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.

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.

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.

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.

Bioinspired Oligo-Urethane Nanoparticles for Delivering Exogenous C-Type Natriuretic Peptide: Synthetic Biomaterial Nanocarrier Complexes and Their Interactions with Cardiac Myofibroblasts.

In a healthy heart, cells naturally secrete C-type natriuretic peptide (CNP), a cytokine that protects against myofibroblast differentiation of cardiac fibroblasts and extracellular matrix deposition leading to fibrosis. CNP availability during myocardial remodeling is important to prevent cardiac fibrosis, but CNP is limited after an injury because of the loss of cardiomyocytes and the activation of cardiac fibroblasts to myofibroblasts. We hypothesized that the sustained release of exogenous CNP from oligo-urethane nanoparticles (NPs) would reduce differentiation of human cardiac fibroblasts toward a myofibrogenic phenotype. Our work used a modified form of a degradable polar hydrophobic ionic (D-PHI) oligo-urethane, which has shown the ability to self-assemble into NPs for the delivery of peptide and oligonucleotide biomolecules. The CNP-loaded NPs (NPCNP) were characterized for a diameter of 129 ± 1.4 nm and a ζ potential of -46 ± 7.8 mV. Treatment of cardiac fibroblasts with NPCNP increased cyclic guanosine-monophosphate (cGMP) synthesis, confirming that exogenous CNP delivered via oligo-urethane NPs is bioactive and can induce downstream signaling that has been implicated in antagonizing transforming growth factor-ß1 (TGF-ß1)-induced myofibrogenic differentiation. It is also shown that treatment with NPCNP attenuated contraction of collagen gels by cardiac myofibroblasts stimulated with TGF-ß1. Coating with heparin on the NPCNP (HEP-NPCNP) exemplified an approach to extend the release of CNP from the NPs. Both HEP-NPCNP and NPCNP show minimal cell toxicity, studied up to 0.25 × 1010 NPs/mL in culture media. These findings support further investigation of CNP delivery via NPs as a future therapy for suppressing cardiac fibrosis.

Serum- and xeno-free culture of human umbilical cord perivascular cells for pediatric heart valve tissue engineering.

BACKGROUND: Constructs currently used to repair or replace congenitally diseased pediatric heart valves lack a viable cell population capable of functional adaptation in situ, necessitating repeated surgical intervention. Heart valve tissue engineering (HVTE) can address these limitations by producing functional living tissue in vitro that holds the potential for somatic growth and remodelling upon implantation. However, clinical translation of HVTE strategies requires an appropriate source of autologous cells that can be non-invasively harvested from mesenchymal stem cell (MSC)-rich tissues and cultured under serum- and xeno-free conditions. To this end, we evaluated human umbilical cord perivascular cells (hUCPVCs) as a promising cell source for in vitro production of engineered heart valve tissue. METHODS: The proliferative, clonogenic, multilineage differentiation, and extracellular matrix (ECM) synthesis capacities of hUCPVCs were evaluated in a commercial serum- and xeno-free culture medium (StemMACS™) on tissue culture polystyrene and benchmarked to adult bone marrow-derived MSCs (BMMSCs). Additionally, the ECM synthesis potential of hUCPVCs was evaluated when cultured on polycarbonate polyurethane anisotropic electrospun scaffolds, a representative biomaterial for in vitro HVTE. RESULTS: hUCPVCs had greater proliferative and clonogenic potential than BMMSCs in StemMACS™ (p < 0.05), without differentiation to osteogenic and adipogenic phenotypes associated with valve pathology. Furthermore, hUCPVCs cultured with StemMACS™ on tissue culture plastic for 14 days synthesized significantly more total collagen, elastin, and sulphated glycosaminoglycans (p < 0.05), the ECM constituents of the native valve, than BMMSCs. Finally, hUCPVCs retained their ECM synthesizing capacity after 14 and 21 days in culture on anisotropic electrospun scaffolds. CONCLUSION: Overall, our findings establish an in vitro culture platform that uses hUCPVCs as a readily-available and non-invasively sourced autologous cell population and a commercial serum- and xeno-free culture medium to increase the translational potential of future pediatric HVTE strategies. This study evaluated the proliferative, differentiation and extracellular matrix (ECM) synthesis capacities of human umbilical cord perivascular cells (hUCPVCs) when cultured in serum- and xeno-free media (SFM) against conventionally used bone marrow-derived MSCs (BMMSCs) and serum-containing media (SCM). Our findings support the use of hUCPVCs and SFM for in vitro heart valve tissue engineering (HVTE) of autologous pediatric valve tissue. Figure created with BioRender.com.

Ascending aortic geometry and its relationship to the biomechanical properties of aortic tissue.

Objective: The objective of this study was to evaluate the relationship between ascending aortic geometry and biomechanical properties. Methods: Preoperative computed tomography scans from ascending aortic aneurysm patients were analyzed using a center line technique (n = 68). Aortic length was measured from annulus to innominate artery, and maximal diameter from this segment was recorded. Biaxial tensile testing of excised tissue was performed to derive biomechanical parameters energy loss (efficiency in performing the Windkessel function) and modulus of elasticity (stiffness). Delamination testing (simulation of dissection) was performed to derive delamination strength (strength between tissue layers). Results: Aortic diameter weakly correlated with energy loss (r 2 = 0.10; P < .01), but not with modulus of elasticity (P = .13) or delamination strength (P = .36). Aortic length was not associated with energy loss (P = .87), modulus of elasticity (P = .13) or delamination strength (P = .90). Using current diameter guidelines, aortas >55 mm (n = 33) demonstrated higher energy loss than those <55 mm (n = 35; P = .05), but no difference in modulus of elasticity (P = .25) or delamination strength (P = .89). A length cutoff of 110 mm was proposed as an indication for repair. Aortas >110 mm (n = 37) did not exhibit a difference in energy loss (P = .40), modulus of elasticity (P = .69), or delamination strength (P = .68) compared with aortas <110 mm (n = 31). Aortas above diameter and length thresholds (n = 21) showed no difference in energy loss (P = .35), modulus of elasticity (P = .55), or delamination strength (P = .61) compared with smaller aortas (n = 47). Conclusions: Aortic geometry poorly reflects the mechanical properties of aortic tissue. Weak association between energy loss and diameter supports intervention at larger diameters. Further research into markers that better capture aortic biomechanics is needed.

Assessing engineered tissues and biomaterials using ultrasound imaging: In vitro and in vivo applications.

Quantitative assessment of the structural, functional, and mechanical properties of engineered tissues and biomaterials is fundamental to their development for regenerative medicine applications. Ultrasound (US) imaging is a non-invasive, non-destructive, and cost-effective technique capable of longitudinal and quantitative monitoring of tissue structure and function across centimeter to sub-micron length scales. Here we present the fundamentals of US to contextualize its application for the assessment of biomaterials and engineered tissues, both in vivo and in vitro. We review key studies that demonstrate the versatility and broad capabilities of US for clinical and pre-clinical biomaterials research. Finally, we highlight emerging techniques that further extend the applications of US, including for ultrafast imaging of biomaterials and engineered tissues in vivo and functional monitoring of stem cells, organoids, and organ-on-a-chip systems in vitro.

High-frequency quantitative ultrasound for the assessment of the acoustic properties of engineered tissues in vitro.

Acoustic properties of biomaterials and engineered tissues reflect their structure and cellularity. High-frequency ultrasound (US) can non-invasively characterize and monitor these properties with sub-millimetre resolution. We present an approach to estimate the speed of sound, acoustic impedance, and acoustic attenuation of cell-laden hydrogels that accounts for frequency-dependent effects of attenuation in coupling media, hydrogel thickness, and interfacial transmission/reflection coefficients of US waves, all of which can bias attenuation estimates. Cell-seeded fibrin hydrogel disks were raster-scanned using a 40 MHz US transducer. Thickness, speed of sound, acoustic impedance, and acoustic attenuation coefficients were determined from the difference in the time-of-flight and ratios of the magnitudes of US signals, interfacial transmission/reflection coefficients, and acoustic properties of the coupling media. With this approach, hydrogel thickness was accurately measured by US, with agreement to confocal microscopy (r2 = 0.97). Accurate thickness measurement enabled acoustic property measurements that were independent of hydrogel thickness, despite up to 60% reduction in thickness due to cell-mediated contraction. Notably, acoustic attenuation coefficients increased with increasing cell concentration (p < 0.001), reflecting hydrogel cellularity independent of contracted hydrogel thickness. This approach enables accurate measurement of the intrinsic acoustic properties of biomaterials and engineered tissues to provide new insights into their structure and cellularity. STATEMENT OF SIGNIFICANCE: High-frequency ultrasound can measure the acoustic properties of engineered tissues non-invasively and non-destructively with µm-scale resolution. Acoustic properties, including acoustic attenuation, are related to intrinsic material properties, such as scatterer density. We developed an analytical approach to estimate the acoustic properties of cell-laden hydrogels that accounts for the frequency-dependent effects of attenuation in coupling media, the reflection/transmission of ultrasound waves at the coupling interfaces, and the dependency of measurements on hydrogel thickness. Despite up to 60% reduction in hydrogel thickness due to cell-mediated contraction, our approach enabled measurements of acoustic properties that were substantially independent of thickness. Acoustic attenuation increased significantly with increasing cell concentration (p < 0.001), demonstrating the ability of acoustic attenuation to reflect intrinsic physical properties of engineered tissues.

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.

Tmem65 is critical for the structure and function of the intercalated discs in mouse hearts.

The intercalated disc (ICD) is a unique membrane structure that is indispensable to normal heart function, yet its structural organization is not completely understood. Previously, we showed that the ICD-bound transmembrane protein 65 (Tmem65) was required for connexin43 (Cx43) localization and function in cultured mouse neonatal cardiomyocytes. Here, we investigate the functional and cellular effects of Tmem65 reductions on the myocardium in a mouse model by injecting CD1 mouse pups (3-7 days after birth) with recombinant adeno-associated virus 9 (rAAV9) harboring Tmem65 shRNA, which reduces Tmem65 expression by 90% in mouse ventricles compared to scrambled shRNA injection. Tmem65 knockdown (KD) results in increased mortality which is accompanied by eccentric hypertrophic cardiomyopathy within 3 weeks of injection and progression to dilated cardiomyopathy with severe cardiac fibrosis by 7 weeks post-injection. Tmem65 KD hearts display depressed hemodynamics as measured echocardiographically as well as slowed conduction in optical recording accompanied by prolonged PR intervals and QRS duration in electrocardiograms. Immunoprecipitation and super-resolution microscopy demonstrate a physical interaction between Tmem65 and sodium channel ß subunit (ß1) in mouse hearts and this interaction appears to be required for both the establishment of perinexal nanodomain structure and the localization of both voltage-gated sodium channel 1.5 (NaV1.5) and Cx43 to ICDs. Despite the loss of NaV1.5 at ICDs, whole-cell patch clamp electrophysiology did not reveal reductions in Na+ currents but did show reduced Ca2+ and K+ currents in Tmem65 KD cardiomyocytes in comparison to control cells. We conclude that disrupting Tmem65 function results in impaired ICD structure, abnormal cardiac electrophysiology, and ultimately cardiomyopathy.

Harnessing conserved signaling and metabolic pathways to enhance the maturation of functional engineered tissues.

The development of induced-pluripotent stem cell (iPSC)-derived cell types offers promise for basic science, drug testing, disease modeling, personalized medicine, and translatable cell therapies across many tissue types. However, in practice many iPSC-derived cells have presented as immature in physiological function, and despite efforts to recapitulate adult maturity, most have yet to meet the necessary benchmarks for the intended tissues. Here, we summarize the available state of knowledge surrounding the physiological mechanisms underlying cell maturation in several key tissues. Common signaling consolidators, as well as potential synergies between critical signaling pathways are explored. Finally, current practices in physiologically relevant tissue engineering and experimental design are critically examined, with the goal of integrating greater decision paradigms and frameworks towards achieving efficient maturation strategies, which in turn may produce higher-valued iPSC-derived tissues.

Immunomagnetic Isolation and Enrichment of Microvascular Endothelial Cells from Human Adipose Tissue.

Human adipose tissue-resident microvascular endothelial cells are not only garnering attention for their emergent role in the pathogenesis of obesity-related metabolic disorders, but are also of considerable interest for vascular tissue engineering due, in part, to the abundant, accessible, and uniquely dispensable nature of the tissue. Here, we delineate a protocol for the acquisition of microvascular endothelial cells from human fat. A cheaper, smaller, and simpler alternative to fluorescence-assisted cell sorting for the immunoselection of cells, our protocol adapts magnet-assisted cell sorting for the isolation of endothelial cells from enzymatically digested adipose tissue and the subsequent enrichment of their primary cultures. Strategies are employed to mitigate the non-specific uptake of immunomagnetic microparticles, enabling the reproducible acquisition of human adipose tissue-resident microvascular endothelial cells with purities ≥98%. They exhibit morphological, molecular, and functional hallmarks of endothelium, yet retain a unique proteomic signature when compared with endothelial cells derived from different vascular beds. Their cultures can be expanded for >10 population doublings and can be maintained at confluence for at least 28 days without being overgrown by residual stromal cells from the cell sorting procedure. The isolation of human adipose tissue-resident microvascular endothelial cells can be completed within 6 hours and their enrichment within 2 hours, following approximately 7 days in culture. Graphical abstract.

Hearts by design.

Scalable biofabrication of heart helical tissue pattern augments pumping function.

An SCPPPQ1/LAM332 protein complex enhances the adhesion and migration of oral epithelial cells: Implications for dentogingival regeneration.

Common periodontal disease treatment procedures often fail to restore the structural integrity of the junctional epithelium (JE), the epithelial attachment of the gum to the tooth, leaving the tooth-gum interface prone to bacterial colonization. To address this issue, we introduced a novel bio-inspired protein complex comprised of a proline-rich enamel protein, SCPPPQ1, and laminin 332 (LAM332) to enhance the JE attachment. Using quartz crystal microbalance with dissipation monitoring (QCM-D), we showed that SCPPPQ1 and LAM332 interacted and assembled into a protein complex with high-affinity adsorption of 5.9e-8 [M] for hydroxyapatite (HA), the main component of the mineralized tooth surfaces. We then designed a unique shear device to study the adhesion strength of the oral epithelial cells to HA. The SCPPPQ1/LAM332 complex resulted in a twofold enhancement in adhesion strength of the cells to HA compared to LAM332 (from 31 dyn/cm2 to 63 dyn/cm2). In addition, using a modified wound-healing assay, we showed that gingival epithelial cells demonstrated a significantly high migration rate of 2.7 ± 0.24 µm/min over SCPPPQ1/LAM332-coated surfaces. Our collective data show that this protein complex has the potential to be further developed in designing a bioadhesive to enhance the JE attachment and protect the underlying connective tissue from bacterial invasion. However, its efficacy for wound healing requires further testing in vivo. STATEMENT OF SIGNIFICANCE: This work is the first functional study towards understanding the combined role of the enamel protein SCPPPQ1 and laminin 332 (LAM332) in the epithelial attachment of the gum, the junctional epithelium (JE), to the tooth hydroxyapatite surfaces. Such studies are essential for developing therapeutic approaches to restore the integrity of the JE in the destructive form of gum infection. We have developed a model system that provided the first evidence of the strong interaction between SCPPPQ1 and LAM332 on hydroxyapatite surfaces that favored protein adsorption and subsequently oral epithelial cell attachment and migration. Our collective data strongly suggested using the SCPPPQ1/LAM332 complex to accelerate the reestablishment of the JE after surgical gum removal to facilitate gum regeneration.

A Carbon-Based Biosensing Platform for Simultaneously Measuring the Contraction and Electrophysiology of iPSC-Cardiomyocyte Monolayers.

Heart beating is triggered by the generation and propagation of action potentials through the myocardium, resulting in the synchronous contraction of cardiomyocytes. This process highlights the importance of electrical and mechanical coordination in organ function. Investigating the pathogenesis of heart diseases and potential therapeutic actions in vitro requires biosensing technologies which allow for long-term and simultaneous measurement of the contractility and electrophysiology of cardiomyocytes. However, the adoption of current biosensing approaches for functional measurement of in vitro cardiac models is hampered by low sensitivity, difficulties in achieving multifunctional detection, and costly manufacturing processes. Leveraging carbon-based nanomaterials, we developed a biosensing platform that is capable of performing on-chip and simultaneous measurement of contractility and electrophysiology of human induced pluripotent stem-cell-derived cardiomyocyte (iPSC-CM) monolayers. This platform integrates with a flexible thin-film cantilever embedded with a carbon black (CB)-PDMS strain sensor for high-sensitivity contraction measurement and four pure carbon nanotube (CNT) electrodes for the detection of extracellular field potentials with low electrode impedance. Cardiac functional properties including contractile stress, beating rate, beating rhythm, and extracellular field potential were evaluated to quantify iPSC-CM responses to common cardiotropic agents. In addition, an in vitro model of drug-induced cardiac arrhythmia was established to further validate the platform for disease modeling and drug testing.

Design of a Mechanobioreactor to Apply Anisotropic, Biaxial Strain to Large Thin Biomaterials for Tissue Engineered Heart Valve Applications.

Repair and replacement solutions for congenitally diseased heart valves capable of post-surgery growth and adaptation have remained elusive. Tissue engineered heart valves (TEHVs) offer a potential biological solution that addresses the drawbacks of existing valve replacements. Typically, TEHVs are made from thin, fibrous biomaterials that either become cell populated in vitro or in situ. Often, TEHV designs poorly mimic the anisotropic mechanical properties of healthy native valves leading to inadequate biomechanical function. Mechanical conditioning of engineered tissues with anisotropic strain application can induce extracellular matrix remodelling to alter the anisotropic mechanical properties of a construct, but implementation has been limited to small-scale set-ups. To address this limitation for TEHV applications, we designed and built a mechanobioreactor capable of modulating biaxial strain anisotropy applied to large, thin, biomaterial sheets in vitro. The bioreactor can independently control two orthogonal stretch axes to modulate applied strain anisotropy on biomaterial sheets from 13 × 13 mm2 to 70 × 40 mm2. A design of experiments was performed using experimentally validated finite element (FE) models and demonstrated that biaxial strain was applied uniformly over a larger percentage of the cell seeded area for larger sheets (13 × 13 mm2: 58% of sheet area vs. 52 × 31 mm2: 86% of sheet area). Furthermore, bioreactor prototypes demonstrated that over 70% of the cell seeding area remained uniformly strained under different prescribed protocols: equibiaxial amplitudes between 5 to 40%, cyclic frequencies between 0.1 to 2.5 Hz and anisotropic strain ratios between 0:1 (constrained uniaxial) to 2:1. Lastly, proof-of-concept experiments were conducted where we applied equibiaxial (εx = εy = 8.75%) and anisotropic (εx = 12.5%, εy = 5%) strain protocols to cell-seeded, electrospun scaffolds. Cell nuclei and F-actin aligned to the vector-sum strain direction of each prescribed protocol (nuclei alignment: equibiaxial: 43.2° ± 1.8°, anisotropic: 17.5° ± 1.7°; p < 0.001). The abilities of this bioreactor to prescribe different strain amplitude, frequency and strain anisotropy protocols to cell-seeded scaffolds will enable future studies into the effects of anisotropic loading protocols on mechanically conditioned TEHVs and other engineered planar connective tissues.

Microengineered platforms for characterizing the contractile function of in vitro cardiac models.

Emerging heart-on-a-chip platforms are promising approaches to establish cardiac cell/tissue models in vitro for research on cardiac physiology, disease modeling and drug cardiotoxicity as well as for therapeutic discovery. Challenges still exist in obtaining the complete capability of in situ sensing to fully evaluate the complex functional properties of cardiac cell/tissue models. Changes to contractile strength (contractility) and beating regularity (rhythm) are particularly important to generate accurate, predictive models. Developing new platforms and technologies to assess the contractile functions of in vitro cardiac models is essential to provide information on cell/tissue physiologies, drug-induced inotropic responses, and the mechanisms of cardiac diseases. In this review, we discuss recent advances in biosensing platforms for the measurement of contractile functions of in vitro cardiac models, including single cardiomyocytes, 2D monolayers of cardiomyocytes, and 3D cardiac tissues. The characteristics and performance of current platforms are reviewed in terms of sensing principles, measured parameters, performance, cell sources, cell/tissue model configurations, advantages, and limitations. In addition, we highlight applications of these platforms and relevant discoveries in fundamental investigations, drug testing, and disease modeling. Furthermore, challenges and future outlooks of heart-on-a-chip platforms for in vitro measurement of cardiac functional properties are discussed.

In Vitro Matured Human Pluripotent Stem Cell-Derived Cardiomyocytes Form Grafts With Enhanced Structure and Function in Injured Hearts.

BACKGROUND: Human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CMs) have tremendous promise for application in cardiac regeneration, but their translational potential is limited by an immature phenotype. We hypothesized that large-scale manufacturing of mature hPSC-CMs could be achieved through culture on polydimethylsiloxane (PDMS)-lined roller bottles and that the transplantation of these cells would mediate better structural and functional outcomes than with conventional immature hPSC-CM populations. METHODS: We comprehensively phenotyped hPSC-CMs after in vitro maturation for 20 and 40 days on either PDMS or standard tissue culture plastic substrates. All hPSC-CMs were generated from a transgenic hPSC line that stably expressed a voltage-sensitive fluorescent reporter to facilitate in vitro and in vivo electrophysiological studies, and cardiomyocyte populations were also analyzed in vitro by immunocytochemistry, ultrastructure and fluorescent calcium imaging, and bulk and single-cell transcriptomics. We next compared outcomes after the transplantation of these populations into a guinea pig model of myocardial infarction using end points including histology, optical mapping of graft- and host-derived action potentials, echocardiography, and telemetric electrocardiographic monitoring. RESULTS: We demonstrated the economic generation of >1×108 mature hPSC-CMs per PDMS-lined roller bottle. Compared with their counterparts generated on tissue culture plastic substrates, PDMS-matured hPSC-CMs exhibited increased cardiac gene expression and more mature structural and functional properties in vitro. More important, intracardiac grafts formed with PDMS-matured myocytes showed greatly enhanced structure and alignment, better host-graft electromechanical integration, less proarrhythmic behavior, and greater beneficial effects on contractile function. CONCLUSIONS: We describe practical methods for the scaled generation of mature hPSC-CMs and provide the first evidence that the transplantation of more mature cardiomyocytes yields better outcomes in vivo.