedented insight into the biofilm killing and prevention efficacy of a copper and diethyldithiocarbamate (DDC) combination nanoparticles¹. The group is currently investigating the use of a Cu-DDC infused gel that can be injected into wounds that are at high risk for chronic infection, such as hernia.
The study of biofilm communities presents unique challenges in experimental design and analysis. While mixed bacterial populations offer fascinating insights into microbial interactions and community dynamics, accurately characterizing and investigating these complex systems has previously required sophisticated and tedious technical approaches. Here, we will discuss advances in technology that have helped to simplify and streamline polymicrobial investigations.
Throughout the last decade, a trend for growing and assaying biofilms under shear flow has gained traction among microbiologists. The rationale for investigating biofilms under flow is multi-dimensional. A recent Cell Microsystems survey asked why microbiologists are using shear flow found that the top responses were:
Of the scientists surveyed, 40% indicated that they use a BioFlux Shear Flow System. This system provides unique advantages over other methods to induce shear stress, such as slide-based parallel plate flow chambers and benchtop rockers.
BioFlux uses microfluidic channels embedded onto the bottom of standard-sized 6, 24, or 48-well plates to create isolated and independent flow chambers where user-defined wall shear stress is initiated and maintained by digitally controlled air pressure. This not only simplifies the setup and experimental processes but also ensures that experiments are contained and do not contaminate the shear flow system.
Although shear flow has been thoroughly demonstrated to improve the biological relevance of biofilm investigations1, an often overlooked aspect of biofilm physiology is the significant role that multicellular aggregates play in biofilm formation2. When investigating Pseudomonas aeruginosa, it has been demonstrated that multicellular aggregates lead to faster biofilm formation and enhanced resistance to antimicrobials and immune responses3. In addition, multispecies microbial aggregates have been shown to dominate single cells in human saliva4. Together, these data show that aggregates are important areas of investigation because they are more likely to represent physiological biofilm formation and growth.
