Identification of congenital aortic valve malformations in juvenile natriuretic peptide receptor 2-deficient mice using high-frequency ultrasound.

Mouse models of congenital aortic valve malformations are useful for studying disease pathobiology, but most models have incomplete penetrance [e.g., ∼2 to 77% prevalence of bicuspid aortic valves (BAVs) across multiple models]. For longitudinal studies of pathologies associated with BAVs and other congenital valve malformations, which manifest over months in mice, it is operationally inefficient, economically burdensome, and ethically challenging to enroll large numbers of mice in studies without first identifying those with valvular abnormalities. To address this need, we established and validated a novel in vivo high-frequency (30 MHz) ultrasound imaging protocol capable of detecting aortic valvular malformations in juvenile mice. Fifty natriuretic peptide receptor 2 heterozygous mice on a low-density lipoprotein receptor-deficient background (Npr2+/-;Ldlr-/-; 32 males and 18 females) were imaged at 4 and 8 wk of age. Fourteen percent of the Npr2+/-;Ldlr-/- mice exhibited features associated with aortic valve malformations, including 1) abnormal transaortic flow patterns on color Doppler (recirculation and regurgitation), 2) peak systolic flow velocities distal to the aortic valves reaching or surpassing ∼1,250 mm/s by pulsed-wave Doppler, and 3) putative fusion of cusps along commissures and abnormal movement elucidated by two-dimensional (2-D) imaging with ultrahigh temporal resolution. Valves with these features were confirmed by ex vivo gross anatomy and histological visualization to have thickened cusps, partial fusions, or Sievers type-0 bicuspid valves. This ultrasound imaging protocol will enable efficient, cost effective, and humane implementation of studies of congenital aortic valvular abnormalities and associated pathologies in a wide range of mouse models.NEW & NOTEWORTHY We developed a high-frequency ultrasound imaging protocol for diagnosing congenital aortic valve structural abnormalities in 4-wk-old mice. Our protocol defines specific criteria to distinguish mice with abnormal aortic valves from those with normal tricuspid valves using color Doppler, pulsed-wave Doppler, and two-dimensional (2-D) imaging with ultrahigh temporal resolution. This approach enables early identification of valvular abnormalities for efficient and ethical experimental design of longitudinal studies of congenital valve diseases and associated pathologies in mice.

Bridging barriers: advances and challenges in modeling biological barriers and measuring barrier integrity in organ-on-chip systems.

Biological barriers such as the blood-brain barrier, skin, and intestinal mucosal barrier play key roles in homeostasis, disease physiology, and drug delivery - as such, it is important to create representative in vitro models to improve understanding of barrier biology and serve as tools for therapeutic development. Microfluidic cell culture and organ-on-a-chip (OOC) systems enable barrier modelling with greater physiological fidelity than conventional platforms by mimicking key environmental aspects such as fluid shear, accurate microscale dimensions, mechanical cues, extracellular matrix, and geometrically defined co-culture. As the prevalence of barrier-on-chip models increases, so does the importance of tools that can accurately assess barrier integrity and function without disturbing the carefully engineered microenvironment. In this review, we first provide a background on biological barriers and the physiological features that are emulated through in vitro barrier models. Then, we outline molecular permeability and electrical sensing barrier integrity assessment methods, and the related challenges specific to barrier-on-chip implementation. Finally, we discuss future directions in the field, as well important priorities to consider such as fabrication costs, standardization, and bridging gaps between disciplines and stakeholders.

Porous Membrane Electrical Cell-Substrate Impedance Spectroscopy for Versatile Assessment of Biological Barriers In Vitro.

Cell culture models of endothelial and epithelial barriers typically use porous membrane inserts (e.g., Transwell inserts) as a permeable substrate on which barrier cells are grown, often in coculture with other cell types on the opposite side of the membrane. Current methods to characterize barrier function in porous membrane inserts can disrupt the barrier or provide bulk measurements that cannot isolate barrier cell resistance alone. Electrical cell-substrate impedance sensing (ECIS) addresses these limitations, but its implementation on porous membrane inserts has been limited by costly manufacturing, low sensitivity, and lack of validation for barrier assessment. Here, we present porous membrane ECIS (PM-ECIS), a cost-effective method to adapt ECIS technology to porous substrate-based in vitro models. We demonstrate high fidelity patterning of electrodes on porous membranes that can be incorporated into well plates of a variety of sizes with excellent cell biocompatibility with mono- and coculture set ups. PM-ECIS provided sensitive, real-time measurement of isolated changes in endothelial cell barrier impedance with cell growth and barrier disruption. Barrier function characterized by PM-ECIS resistance correlated well with permeability coefficients obtained from simultaneous molecular tracer permeability assays performed on the same cultures, validating the device. Integration of ECIS into conventional porous cell culture inserts provides a versatile, sensitive, and automated alternative to current methods to measure barrier function in vitro, including molecular tracer assays and transepithelial/endothelial electrical resistance.

Sensitivity and Validation of Porous Membrane Electrical Cell Substrate Impedance Spectroscopy (PM-ECIS) for Measuring Endothelial Barrier Properties.

Measurement of endothelial and epithelial barrier integrity is important for a variety of in vitro models, including Transwell assays, cocultures, and organ-on-chip platforms. Barrier resistance is typically measured by trans-endothelial electrical resistance (TEER), but TEER is invasive and cannot accurately measure isolated monolayer resistance in coculture or most organ-on-chip devices. These limitations are addressed by porous membrane electrical cell-substrate impedance sensing (PM-ECIS), which measures barrier integrity in cell monolayers grown directly on permeable membranes patterned with electrodes. Here, we advanced the design and utility of PM-ECIS by investigating its sensitivity to working electrode size and correlation with TEER. Gold electrodes were fabricated on porous membrane inserts using hot embossing and UV lithography, with working electrode diameters of 250, 500, and 750 µm within the same insert. Sensitivity to resistance changes (4 kHz) during endothelial barrier formation was inversely proportional to electrode size, with the smallest being the most sensitive (p < 0.001). Similarly, smaller electrodes were most sensitive to changes in impedance (40 kHz) corresponding to cell spreading and proliferation (p < 0.001). Barrier disruption with both EGTA and thrombin was detectable by all electrode sizes. Resistances measured by PM-ECIS vs TEER for sodium chloride solutions were positively and significantly correlated for all electrode sizes (r > 0.9; p < 0.0001), but only with 750 µm electrodes for endothelial monolayers (r = 0.71; p = 0.058). These data inform the design and selection of PM-ECIS electrodes for specific applications and support PM-ECIS as a promising alternative to conventional TEER for direct, noninvasive, real-time assessment of cells cultured on porous membranes in conventional and organ-on-chip barrier models.

Interrogating Matrix Stiffness and Metabolomics in Pancreatic Ductal Carcinoma Using an Openable Microfluidic Tumor-on-a-Chip.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stroma that contributes to aggressive tumor biology and therapeutic resistance. Current in vitro PDAC models lack sufficient optical and physical access for fibrous network visualization, in situ mechanical stiffness measurement, and metabolomic profiling. Here, we describe an openable multilayer microfluidic PDAC-on-a-chip platform that consists of pancreatic tumor cells (PTCs) and pancreatic stellate cells (PSCs) embedded in a 3D collagen matrix that mimics the stroma. Our system allows fibrous network visualization via reflected light confocal (RLC) microscopy, in situ mechanical stiffness testing using atomic force microscopy (AFM), and compartmentalized hydrogel extraction for PSC metabolomic profiling via mass spectrometry (MS) analysis. In comparing cocultures of gel-embedded PSCs and PTCs with PSC-only monocultures, RLC microscopy identified a significant decrease in pore size and corresponding increase in fiber density. In situ AFM indicated significant increases in stiffness, and hallmark characteristics of PSC activation were observed using fluorescence microscopy. PSCs in coculture also demonstrated localized fiber alignment and densification as well as increased collagen production. Finally, an untargeted MS study putatively identified metabolic contributions consistent with in vivo PDAC studies. Taken together, this platform can potentially advance our understanding of tumor-stromal interactions toward the discovery of novel therapies.

Noninvasive Quantification of Contractile Dynamics in Cardiac Cells, Spheroids, and Organs-on-a-Chip Using High-Frequency Ultrasound.

Cell-based models that mimic in vivo heart physiology are poised to make significant advances in cardiac disease modeling and drug discovery. In these systems, cardiomyocyte (CM) contractility is an important functional metric, but current measurement methods are inaccurate and low-throughput or require complex setups. To address this need, we developed a standalone noninvasive, label-free ultrasound technique operating at 40-200 MHz to measure the contractile kinetics of cardiac models, ranging from single adult CMs to 3D microtissue constructs in standard cell culture formats. The high temporal resolution of 1000 fps resolved the beat profile of single mouse CMs paced at up to 9 Hz, revealing limitations of lower speed optical based measurements to resolve beat kinetics or characterize aberrant beats. Coupling of ultrasound with traction force microscopy enabled the measurement of the CM longitudinal modulus and facile estimation of adult mouse CM contractile forces of 2.34 ± 1.40 µN, comparable to more complex measurement techniques. Similarly, the beat rate, rhythm, and drug responses of CM spheroid and microtissue models were measured, including in configurations without optical access. In conclusion, ultrasound can be used for the rapid characterization of CM contractile function in a wide range of commonly studied configurations ranging from single cells to 3D tissue constructs using standard well plates and custom microdevices, with applications in cardiac drug discovery and cardiotoxicity evaluation.

Myosin inhibitor reverses hypertrophic cardiomyopathy in genotypically diverse pediatric iPSC-cardiomyocytes to mirror variant correction.

Pathogenic variants in MYH7 and MYBPC3 account for the majority of hypertrophic cardiomyopathy (HCM). Targeted drugs like myosin ATPase inhibitors have not been evaluated in children. We generate patient and variant-corrected iPSC-cardiomyocytes (CMs) from pediatric HCM patients harboring single variants in MYH7 (V606M; R453C), MYBPC3 (G148R) or digenic variants (MYBPC3 P955fs, TNNI3 A157V). We also generate CMs harboring MYBPC3 mono- and biallelic variants using CRISPR editing of a healthy control. Compared with isogenic and healthy controls, variant-positive CMs show sarcomere disorganization, higher contractility, calcium transients, and ATPase activity. However, only MYH7 and biallelic MYBPC3 variant-positive CMs show stronger myosin-actin binding. Targeted myosin ATPase inhibitors show complete rescue of the phenotype in variant-positive CMs and in cardiac Biowires to mirror isogenic controls. The response is superior to verapamil or metoprolol. Myosin inhibitors can be effective in genotypically diverse HCM highlighting the need for myosin inhibitor drug trials in pediatric HCM.

Biomechanical properties of the aortic root are distinct from those of the ascending aorta in both normal and aneurysmal states.

Background: Although aneurysms of the ascending aorta and the aortic root are treated similarly in clinical guidelines, how biomechanical properties differ between these 2 segments of aorta is poorly defined. Methods: Biomechanical testing was performed on tissue collected from the aortic root (normal = 11, aneurysm = 51) and the ascending aorta (normal = 21, aneurysm = 76). Energy loss, tangent modulus of elasticity, and delamination strength were evaluated. These biomechanical properties were then compared between (1) normal ascending and normal root tissue, (2) normal and aneurysmal root tissue, (3) normal and aneurysmal ascending tissue, and (4) aneurysmal root and aneurysmal ascending tissue. Propensity score matching was performed to further compare aneurysmal root and aneurysmal ascending aortic tissue. Clinical and biomechanical variables associated with decreased delamination strength in the aortic root were evaluated. Results: The normal aortic root demonstrated greater viscoelastic behavior (energy loss 0.08 [0.06, 0.10] vs 0.05 [0.04, 0.06], P = .008), and greater resistance against delamination (93 [58, 126] mN/mm vs 54 [40, 63] mN/mm, P = .05) compared with the ascending aorta. Delamination strength was significantly reduced in aneurysms in both the root and the ascending aorta compared with their normal states. Aneurysms of the aortic root matched to the ascending aortic aneurysms in terms of baseline characteristics including size, were characterized by a larger decrease in delamination strength from baseline (Δ -59 mN/mm vs Δ -24 mN/mm). Aging (P = .003) and the presence of hypertension (P = .02) were associated with weakening of the aortic root, while diameter did not have this association (P = .29). Conclusions: The normal aortic root was found to have distinct biomechanical properties compared with the ascending aorta. When aneurysms form in the aortic root, there is less strength against delamination, without other biomechanical changes such as increased energy loss observed in aneurysmal ascending aortas. Age and hypertension were associated decreased aortic wall strength in the aortic root, whereas diameter had no such association.