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Microfluidic lab-on-a-chip technologies enable the analysis and manipulation of small fluid volumes and particles at small scales and the control of fluid flow and transport processes at the microscale, leading to the development of new methods to address a broad range of scientific and medical challenges. Microfluidic and lab-on-a-chip technologies have made a noteworthy impact in basic, preclinical, and clinical research, especially in hematology and vascular biology due to the inherent ability of microfluidics to mimic physiologic flow conditions in blood vessels and capillaries. With the potential to significantly impact translational research and clinical diagnostics, technical issues and incentive mismatches have stymied microfluidics from fulfilling this promise. We describe how accessibility, usability, and manufacturability of microfluidic technologies should be improved and how a shift in mindset and incentives within the field is also needed to address these issues. In this report, we discuss the state of the microfluidic field regarding current limitations and propose future directions and new approaches for the field to advance microfluidic technologies closer to translation and clinical use. While our report focuses on using blood as the prototypical biofluid sample, the proposed ideas and research directions can be extrapolated to other areas of hematology, oncology, biology, and medicine.

While microscopy-based cellular assays, including microfluidics, have significantly advanced over the last several decades, there has not been concurrent development of widely-accessible techniques to analyze time-dependent microscopy data incorporating phenomena such as fluid flow and dynamic cell adhesion. As such, experimentalists typically rely on error-prone and time-consuming manual analysis, resulting in lost resolution and missed opportunities for innovative metrics. We present a user-adaptable toolkit packaged into the open-source, standalone Interactive Cellular assay Labeled Observation and Tracking Software (iCLOTS). We benchmark cell adhesion, single-cell tracking, velocity profile, and multiscale microfluidic-centric applications with blood samples, the prototypical biofluid specimen. Moreover, machine learning algorithms characterize previously imperceptible data groupings from numerical outputs. Free to download/use, iCLOTS addresses a need for a field stymied by a lack of analytical tools for innovative, physiologically-relevant assays of any design, democratizing use of well-validated algorithms for all end-user biomedical researchers who would benefit from advanced computational methods., (© 2023. Springer Nature Limited.)

Sickle cell disease (SCD) is caused by a single point mutation in the β-globin gene of hemoglobin, which produces an altered sickle hemoglobin (HbS). The ability of HbS to polymerize under deoxygenated conditions gives rise to chronic hemolysis, oxidative stress, inflammation, and vaso-occlusion. Herein, we review recent findings using microfluidic technologies that have elucidated mechanisms of oxygen-dependent and -independent induction of HbS polymerization and how these mechanisms elicit the biophysical and inflammatory consequences in SCD pathophysiology. We also discuss how validation and use of microfluidics in SCD provides the opportunity to advance development of numerous therapeutic strategies, including curative gene therapies., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Numerous studies have demonstrated the importance of altered hyaluronan metabolism to malignant progression of multiple tumor types, including breast carcinomas. Increased hyaluronan (HA) metabolism in the stroma of primary tumors promotes activation of oncogenic signaling pathways that impact tumor initiation, growth, and invasion. Carcinoma cell synthesis and assembly of HA-rich pericellular matrices induces a stromal-independent phenotype, which is associated with cancer progression. Although the pro-tumorigenic role of stromal HA is well established, a novel but unexplored hypothesis is that carcinoma cell-associated HA pericellular matrices promote metastasis of circulating tumor cells. Here, we report the development of an in vitro assay that employs microfluidic techniques to directly measure the importance of an HA-rich pericellular matrix in the entry of carcinoma cells into ectopic sites. This model provides the capability to visualize specific steps in metastasis, which is difficult using animal models. The results show that the presence of a HA-rich pericellular matrix correlates to the invasive and metastatic potential of breast carcinoma cells. Furthermore, enzymatic removal or pharmacologic inhibition of HA synthesis significantly inhibits carcinoma cell extravasation and invasion in this model system. These results implicate pericellular HA-rich carcinoma cell associated pericellular matrices in colonization of ectopic sites by circulating tumor cells and support specific targeting of this matrix to limit metastasis in patients.

Our goal was to develop a model of the microvasculature that would allow us to quantify changes in the rheology of sickle blood as it traverses the varying vessel sizes and oxygen tensions in the microcirculation. We designed and implemented a microfluidic model of the microcirculation that comprises a branching microvascular network and physiologic oxygen gradients. We used computational modeling to determine the parameters necessary to generate stable, linear gradients in our devices. Sickle blood from six unique patients was perfused through the microvascular network and subjected to varying oxygen gradients while we observed and quantified blood flow. We found that all sickle blood samples fully occluded the microvascular network when deoxygenated, and we observed that sickle blood could cause vaso-occlusions under physiologic oxygen gradients during the microvascular transit time. The number of occlusions observed under five unique oxygen gradients varied among the patient samples, but we generally observed that the number of occlusions decreased with increasing inlet oxygen tension. The model system we have developed is a valuable tool to address fundamental questions about where in the circulation sickle-cell vaso-occlusions are most likely to occur and to test new therapies., (© 2017 John Wiley & Sons Ltd.)

With noncommunicable diseases (NCDs) now constituting the majority of global mortality, there is a growing need for low-cost, noninvasive methods to diagnose and treat this class of diseases, especially in resource-limited settings. Molecular biomarkers combined with low-cost point-of-care assays constitute a potential solution for diagnosing NCDs, but the dearth of naturally occurring, predictive markers limits this approach. Here, we describe the design of exogenous agents that serve as synthetic biomarkers for NCDs by producing urinary signals that can be quantified by a companion paper test. These synthetic biomarkers are composed of nanoparticles conjugated to ligand-encoded reporters via protease-sensitive peptide substrates. Upon delivery, the nanoparticles passively target diseased sites, such as solid tumors or blood clots, where up-regulated proteases cleave the peptide substrates and release reporters that are cleared into urine. The reporters are engineered for detection by sandwich immunoassays, and we demonstrate their quantification directly from unmodified urine; furthermore, capture antibody specificity allows the probes to be multiplexed in vivo and quantified simultaneously by ELISA or paper lateral flow assay (LFA). We tailor synthetic biomarkers specific to colorectal cancer, a representative solid tumor, and thrombosis, a common cardiovascular disorder, and demonstrate urinary detection of these diseases in mouse models by paper diagnostic. Together, the LFA and injectable synthetic biomarkers, which could be tailored for multiple diseases, form a generalized diagnostic platform for NCDs that can be applied in almost any setting without expensive equipment or trained medical personnel.

The complex, systemic pathology of sickle cell disease is driven by multiple mechanisms including red blood cells (RBCs) stiffened by polymerized fibers of deoxygenated sickle hemoglobin. A critical step toward understanding the pathologic role of polymer-containing RBCs is quantifying the biophysical changes in these cells in physiologically relevant oxygen environments. We have developed a microfluidic platform capable of simultaneously measuring single RBC deformability and oxygen saturation under controlled oxygen and shear stress. We found that RBCs with detectable amounts of polymer have decreased oxygen affinity and decreased deformability. Surprisingly, the deformability of the polymer-containing cells is oxygen-independent, while the fraction of these cells increases as oxygen decreases. We also find that some fraction of these cells is present at most physiologic oxygen tensions, suggesting a role for these cells in the systemic pathologies. Additionally, the ability to measure these pathological cells should provide clearer targets for evaluating therapies. [ABSTRACT FROM AUTHOR]

Summary: Sickle cell trait (SCT) is the benign heterozygous carrier state for the sickle variant of the HBB gene. Most of the ~300 million people with SCT worldwide will not experience any significant complications. However, accumulating evidence finds SCT associated with increased risk for the common conditions of chronic kidney disease and venous thromboembolism, and severe but rare renal medullary carcinoma and exercise‐induced rhabdomyolysis. The mechanism is uncertain, but probably involves pathological rheology of SCT blood in regions of low oxygen tension, resulting from sickle haemoglobin polymerization in SCT red cells and leading to reduced blood flow and further tissue hypoxia and damage. Here, we used an in vitro microfluidic flow system to study the oxygen‐dependent rheology of SCT blood and show that 5‐(hydroxymethyl)furfural, a natural breakdown product of glucose and fructose‐containing foods, such as fruit juices, can reduce the effects of hypoxia on SCT blood rheology in vitro, restoring near‐normal flow velocities at very low oxygen. While opinions regarding the clinical significance of the risks associated with SCT are still evolving, these results suggest that a compound present in some food may provide a potential approach for managing risks that may be associated with SCT. [ABSTRACT FROM AUTHOR]