Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Abstract

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro-and nanoscales 1,2 . In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions [3][4][5][6] . As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions 6-13 .However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

Cellular softening mediates leukocyte demargination and trafficking, thereby increasing clinical blood counts

“…In addition, we observed that glucocorticoid and catecholamine-mediated leukocyte demargination occurs in more physiologic microfluidic devices cultured with a confluent endothelial monolayer that spans the entire inner surface area of the device, providing further evidence that this demargination effect likely also occurs in vivo (Fig. S3) (25).…”

“…A custom-written MATLAB code (MathWorks) extracted individual leukocyte positions from the series of confocal images. For endothelialized experiments, devices were endothelialized as previously described (25). For the capillary bed experiments, isolated granulocytes in PBS (1-3 × 10 6 cells per milliliter) were perfused through a microfluidic model of a capillary bed (23) at 0.250-1 μL/min.…”

Development and Characterization of <em>In Vitro</em> Microvessel Network and Quantitative Measurements of Endothelial [Ca²⁺]_iand Nitric Oxide Production

“…Some advanced microfluidic systems, such as reconstruction of lung-on-chip model demonstrated that this technology is capable to reproduce complex integrated organ-level responses to bacteria and inflammatory cytokines involving microvascular channels 25,26 . The most widely used material for microfluidic applications is PDMS, as it is an optically transparent, nontoxic, and gas permeable soft elastomer 27–29 . Various hydrogel devices, or hydrogel/PDMS hybrid devices have also been used to promote cell adhesion, or serve as a physical barrier to induce chemical gradient 21,30–32 .…”

Resolving the multifaceted mechanisms of the ferric chloride thrombosis model using an interdisciplinary microfluidic approach

“…Our laboratory has shown that vascular phenomenon relevant to hematologic processes can be studied in vitro by culturing endothelial cells to a confluent monolayer in microchannels. 13,14 These "endothelial-ized" microfluidics allow us to recapitulate the in vivo endothelium-bloodFeCl 3 interface and monitor the status of the endothelium during FeCl 3 application, while controlling the flow conditions and cellular and molecular "inputs" to the system. We find that all isolated blood components and molecules aggregate in a FeCl 3 concentration-dependent manner, with and without endothelial cells.…”