Physiological Flow Conditions Research Articles

Intraplatelet Calcium Signaling Regulates Thrombus Growth under Flow: Insights from a Multiscale Model

In injured arteries, platelets adhere to the subendothelium and initiate the coagulation process. They recruit other platelets and form a plug that stops blood leakage. The formation of the platelet plug depends on platelet activation, a process that is regulated by intracellular calcium signaling. Using an improved version of a previous multiscale model, we study the effects of changes in calcium signaling on thrombus growth. This model utilizes the immersed boundary method to capture the interplay between platelets and the flow. Each platelet can attach to other platelets, become activated, express proteins on its surface, detach, and/or become non-adhesive. Platelet activation is captured through a specific calcium signaling model that is solved at the intracellular level, which considers calcium activation by agonists and contacts. Simulations reveal a contact-dependent activation threshold necessary for the formation of the thrombus core. Next, we evaluate the effect of knocking out the P2Y and PAR receptor families. Further, we show that blocking P2Y receptors reduces platelet numbers in the shell while slightly increasing the core size. An analysis of the contribution of P2Y and PAR activation to intraplatelet calcium signaling reveals that each of the ADP and thrombin agonists promotes the activation of platelets in different regions of the thrombus. Finally, the model predicts that the heterogeneity in platelet size reduces the overall number of platelets recruited by the thrombus. The presented framework can be readily used to study the effect of antiplatelet therapy under different physiological and pathological blood flow, platelet count, and activation conditions.

Induction of Controllable Vortex Flow in a New Aorta Stenosis Model for Potential Aorta Mechanobiology Study: A Flow Simulation Platform

Background: Abdominal aortic aneurysms (AAA) are common, particularly among individuals over age 65, with a prevalence of 9%. To better understand the natural history of AAAs, one critical knowledge gap must be filled to elucidate the role of vortical flow in vascular degradation and thrombosis formation. We aimed to induce various types of vortical flow with aneurysm-like hemodynamic environments using a novel aorta stenosis model. Such a model will enable us to investigate the interplays between disturbed hemodynamics and vascular pathological remodeling. Methods: In this study, the hemodynamics and geometry of the aorta in rabbits were used to simulate the physiological flow conditions after two adjacent stenoses were created virtually with varying degrees of area reduction (30-70%) and spacing (5-6.8 mm). A physiological blood flow (rate) waveform measured in rabbits using ultrasound Doppler (normalized to 3.0 mL/s) was prescribed to the inlet (i.e., descending aorta), and zero pressure boundary conditions were applied at two outlets (i.e., iliac arteries). 3D computational fluid dynamics (CFD) simulation in five different stenosis configurations was performed using commercial software (Fluent, Version 20, Ansys Inc., PA). In-house software assessed the evolution of flow vortices over three distinct regions post stenosis using an established method. The total volume of vortex flow, the number of vortices, and the phase-to-phase overlap of vortex flows within different post-stenosis regions were derived to characterize the vortex flow. Results: Quantitative and qualitative flow assessments were performed for all five CFD models. Firstly, in all models, we found consistent patterns of the three vortex flow parameters, indicating that the adjacent stenoses induced three different hemodynamic zones in the post-stenosis regions, namely, stable vortical flow (after first stenosis), transient flow (after second stenosis), unstable vortical flow (further distal to second stenosis). Moreover, increased stenosis degree or reduced spacing led to reduced vortex flow number but increased total volume of vortex flow. The distinctions of the three regions were most significant in the model with 50% and then 30% stenoses, and with a 5 mm (center-to-center) spacing between two stenoses. Conclusions: CFD simulations can offer a powerful tool to predict complex flows using subject-specific vasculature. Such a flow simulation platform is valuable for guiding experimental design or therapy in various diseases. We demonstrated that it is feasible to adjust and achieve multiple types of vortical flows with the novel stenosis model. The three zones with distinct hemodynamic environments allow us to investigate the vascular remodeling regulated by the specifics of the eddies. Our ongoing work is implementing optimized aorta stenosis models to preclinical or in vitro studies to reveal the interplays between varying disturbed flow and aneurysm remodeling. We acknowledge funding support from NIH/NIBIB (R01-EB029570A1) and the Health Research Institute of Michigan Technological University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Versatile Microfluidic Platform for Extravasation Studies Based on DNA Origami - Cell Interactions.

The adhesion of circulating tumor cells (CTCs) to the endothelial lumen and their extravasation to surrounding tissues are crucial in the seeding of metastases and remain the most complex events of the metastatic cascade to study. Integrins expressed on CTCs are major regulators of the extravasation process. This knowledge is primarily derived from animal models and biomimetic systems based on artificial endothelial layers, but these methods have ethical or technical limitations. We present a versatile microfluidic device to study cancer cell extravasation that mimics the endothelial barrier by using a porous membrane functionalized with DNA origami nanostructures (DONs) that display nanoscale patterns of adhesion peptides to circulating cancer cells. The device simulates physiological flow conditions and allows direct visualization of cell transmigration through microchannel pores using 3D confocal imaging. Using this system, we studied integrin-specific adhesion in the absence of other adhesive events. Specifically, we show that the transmigration ability of the metastatic cancer cell line MDA-MB-231 is influenced by the type, distance, and density of adhesion peptides present on the DONs. Furthermore, studies with mixed ligand systems indicate that integrins binding to RGD (arginine-glycine-aspartic acid) and IDS (isoleucine-aspartic acid-serine) did not synergistically enhance the extravasation process of MDA-MB-231 cells.

A Versatile Microfluidic Platform for Extravasation Studies Based on DNA Origami – Cell Interactions

The adhesion of circulating tumor cells (CTCs) to the endothelial lumen and their extravasation to surrounding tissues are crucial in the seeding of metastases and remain the most complex events of the metastatic cascade to study. Integrins expressed on CTCs are major regulators of the extravasation process. This knowledge is primarily derived from animal models and biomimetic systems based on artificial endothelial layers, but these methods have ethical or technical limitations. We present a versatile microfluidic device to study cancer cell extravasation that mimics the endothelial barrier by using a porous membrane functionalized with DNA origami nanostructures (DONs) that display nanoscale patterns of adhesion peptides to circulating cancer cells. The device simulates physiological flow conditions and allows direct visualization of cell transmigration through microchannel pores using 3D confocal imaging. Using this system, we studied integrin‐specific adhesion in the absence of other adhesive events. Specifically, we show that the transmigration ability of the metastatic cancer cell line MDA‐MB‐231 is influenced by the type, distance, and density of adhesion peptides present on the DONs. Furthermore, studies with mixed ligand systems indicate that integrins binding to RGD (arginine‐glycine‐aspartic acid) and IDS (isoleucine‐aspartic acid‐serine) did not synergistically enhance the extravasation process of MDA‐MB‐231 cells.

A 3D bioprinted hydrogel gut-on-chip with integrated electrodes for transepithelial electrical resistance (TEER) measurements

Conventional gut-on-chip (GOC) models typically represent the epithelial layer of the gut tissue, neglecting other important components such as the stromal compartment and the extracellular matrix (ECM) that play crucial roles in maintaining intestinal barrier integrity and function. These models often employ hard, flat porous membranes for cell culture, thus failing to recapitulate the soft environment and complex 3D architecture of the intestinal mucosa. Alternatively, hydrogels have been recently introduced in GOCs as ECM analogs to support the co-culture of intestinal cells in in vivo-like configurations, and thus opening new opportunities in the organ-on-chip field. In this work, we present an innovative GOC device that includes a 3D bioprinted hydrogel channel replicating the intestinal villi architecture containing both the epithelial and stromal compartments of the gut mucosa. The bioprinted hydrogels successfully support both the encapsulation of fibroblasts and their co-culture with intestinal epithelial cells under physiological flow conditions. Moreover, we successfully integrated electrodes into the microfluidic system to monitor the barrier formation in real time via transepithelial electrical resistance measurements.

Abstract 6764: Vascularized tumor-on-chip model for evaluating chemotherapeutic-mediated damage to adjacent healthy tissue

Abstract Introduction: Systemic chemotherapy is an effective anticancer treatment for all stages of breast cancer, with intravenous (IV) infusion being the principal method of administration. In addition to targeting cancer, systemic chemotherapy can impact the viability and function of surrounding normal tissues and vasculature. Early-discovery research tools comparing novel and existing systemic treatment strategies are urgently needed to support predictive efficacy and toxicity efforts. Microfluidic systems have the potential to fill this need while simultaneously reducing animal use. The overall goal of the study is to use an advanced microfluidic tumor-on-chip system to simultaneously evaluate the therapeutic and toxic effects of IV chemotherapy on normal and cancerous tissues. Materials and Methods: Synthetic Tumor Networks, which are comprised of primary and secondary tissue sites separated by an interconnected vasculature, were developed using in vivo images and fabricated using soft lithography. Primary vascular endothelial cells were used to establish microvasculature. A normal breast cell line (MCF 10A), or a GFP-labeled metastatic human breast cancer cell line (MDA-MB-231/GFP), were cultured in the tissue sites in a three-dimensional (3D) environment using a human-derived hydrogel. The vascular networks were perfused with endothelial cell media under physiological fluid flow conditions to establish the model. An EZH2 methyltransferase inhibitor (EZH2 inhibitor III) was delivered by IV infusion to the model tissues. Real-time monitoring of cellular growth, tumor invasion and extravasation, and cellular viability were performed using fluorescence microscopy over a two-week period. Results and Discussion: Metastatic MDA-MB-231/GFP breast cancer cells proliferated rapidly in the primary tumor site. Tumor intravasation into the vascular channels was observed in models that received no anticancer treatment, as well as extravasation into the secondary tissue site and invasion of the healthy breast tissue. Differences in tumor viability and migratory behavior were observed between untreated models or models receiving IV infusions of the EZH2 inhibitor through the vascular networks. Vascular barrier integrity and normal breast tissue viability were monitored at early (24-72 hr) and late (7-14 days) timepoints after anticancer treatment to measure short- and long-term adverse drug impacts. These results provide a unique perspective on the in vivo realism of an in vitro system for monitoring both therapeutic and toxic effects of systemic chemotherapies. Conclusions: We have developed a 3D vascularized tumor-on-chip model for monitoring the impacts of systemic chemotherapeutic agents on tumor and healthy tissues. This model can be used to investigate therapeutic and toxic effects on multiple tissue types simultaneously using real-time imaging techniques. Citation Format: Deborah Ramsey, Jenna Rosano, Cristabel Gordon, Gwen Fewell. Vascularized tumor-on-chip model for evaluating chemotherapeutic-mediated damage to adjacent healthy tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6764.

Unveiling the Impact of Hemodynamics on Endothelial Inflammation-Mediated Hepatocellular Carcinoma Metastasis Using a Biomimetic Vascular Flow Model.

Hepatocellular carcinoma (HCC) hematogenous dissemination is a leading cause of HCC-related deaths. The inflammatory facilitates this process by promoting the adhesion and invasion of tumor cells in the circulatory system. But the contribution of hemodynamics to this process remains poorly understood due to the lack of a suitable vascular flow model for investigation. This study develops a vascular flow model to examine the impact of hemodynamics on endothelial inflammation-mediated HCC metastasis. This work finds the increasing shear stress will reduce the recruitment of HCC cells by disturbing adhesion forces between endothelium and HCC cells. However, this reduction will be restored by the inflammation. When applying high FSS (4-6dyn cm-2) to the inflammatory endothelium, there will be a 4.8-fold increase in HCC cell adhesions compared to normal condition. Nevertheless, the increase fold of cell adhesions is inapparent, around 1.5-fold, with low and medium FSS. This effect can be attributed to the FSS-induced upregulation of ICAM-1 and VCAM-1 of the inflammatory endothelium, which serve to strengthen cell binding forces. These findings indicate that hemodynamics plays a key role in HCC metastasis during endothelial inflammation by regulating the expression of adhesion-related factors.

SARS-CoV-2 infection of endothelial cells, dependent on flow-induced ACE2 expression, drives hypercytokinemia in a vascularized microphysiological system.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for COVID-19, has caused nearly 7 million deaths worldwide. Severe cases are marked by an aggressive inflammatory response known as hypercytokinemia, contributing to endothelial damage. Although vaccination has reduced hospitalizations, hypercytokinemia persists in breakthrough infections, emphasizing the need for disease models mimicking this response. Using a 3D microphysiological system (MPS), we explored the vascular role in SARS-CoV-2-induced hypercytokinemia. The vascularized micro-organ (VMO) MPS, consisting of human-derived primary endothelial cells (ECs) and stromal cells within an extracellular matrix, was used to model SARS-CoV-2 infection. A non-replicative pseudotyped virus fused to GFP was employed, allowing visualization of viral entry into human ECs under physiologic flow conditions. Expression of ACE2, TMPRSS2, and AGTR1 was analyzed, and the impact of viral infection on ACE2 expression, vascular inflammation, and vascular morphology was assessed. The VMO platform facilitated the study of COVID-19 vasculature infection, revealing that ACE2 expression increased significantly in direct response to shear stress, thereby enhancing susceptibility to infection by pseudotyped SARS-CoV-2. Infected ECs secreted pro-inflammatory cytokines, including IL-6 along with coagulation factors. Cytokines released by infected cells were able to activate downstream, non-infected EC, providing an amplification mechanism for inflammation and coagulopathy. Our findings highlight the crucial role of vasculature in COVID-19 pathogenesis, emphasizing the significance of flow-induced ACE2 expression and subsequent inflammatory responses. The VMO provides a valuable tool for studying SARS-CoV-2 infection dynamics and evaluating potential therapeutics.

A Microfluidic Platform for Evaluating the Internalization of Liposome Drug Carriers in Tumor Spheroids.

The development of in vitro models recapitulating nanoparticle transport under physiological flow conditions is of great importance for predicting the efficacy of nanoparticle drug carriers. Liposomes are extensively used for drug delivery owing to their biocompatibility and biodegradability and the ability to carry both hydrophilic and hydrophobic compounds. Here, we used a library of liposomes with various dimensions and a microfluidic platform comprising a large array of uniformly sized breast cancer spheroids to explore size-dependent liposome internalization and retention in the spheroids under close-to-physiological interstitial conditions. Such a platform showed promising applications in the preclinical screening of small molecule drugs; however, the capability to deliver nanoparticles in the spheroid interior under close-to-physiological flow conditions was not explored. For the liposomes with diameters in the range of 45-200 nm, we show experimentally and by simulations that in comparison with liposome delivery solely by diffusion, flow significantly enhances liposome internalization in the microgels and mitigates the size-dependent spheroid penetration by the liposomes. The utility of the microfluidic platform was validated by evaluating the efficacy of clinically approved doxorubicin-loaded liposomes (Doxil), which exhibited superior retention in the spheroids under flow conditions, in comparison with free doxorubicin. This MF platform can serve as an in vitro model for screening the efficacy of drugs encapsulated in liposomes and find applications for screening other types of nanoparticle carriers for vaccine delivery, diagnostics, and skincare.

Hemodynamic Insights into Abdominal Aortic Aneurysms: Bridging the Knowledge Gap for Improved Patient Care

Background: Abdominal aortic aneurysms (AAAs) present a formidable public health concern due to their propensity for localized, anomalous expansion of the abdominal aorta. These insidious dilations, often in their early stages, mask the life-threatening potential for rupture, which carries a grave prognosis. Understanding the hemodynamic intricacies governing AAAs is paramount for predicting aneurysmal growth and the imminent risk of rupture. Objective: Our extensive investigation delves into this complex hemodynamic environment intrinsic to AAAs, utilizing comprehensive numerical analyses of the physiological pulsatile blood flow and realistic boundary conditions to explore the multifaceted dynamics influencing aneurysm rupture risk. Our study introduces novel elements by integrating these parameters into the overall context of aneurysm pathophysiology, thus advancing our understanding of the intricate mechanics governing their evolution and rupture. Methods: Conservation of mass and momentum equations are used to model the blood flow in an AAAs, and these equations are solved using a finite volume-based ANSYS Fluent solver. Resistance pressure outlets following a three-element Windkessel model were imposed at each outlet to accurately model the blood flow and the AAAs’ shear stress. Results: Our results uncover elevated blood flow velocities within an aneurysm, suggesting an augmented risk of future rupture due to increased stress in the aneurysm wall. During the systole phase, high wall shear stress (WSS) was observed, typically associated with a lower risk of rupture, while a low oscillatory shear index (OSI) was noted, correlating with a decreased risk of aneurysm expansion. Conversely, during the diastole phase, low WSS and a high OSI were identified, potentially weakening the aneurysm wall, thereby promoting expansion and rupture. Conclusion: Our study underscores the indispensable role of computational fluid dynamic (CFD) techniques in the diagnostic, therapeutic, and monitoring realms of AAAs. This body of research significantly advances our understanding of aneurysm pathophysiology, thus offering pivotal insights into the intricate mechanics underpinning their progression and rupture, informing clinical interventions and enhancing patient care.

Next generation microfluidics: fulfilling the promise of lab-on-a-chip technologies.

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.

Cold-stored platelet hemostatic capacity is maintained for three weeks of storage and associated with taurine metabolism

BackgroundPlatelet (PLT) product transfusion is a life-saving therapy for actively bleeding patients. There is an urgent need to maintain PLT function and extend shelf life to improve outcomes in these patients. Cold-stored PLT (CS-PLT) maintain hemostatic potential better than room temperature–stored PLT (RT-PLT). However, whether function in long-term CS-PLT is maintained under physiological flow regimes and/or determined by cold-induced metabolic changes is unknown. ObjectivesThis study aimed to (i) compare the function of RT-PLT and CS-PLT under physiological flow conditions, (ii) determine whether CS-PLT maintain function after 3 weeks of storage, and (iii) identify metabolic pathways associated with the CS-PLT lesion. MethodsWe performed phenotypic and functional assessments of RT- and CS-PLT (22 °C and 4 °C storage, respectively; N = 10 unique donors) at storage days 0, 5, and/or 21 via metabolomics, flow cytometry, aggregation, thrombin generation, viscoelastic testing, and a microfluidic assay to measure primary hemostatic function. ResultsDay 21 4 °C PLT formed an occlusive thrombus under arterial shear at a similar rate to day 5 22 °C PLT. Day 21 4 °C PLTs had enhanced thrombin generation capacity compared with day 0 PLT and maintained functionality comparable to day RT-PLT across all assays performed. Key metrics from microfluidic assessment, flow cytometry, thrombin generation, and aggregation were associated with 4 °C storage, and metabolites involved in taurine and purine metabolism significantly correlated with these metrics. Taurine supplementation of PLT during storage improved hemostatic function under flow. ConclusionCS-PLT stored for 3 weeks maintain hemostatic activity, and storage-induced phenotype and function are associated with taurine and purine metabolism.

Bioreactor Design for Culturing Vascularized Engineered Tissue in Flow Conditions.

Background: Current treatments for congenital heart defects often require surgery and implantation of a synthetic patch or baffle that becomes a fibrous scar and leads to a high number of reoperations. Previous studies in rats have shown that a prevascularized scaffold can integrate into the heart and result in regions of vascularized and muscularized tissue. However, increasing the thickness of this scaffold for use in human hearts requires a method to populate the thick scaffold and mature it under physiologic flow and electrical conditions. Experiment: We developed a bioreactor system that can perfuse up to six 7 mm porous scaffolds with tunable gravity-mediated flow and chronic electrical stimulation. Three polymers, which have been reported to be biocompatible, were evaluated for effects on the viability of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM). Bioreactor flow and electrical stimulation functions were tested, and the bioreactor was operated for up to 7 days to ensure reliability and lack of leaks in a 37°C, humidified incubator. Height and flow relationships were measured for perfusion through an electrospun polycaprolactone and gelatin scaffold, previously reported by our laboratory. Culture with cells was evaluated by plating human umbilical vein endothelial cells and human dermal fibroblasts on top of the scaffolds in both static and flow conditions for 2, 5, and 7 days. As a proof-of concept, scaffolds were cryosectioned and cell infiltration was quantified using immunofluorescence staining. Results: Neither MED610 (Stratasys), Vero (Stratasys), nor FORMLAB materials affected the viability of iPSC-CM, and MED610 was chosen for manufacture due to familiarity of 3D printing from this material. The generation of electrical field stimulation from 0 to 5 V and physiological ranges of pump capacities were verified. The relationship between height and flow was calculated for scaffolds with and without cells. Finally, we demonstrated evaluation of cell depth and structure in scaffolds cultured for 2, 5, and 7 days. Conclusion: The gravity-mediated flow bioreactor system we developed can be used as a platform for 3D cell culture particularly designed for perfusing vascularized tissue constructs with electrical stimulation for cardiac maturation.

Antiplatelet Agents Inhibit Platelet Adhesion and Aggregation on Glass Surface Under Physiological Flow Conditions: Toward a Microfluidic Platelet Functional Assay Without Additional Adhesion Protein Modification.

As the pathogenesis of arterial thrombosis often includes platelet adhesion and aggregation, antiplatelet agents are commonly used to prevent thromboembolic events. Here, a new microfluidic method without additional adhesion protein modification was developed to quantify the inhibitory effect of antiplatelet drugs on the adhesion and aggregation behavior of platelets on glass surfaces under physiological flow conditions. Polydimethylsiloxane-glass microfluidic chips were fabricated by soft photolithography. Blood samples from healthy volunteers or patients before and after taking antiplatelet drugs flowed through the microchannels at wall shear rates of 300 and 1500 second -1 , respectively. The time to reach 2.5% platelet aggregation surface coverage (Ti), surface coverage (A 150s ), and mean fluorescence intensity (F 150s ) were used as quantitative indicators. Aspirin (80 μM) prolonged Ti and reduced F 150s . Alprostadil, ticagrelor, eptifibatide, and tirofiban prolonged Ti and reduced A 150s and F 150s in a concentration-dependent manner, whereas high concentrations of alprostadil did not completely inhibit platelet aggregation. Aspirin combined with ticagrelor synergistically inhibited platelet adhesion and aggregation; GPIb-IX-von Willebrand factor inhibitors partially inhibited platelet aggregation, and the inhibition was more pronounced at 1500 than at 300 second -1 . Patient administration of aspirin or (and) clopidogrel inhibited platelet adhesion and aggregation on the glass surface under flow conditions. This technology is capable of distinguishing the pharmacological effects of various antiplatelet drugs on inhibition of platelet adhesion aggregation on glass surface under physiological flow conditions, which providing a new way to develop microfluidic platelet function detection method without additional adhesive protein modification for determining the inhibitory effects of antiplatelet drugs in the clinical setting.

GİRİŞ HIZ PROFİLİ VE GİRİŞ UZUNLUĞUNUN ABDOMİNAL AORT ANEVRİZMASI HEMODİNAMİĞİ SİMÜLASYONLARINA ETKİSİ

In computational Abdominal Aortic Aneurysm (AAA) hemodynamics studies, along with adjusting the problem geometry, mesh, transport, turbulence and rheology models; setting up boundary conditions (BC) is also a very important step which affect the reliability and accuracy of the hemodynamic assessment. The transient effects of physiological flow are well described by the Womersley profile, though its application might be difficult due to the complex nature of functions involved. Conversely, in literature, studies utilizing Plug or Parabolic profiles as inlet boundary conditions generally requires large entrance lengths to obtain the exact characteristics of the Womersley profile. In the current study, the differences arising between those boundary conditions, Womersley, Parabolic and Plug, with different entrance lengths, L_(ent )=D,3D and 11D, are examined by comparing the results with a Base condition, which is a solution obtained with ensured fully-developed flow before entering the aneurysm sac at two physiological flow conditions with mean Reynolds numbers, 〖Re〗_m=340 and 1160. The results reveal that with increasing mean flow rate, applying the complex Womersley equation might not be necessary. For the inlet flow waveform with 〖Re〗_m=1160, the Parabolic profile can be used instead of the Womersley profile by supplying an entrance length L_(ent )= 3D. On the other hand, the Plug profile requires an entrance length at least L_(ent )= 11D to replicate the Base condition for waveform with 〖Re〗_m=340.

Apoptotic Pathway Inhibition in Cold-Stored Platelet Concentrates Facilitates Retained Ability to Form Thrombi

Introduction: Platelet concentrates (PCs) are used to treat or prevent bleeding in patients with impaired platelet function or after injury. However, the standard storage of PCs at room-temperature (RT) increases the risk of bacterial contamination and is associated with reduced platelet functionality due to storage lesions. Our group reported that cold-stored PCs have better functionality compared to RT-stored PCs but reduced in vivo circulation time due to cold-induced apoptosis. In order to investigate cold-stored PCs hemostatic functionality with and without apoptosis inhibition, we established an ex vivo model simulating physiological conditions of blood flow to evaluate the contribution of cold-stored PCs in thrombus formation. Methods: Apheresis-derived PCs were stored for 1, 4, 7 and 10 days under agitation at 4°C or RT either with or without the apoptosis inhibitor G04 (RhoA GTPase inhibitor). Next, PCs were stained with CD41 antibody, recalcified and incubated with TRAP6 (thrombin receptor-activating peptide 6) to initiate thrombus formation. Platelet-depleted whole blood samples from healthy donors were spiked-in with PCs and administered to the microfluidic channels of the ex vivo system (Bioflux), under physiological shear pressure. After five minutes of perfusion the resulting thrombi were imaged and analyzed. Ability to form thrombi was further assessed by thromboelastography and impedance aggregometry. Results: Platelets from 24h storage PCs showed stable thrombus formation upon activation with TRAP6. Unstimulated platelets did not form any thrombi during the entire period of perfusion. To evaluate the effect of cold storage on platelet functions we analyzed the extent of thrombus formation of PCs stored at RT and 4°C upon CD41 staining. Our results indicate that cold-stored platelets form larger clots under flow conditions and maintain higher functionality compared to PCs stored at RT. Moreover, incubation of cold-stored PCs with the apoptosis inhibitor G04 improved ex vivo thrombus formation compared to PCs stored without the inhibitor, without inducing any adverse effects (Figure 1). Conclusion: The results indicate that our ex vivo assay, which simulates PC transfusion in thrombocytopenic patients, is suitable to test the hemostatic functions of PCs under physiological flow conditions. We demonstrated that PCs stored at 4°C show better thrombus formation ability upon stimulation by agonists compared to RT-stored PCs and that the ability to form clots under shear stress is still preserved after incubation with the apoptosis inhibitor. To conclude, cold-storage of PCs in combination with apoptosis inhibition is a promising stragedy to reduce the risk of bacterial contamination and prolong in vivo survival time while preserving platelet's hemostatic functionality.

Design and use of an ex vivo peripheral simulating bioreactor system for pharmacokinetic analysis of a drug coated stent.

Currently, there are no ex vivo systems that can model the motion of peripheral arteries and allow for the evaluation of pharmacokinetics (PK) of endovascular devices. The objective of this study was to develop a novel peripheral simulating bioreactor system to evaluate drug pharmacokinetics of stents. We utilized 3D-printed and off-the-shelf components to construct a peripheral-simulating bioreactor system capable of mimicking the motion of peripheral arteries. Servo motors were primarily used to shorten/elongate, twist, and bend explanted porcine carotid arteries. To evaluate the pharmacokinetics in the bioreactor, drug-eluting stents were deployed within explanted arteries and subjected to vascular motion along with pulsatile flow conditions. Following 30 min and 24 h, the arteries were removed, and paclitaxel levels were measured. Scanning electron microscopy was also performed to evaluate the stent surface. Arterial paclitaxel levels of the stent-treated arteries were found to be higher at 30 min than at 24 h following pulsatile and no vascular motion and even higher at 24 h following pulsatile flow and vascular motion. The residual drug on the stent significantly decreased from 30 min to 24 h. Scanning electron microscopy confirmed the loss of paclitaxel coating at 24 h and greater disturbance in stents under peripheral motion versus pulsatile only. This system represents the first ex vivo system to determine the PK of drug-eluting stents under physiological flow and vascular motion conditions. This work provides a novel system for a quick and inexpensive preclinical tool to study acute drug tissue concentration kinetics of drug-releasing interventional vascular devices designed for peripheral applications.

Dual Affinity Nanoparticles for the Transport of Therapeutics from Carrier Cells to Target Cells under Physiological Flow Conditions.

In this study, a novel two-stage nanoparticle delivery platform was developed based on the dual functionalization of a liposome with moieties that have fundamentally different strengths of adhesion and binding kinetics. The essential concept of this system is that the nanoparticles are designed to loosely bind to the carrier cell until they come into contact with the target cell, to which they bind with greater strength. This allows the nanoparticle to be transferred from one cell to another, circulating for longer periods of time in the blood and delivering the therapeutic agent to the target circulating tumor cell. Liposomes were prepared using the lipid cake and extrusion technique, then functionalized with E-selectin (ES), anti-cell surface vimentin antibody fragments, and TRAIL via click chemistry. The binding of dual affinity (DA) liposomes was confirmed with the neutrophil-like cell line PLB985, the colorectal cancer cell line HCT116, and healthy granulocytes isolated from peripheral whole blood under physiologically relevant fluid shear stress (FSS) in a cone-and-plate viscometer. Transfer of the DA liposomes from PLB985 to HCT116 cells under FSS was greater compared to all of the control liposome formulations. Additionally, DA liposomes demonstrated enhanced apoptotic effects on HCT116 cells in whole blood under FSS, surpassing the efficacy of the ES/TRAIL liposomes previously developed by the King Lab.

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research.

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

Pre-clinical investigation of liquid sirolimus for local drug delivery.

Sirolimus is currently being explored as an alternative drug to paclitaxel for the treatment of peripheral artery disease (PAD). To date, sirolimus has only been used as drug coatings for stents and balloons and no studies have yet demonstrated the delivery of sirolimus in liquid form. The purpose of this pilot study was to investigate the feasibility of the delivery of liquid sirolimus into arterial segments in a benchtop peripheral artery bioreactor. The feasibility to deliver liquid therapy was first tested on four drug delivery devices using a fluorescently tagged liquid drug and an ex vivo porcine artery benchtop model. The four devices included the Bullfrog micro-infusion device, ClearWay RX catheter, Occlusion perfusion catheter (OPC), and the targeted adjustable pharmaceutical administration system (TAPAS). Penetration of the fluorescently tagged drug was measured via microscopic imaging and quantification of the depth of drug penetration into all device-treated tissue. Based on the penetration outcome, we then selected a single device to deliver liquid sirolimus into the ex vivo porcine artery model undergoing physiological flow and pressure conditions. The liquid sirolimus-treated arteries were collected from the ex vivo bioreactor at 1- and 24-hour post-delivery and arterial drug retention analyzed by liquid chromatography-tandem mass spectrometry. Fluorescent microscopy demonstrated that drug delivery with the OPC had greater drug penetration into the medial wall as compared to other devices (OPC: 234 ± 161 µm; TAPAS: 127 ± 68 µm; ClearWay: 118 ± 77 µm; Bullfrog: 2.12 ± 3.78 µm; p = 0.098). The results of the ex vivo flow-circuit bench top model showed that the OPC device successfully delivered the liquid sirolimus at 1-hour (5.17 ± 4.48 ng/mg) and 24-hour (0.78 ± 0.55 ng/mg). These results demonstrate for the first time the ability to deliver liquid sirolimus directly to the medial layer of an artery via a liquid delivery catheter.