Flow-Based Assays Offer Alternative to Animal Experiments
By Shrey Kohli, Ph.D.
The blood vascular system lined by a monolayer of endothelial cells is constantly under flow conditions. These flow conditions create a physiological shear stress not only on the endothelium but also to the blood cells including immune cells and platelets. This shear stress creates a mechanical force affecting the morphology, physiology and development of these cells. Therefore in vitro experiments designed to study endothelial cells per se or an association between endothelial cells with other cell types in the blood stream would require culturing them under flow conditions. Static culture conditions provide simplicity, ease of use and lesser costs, however, they are less likely to mimic the physiological conditions. In this regard, flow-based assays can offer a closer in vitro alternative to animal experiments, leading to approaches that can replace, reduce and/or refine animal experiments.
The flow assays are in particular beneficial to the thrombosis and hemostasis field. Current diagnostic assays largely measure clot formation in isolated aspects. In the context of thrombus formation, development of in vitro flow-based assays dates back to the 1980s and provided an alternative to the study of platelet aggregation and platelet interactions with other cell types. Perfusion of whole blood on collagen-coated micro slides in these experiments provided a basic framework of thrombus formation assay. Microfabrication and availability of microfluidic devices has broadened the field to develop more sophisticated and clinically relevant in vitro flow assays to study aspects of thrombosis, hemostasis and vascular biology.
However, there is an opportunity to further develop these assays to mimic the complexity of both physiological and pathological conditions. One challenge to developing a mimic is to provide a suitably placed endothelial cell layer in the microfluidic devices. However, the layer is limited to discreet areas, and therefore blood flow in these devices is in direct contact with plastic ware and possibly other areas of the device which could potentially activate the thrombotic system.
Another challenge is associated with the complexity and plasticity of organ-specific endothelial cells making each type of endothelial cell unique, and therefore difficult to mimic in vivo. Additionally, smooth muscle cells, which are a major source of tissue factor and therefore activation of the clotting cascade, are difficult to integrate in the currently available perfusion systems and microfluidic devices. Lack of smooth muscle cells, therefore, limits the study of cell-cell and organ-organ communication. However, technological advancements have made it possible to incorporate various cell types in different channels. Another method addressing these challenges include culture of cells onto a matrix or a semi-permeable membrane, which allows the study of cellular cross-talk and barrier disruption.
Beyond thrombus formation, flow-based assays help advance the field in several other aspects. Cardiovascular complications often result in alterations of shear stress, thereby modifying endothelial cell physiology. Flow-based assays enable us to regulate the type (pulsatile, laminar, or oscillating) and level (high or low) of shear stress on cells. Such modulations are especially beneficial to study the effect on organ-specific endothelial cells. Beyond this, rolling and adhesion of immune cells and platelets on the endothelial surface, an important aspect of inflammation, can be easily studied using microfluidic devices.
Furthermore, flow assays enable us to study cross-talk between the immune cells or between immune-cell and platelets in a more physiological conditions. Static assays involving co-culture would induce a forced interaction, whereas studying them under flow conditions mimics the physiological cell-cell conjugate formation.
These in vitro flow-based assays are a valuable tool to study aspects of the blood-vascular system. They provide a system that more closely resembles physiological and hemodynamic conditions in health and disease. The benefit and relevance for scientific advancement in the fields of thrombosis and hemostasis, vascular biology, immunology, hematology, and other allied fields is just beginning and will continue to be seen with future advances.