iCLOTS: open-source, artificial intelligence-enabled software for analyses of blood cells in microfluidic and microscopy-based assays.

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.

A novel mobile health application to support cancer surveillance needs of patients and families with cancer predisposition syndromes.

BACKGROUND: At least 5%-10% of malignancies occur secondary to an underlying cancer predisposition syndrome (CPS). For these families, cancer surveillance is recommended with the goal of identifying malignancy earlier, in a presumably more curable form. Surveillance protocols, including imaging studies, bloodwork, and procedures, can be complex and differ based on age, gender, and syndrome, which adversely affect adherence. Mobile health (mHealth) applications (apps) have been utilized in oncology and could help to facilitate adherence to cancer surveillance protocols. METHODS: Applying a user-centered mobile app design approach, patients with a CPS and/or primary caregivers were interviewed to identify current methods for care management and barriers to compliance with recommended surveillance protocols. Broad themes from these interviews informed the design of the mobile app, HomeTown, which was subsequently evaluated by usability experts. The design was then converted into software code in phases, evaluated by patients and caregivers in an iterative fashion. User population growth and app usage data were assessed. RESULTS: Common themes identified included general distress surrounding surveillance protocol scheduling and results, difficulty remembering medical history, assembling a care team, and seeking resources for self-education. These themes were translated into specific functional app features, including push reminders, syndrome-specific surveillance recommendations, ability to annotate visits and results, storage of medical histories, and links to reliable educational resources. CONCLUSIONS: Families with CPS demonstrate a desire for mHealth tools to facilitate adherence to cancer surveillance protocols, reduce related distress, relay medical information, and provide educational resources. HomeTown may be a useful tool for engaging this patient population.

The critical role of engineering in the rapid development of COVID-19 diagnostics: Lessons from the RADx Tech Test Verification Core.

Engineering plays a critical role in the development of medical devices, and this has been magnified since 2020 as severe acute respiratory syndrome coronavirus 2 swept over the globe. In response to the coronavirus disease 2019, the National Institutes of Health launched the Rapid Acceleration of Diagnostics (RADx) initiative to help meet the testing needs of the United States and effectively manage the pandemic. As the Engineering and Human Factors team for the RADx Tech Test Verification Core, we directly assessed more than 30 technologies that ultimately contributed to an increase of the country's total testing capacity by 1.7 billion tests to date. In this review, we present central lessons learned from this "apples-to-apples" comparison of novel, rapidly developed diagnostic devices. Overall, the evaluation framework and lessons learned presented in this review may serve as a blueprint for engineers developing point-of-care diagnostics, leaving us better prepared to respond to the next global public health crisis rapidly and effectively.

Navigating the complexities of mobile medical app development from idea to launch, a guide for clinicians and biomedical researchers.

With today's pace of rapid technological advancement, many patient issues in modern medicine are increasingly solvable by mobile app solutions, which also have the potential to transform how clinical research is conducted. However, many critical challenges in the app development process impede bringing these translational technologies to patients, caused in large part by the lack of knowledge among clinicians and biomedical researchers of "what it takes" to design, develop, and maintain a successful medical app. Indeed, problems requiring mobile app solutions are often nuanced, requiring more than just clinical expertise, and issues such as the cost and effort required to develop and maintain a well-designed, sustainable, and scalable mobile app are frequently underestimated. To bridge this skill set gap, we established an academic unit of designers, software engineers, and scientists that leverage human-centered design methodologies and multi-disciplinary collaboration to develop clinically viable smartphone apps. In this report, we discuss major misconceptions clinicians and biomedical researchers often hold regarding medical app development, the steps we took to establish this unit to address these issues and the best practices and lessons learned from successfully ideating, developing, and launching medical apps. Overall, this report will serve as a blueprint for clinicians and biomedical researchers looking to better benefit their patients or colleagues via medical mobile apps.

Don't forget about human factors: Lessons learned from COVID-19 point-of-care testing.

During the COVID-19 pandemic, the development of point-of-care (POC) diagnostic testing accelerated in an unparalleled fashion. As a result, there has been an increased need for accurate, robust, and easy-to-use POC testing in a variety of non-traditional settings (i.e., pharmacies, drive-thru sites, schools). While stakeholders often express the desire for POC technologies that are "as simple as digital pregnancy tests," there is little discussion of what this means in regards to device design, development, and assessment. The design of POC technologies and systems should take into account the capabilities and limitations of the users and their environments. Such "human factors" are important tenets that can help technology developers create POC technologies that are effective for end-users in a multitude of settings. Here, we review the core principles of human factors and discuss lessons learned during the evaluation process of SARS-CoV-2 POC testing.

A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits.

Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

A blueprint for academic labs to produce SARS-CoV-2 RT-qPCR test kits.

Widespread testing for the presence of the novel coronavirus SARS-CoV-2 in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise and/or instrumentation necessary to detect the virus by quantitative reverse transcription polymerase chain reaction (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably to a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces across various campus laboratories for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

Enabling mesenchymal stromal cell immunomodulatory analysis using scalable platforms.

Human mesenchymal stromal cells (hMSCs) are a promising cell source for numerous regenerative medicine and cell therapy-based applications. However, MSC-based therapies have faced challenges in translation to the clinic, in part due to the lack of sufficient technologies that accurately predict MSC potency and are viable in the context of cell manufacturing. Microfluidic platforms may provide an innovative opportunity to address these challenges by enabling multiparameter analyses of small sample sizes in a high throughput and cost-effective manner, and may provide a more predictive environment in which to analyze hMSC potency. To this end, we demonstrate the feasibility of incorporating 3D culture environments into microfluidic platforms for analysis of hMSC secretory response to inflammatory stimuli and multi-parameter testing using cost-effective and scalable approaches. We first find that the cytokine secretion profile for hMSCs cultured within synthetic poly(ethylene glycol)-based hydrogels is significantly different compared to those cultured on glass substrates, both in growth media and following stimulation with IFN-γ and TNF-α, for cells derived from two donors. For both donors, perfusion with IFN-γ and TNF-α leads to differences in secretion of interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), macrophage colony-stimulating factor (M-CSF), and interleukin-1 receptor antagonist (IL-1ra) between hMSCs cultured in hydrogels and those cultured on glass substrates. We then demonstrate the feasibility of analyzing the response of hMSCs to a stable concentration gradient of soluble factors such as inflammatory stimuli for potential future use in potency analyses, minimizing the amount of sample required for dose-response testing.

Integrated automated particle tracking microfluidic enables high-throughput cell deformability cytometry for red cell disorders.

Investigating individual red blood cells (RBCs) is critical to understanding hematologic diseases, as pathology often originates at the single-cell level. Many RBC disorders manifest in altered biophysical properties, such as deformability of RBCs. Due to limitations in current biophysical assays, there exists a need for high-throughput analysis of RBC deformability with single-cell resolution. To that end, we present a method that pairs a simple in vitro artificial microvasculature network system with an innovative MATLAB-based automated particle tracking program, allowing for high-throughput, single-cell deformability index (sDI) measurements of entire RBC populations. We apply our technology to quantify the sDI of RBCs from healthy volunteers, Sickle cell disease (SCD) patients, a transfusion-dependent beta thalassemia major patient, and in stored packed RBCs (pRBCs) that undergo storage lesion over 4 weeks. Moreover, our system can also measure cell size for each RBC, thereby enabling 2D analysis of cell deformability vs cell size with single cell resolution akin to flow cytometry. Our results demonstrate the clear existence of distinct biophysical RBC subpopulations with high interpatient variability in SCD as indicated by large magnitude skewness and kurtosis values of distribution, the "shifting" of sDI vs RBC size curves over transfusion cycles in beta thalassemia, and the appearance of low sDI RBC subpopulations within 4 days of pRBC storage. Overall, our system offers an inexpensive, convenient, and high-throughput method to gauge single RBC deformability and size for any RBC population and has the potential to aid in disease monitoring and transfusion guidelines for various RBC disorders.

Smartphone app for non-invasive detection of anemia using only patient-sourced photos.

We introduce a paradigm of completely non-invasive, on-demand diagnostics that may replace common blood-based laboratory tests using only a smartphone app and photos. We initially targeted anemia, a blood condition characterized by low blood hemoglobin levels that afflicts >2 billion people. Our app estimates hemoglobin levels by analyzing color and metadata of fingernail bed smartphone photos and detects anemia (hemoglobin levels <12.5 g dL-1) with an accuracy of ±2.4 g dL-1 and a sensitivity of 97% (95% CI, 89-100%) when compared with CBC hemoglobin levels (n = 100 subjects), indicating its viability to serve as a non-invasive anemia screening tool. Moreover, with personalized calibration, this system achieves an accuracy of ±0.92 g dL-1 of CBC hemoglobin levels (n = 16), empowering chronic anemia patients to serially monitor their hemoglobin levels instantaneously and remotely. Our on-demand system enables anyone with a smartphone to download an app and immediately detect anemia anywhere and anytime.

Microvasculature-on-a-chip for the long-term study of endothelial barrier dysfunction and microvascular obstruction in disease.

Alterations in the mechanical properties of erythrocytes occurring in inflammatory and hematologic disorders such as sickle cell disease (SCD) and malaria often lead to increased endothelial permeability, haemolysis, and microvascular obstruction. However, the associations among these pathological phenomena remain unknown. Here, we report a perfusable, endothelialized microvasculature-on-a-chip featuring an interpenetrating-polymer-network hydrogel that recapitulates the stiffness of blood-vessel intima, basement membrane self-deposition and self-healing endothelial barrier function for longer than 1 month. The microsystem enables the real-time visualization, with high spatiotemporal resolution, of microvascular obstruction and endothelial permeability under physiological flow conditions. We found how extracellular heme, a hemolytic byproduct, induces delayed but reversible endothelial permeability in a dose-dependent manner, and demonstrate that endothelial interactions with SCD or malaria-infected erythrocytes cause reversible microchannel occlusion and increased in situ endothelial permeability. The microvasculature-on-a-chip enables mechanistic insight into the endothelial barrier dysfunction associated with SCD, malaria and other inflammatory and haematological diseases.

Interdigitated microelectronic bandage augments hemostasis and clot formation at low applied voltage in vitro and in vivo.

Hemorrhage or uncontrolled bleeding can arise either due to a medical condition or from a traumatic injury and are typically controlled with the application of a hemostatic agent. Hemostatic agents are currently derived from animal or human products, which carry risks of blood borne infections and immune dysregulation. Therefore, the need exists for novel biomedical therapies not derived from animal or human products to achieve hemostasis. Accordingly, we created an interdigitated microelectronic bandage that applies low voltage electrical stimulation to an injury site, resulting in faster clot formation without excessive heating, accelerated fibrin formation, and hemostasis overall. Our interdigitated microelectronic bandage found fibrin formed 1.5× faster in vitro. In vivo, total cessation of bleeding was 2.5× faster, resulting in 2× less blood loss. Electricity has been used in medical applications such as defibrillation, cauterization, and electrosurgery, but scant research has focused on hemostasis. Here we report a novel surface treatment using an interdigitated microelectronic device that creates rapid hemostasis in both in vitro and in vivo bleeding models with low applied voltages, representing a new and novel class of hemostatic agents that are electrically-based.

3D in vitro microvascular model-based lymphoma model.

Diffuse large B-cell lymphoma (DLBCL) is a particularly aggressive cancer, impacting the lives of approximately 20,000 people annually in the United States. Elucidating cellular interactions that occur within the microenvironment of DLBCL tumors is crucial to the successful development of therapeutic strategies for this condition. As the in vivo microenvironment of DLBCL is quite complex and variable, in vitro platforms that can sufficiently recapitulate these multifaceted cellular interactions without introducing the complexities of in vivo systems are vital for understanding the pathophysiology of this disease. In this chapter, we present a method for fabrication and development of an in vitro DLBCL-on-chip model in which a fully vascularized, perfusable, microfluidic traverses a DLBCL tumor cell-laden hydrogel that successfully recapitulates hallmark attributes and cellular interaction that occur within the DLBCL tumor microenvironment. As this microfluidic approach makes use of common laboratory items and does not require traditional photolithography to fabricate, this system represents a vital tool that can unlock previously inaccessible research areas of the DLBCL tumor microenvironment to researchers across numerous fields.

Endothelial cell culture in microfluidic devices for investigating microvascular processes.

Numerous conditions and disease states such as sickle cell disease, malaria, thrombotic microangiopathy, and stroke significantly impact the microvasculature function and its role in disease progression. Understanding the role of cellular interactions and microvascular hemodynamic forces in the context of disease is crucial to understanding disease pathophysiology. In vivo models of microvascular disease using animal models often coupled with intravital microscopy have long been utilized to investigate microvascular phenomena. However, these methods suffer from some major drawbacks, including the inability to tightly and quantitatively control experimental conditions, the difficulty of imaging multiple microvascular beds within a living organism, and the inability to isolate specific microvascular geometries such as bifurcations. Thus, there exists a need for in vitro microvascular models that can mitigate the drawbacks associated with in vivo systems. To that end, microfluidics has been widely used to develop such models, as it allows for tight control of system inputs, facile imaging, and the ability to develop robust and repeatable systems with well-defined geometries. Incorporating endothelial cells to branching microfluidic models allows for the development of "endothelialized" systems that accurately recapitulate physiological microvessels. In this review, we summarize the field of endothelialized microfluidics, specifically focusing on fabrication methods, limitations, and applications of these systems. We then speculate on future directions and applications of these cutting edge technologies. We believe that this review of the field is of importance to vascular biologists and bioengineers who aim to utilize microfluidic technologies to solve vascular problems.

Engineering "Endothelialized" Microfluidics for Investigating Vascular and Hematologic Processes Using Non-Traditional Fabrication Techniques.

Investigating the complex interplay between blood cells and the endothelium is crucial in understanding the pathophysiology of many diseases. Observation of the in vivo vasculature is difficult due to the complexities of vessel geometry, limited visualization capability, as well as variability and complexity inherent to biologic systems. Therefore, in vitro systems serve as ideal tools to study these cellular interactions. Microfluidic technologies are an ideal tool for recapitulating the vasculature in vivo as they can be used to fabricate fluidic channels on the size scale capillaries using gas permeable, biologically inert, and optically transparent substrates. Microfluidic channels can be vascularized by coating the inner surface of the microchannels with a confluent monolayer of endothelial cells, representing a reductionist, tightly controlled, in vitro model of the microvasculature. In this review, we present advances in the field of "endothelialized" microfluidics, focusing specifically on non-traditional fabrication and endothelialization techniques. We then summarize the various applications of endothelialized microfluidics, and speculate on the future directions of the field, including the exciting applications to personalized medicine.

Protein Corona in Response to Flow: Effect on Protein Concentration and Structure.

Nanoparticles used in cellular applications encounter free serum proteins that adsorb onto the surface of the nanoparticle, forming a protein corona. This protein layer controls the interaction of nanoparticles with cells. For nanomedicine applications, it is important to consider how intravenous injection and the subsequent shear flow will affect the protein corona. Our goal was to determine if shear flow changed the composition of the protein corona and if these changes affected cellular binding. Colorimetric assays of protein concentration and gel electrophoresis demonstrate that polystyrene nanoparticles subjected to flow have a greater concentration of serum proteins adsorbed on the surface, especially plasminogen. Plasminogen, in the absence of nanoparticles, undergoes changes in structure in response to flow, characterized by fluorescence and circular dichroism spectroscopy. The protein-nanoparticle complexes formed from fetal bovine serum after flow had decreased cellular binding, as measured with flow cytometry. In addition to the relevance for nanomedicine, these results also highlight the technical challenges of protein corona studies. The composition of the protein corona was highly dependent on the initial mixing step: rocking, vortexing, or flow. Overall, these results reaffirm the importance of the protein corona in nanoparticle-cell interactions and point toward the challenges of predicting corona composition based on nanoparticle properties.

A microengineered vascularized bleeding model that integrates the principal components of hemostasis.

Hemostasis encompasses an ensemble of interactions among platelets, coagulation factors, blood cells, endothelium, and hemodynamic forces, but current assays assess only isolated aspects of this complex process. Accordingly, here we develop a comprehensive in vitro mechanical injury bleeding model comprising an "endothelialized" microfluidic system coupled with a microengineered pneumatic valve that induces a vascular "injury". With perfusion of whole blood, hemostatic plug formation is visualized and "in vitro bleeding time" is measured. We investigate the interaction of different components of hemostasis, gaining insight into several unresolved hematologic issues. Specifically, we visualize and quantitatively demonstrate: the effect of anti-platelet agent on clot contraction and hemostatic plug formation, that von Willebrand factor is essential for hemostasis at high shear, that hemophilia A blood confers unstable hemostatic plug formation and altered fibrin architecture, and the importance of endothelial phosphatidylserine in hemostasis. These results establish the versatility and clinical utility of our microfluidic bleeding model.

Thrombosis-on-a-chip: Prospective impact of microphysiological models of vascular thrombosis.

The most common pathology of the blood-vessel organ system is thrombosis or undesirable clotting of the blood. Thrombosis is life threatening as more than 25% of such cases lead to sudden death from stroke and myocardial infarction. Even though the process of thrombosis has been extensively investigated with animal models, its exact pathobiology in different blood vessels is not yet fully understood and drug assessment remains unpredictable. This is primarily because the cause for thrombus formation is multifactorial and depends on the interplay of flow patterns within the blood vessel, the vessel wall or endothelium, extracellular matrix, parenchymal tissue, and the cellular and plasma components of the blood. Current in vitro and animal models do not mimic or dissect this organ-level complexity faithfully. However, microfluidic technology has recently been deployed to effectively recapitulate blood-endothelial-epithelial interactions in the onset of thrombosis in blood vessels. This technology is promising because it permits inclusion of primary human cells and blood obtained from patients, which is currently lacking in other in vitro models of thrombosis. In this review, we summarize the current state-of-the-art and practices in microfluidics and expected improvements in this field that will impact basic understanding of thrombosis, drug discovery and personalized medicine.

Extracellular fluid tonicity impacts sickle red blood cell deformability and adhesion.

Abnormal sickle red blood cell (sRBC) biomechanics, including pathological deformability and adhesion, correlate with clinical severity in sickle cell disease (SCD). Clinical intravenous fluids (IVFs) of various tonicities are often used during treatment of vaso-occlusive pain episodes (VOE), the major cause of morbidity in SCD. However, evidence-based guidelines are lacking, and there is no consensus regarding which IVFs to use during VOE. Further, it is unknown how altering extracellular fluid tonicity with IVFs affects sRBC biomechanics in the microcirculation, where vaso-occlusion takes place. Here, we report how altering extracellular fluid tonicity with admixtures of clinical IVFs affects sRBC biomechanical properties by leveraging novel in vitro microfluidic models of the microcirculation, including 1 capable of deoxygenating the sRBC environment to monitor changes in microchannel occlusion risk and an "endothelialized" microvascular model that measures alterations in sRBC/endothelium adhesion under postcapillary venular conditions. Admixtures with higher tonicities (sodium = 141 mEq/L) affected sRBC biomechanics by decreasing sRBC deformability, increasing sRBC occlusion under normoxic and hypoxic conditions, and increasing sRBC adhesion in our microfluidic human microvasculature models. Admixtures with excessive hypotonicity (sodium = 103 mEq/L), in contrast, decreased sRBC adhesion, but overswelling prolonged sRBC transit times in capillary-sized microchannels. Admixtures with intermediate tonicities (sodium = 111-122 mEq/L) resulted in optimal changes in sRBC biomechanics, thereby reducing the risk for vaso-occlusion in our models. These results have significant translational implications for patients with SCD and warrant a large-scale prospective clinical study addressing optimal IVF management during VOE in SCD.