Cardiac Spheroids Research Articles

Amyloidogenic immunoglobulin light chains disturb contractile function and calcium transients in a human cardiac spheroid model of light chain (AL) amyloidosis

Light chain (AL) amyloidosis is a serious systemic disease caused by the deposition of free misfolded immunoglobulin light chains (LCs) in the form of amyloid fibrils within tissues. Cardiac involvement determines prognosis and mortality. An important cytotoxic impact of amyloidogenic prefibrillar LC oligomers on cardiomyocytes is by now established in isolated rodent cardiomyocytes, simple animal models, or cardiomyocyte-like cell lines. However, the response of human cardiomyocytes to this pathogenic condition is currently unknown. In this work, we have set up a human cellular disease model of AL cardiac amyloidosis (AL-CA) in the form of cardiac spheroids, to study the cytotoxic effects of amyloidogenic LCs with regard to contractile function and calcium handling. To mimic the disease in a reconstituted system, soluble amyloidogenic LCs purified from urine of AL-CA patients were added to a mixture of induced pluripotent stem cell-issued human cardiomyocytes (hiPSC-CM) and human primary cardiac fibroblasts, which resulted in formation of spheroids within 7 days. This procedure ensured a uniform pericellular LC distribution within spheroids. LC-treated hiPSC-CM cultures and LC-containing spheroids presented structural and functional defects including: (1) decreased levels and subcellular disorganization of sarcomeric protein alpha-actinin; (2) abnormal accumulation of calcium handling SERCA2a protein; (3) impaired contractility of spheroids and altered calcium transients. Three independent patient-derived LCs had similar effects, albeit to varying degrees, highlighting the patient-specific properties of this type of amyloids. Taken together, these results indicate that the present cardiac spheroid disease model could be appropriate to the study of cardiac cytotoxicity caused by different amyloidogenic LCs in AL-CA patients, contributing to a better understanding and therapeutic handling of the disease.

Optogenetic stimulation and simultaneous optical mapping of membrane potential and calcium transients in human engineered cardiac spheroids.

Optogenetic stimulation and simultaneous optical mapping of membrane potential and calcium transients in human engineered cardiac spheroids.

Deep phenotyping of testosterone-prompted fibrosis in arrhythmogenic right ventricular cardiomyopathy using iPSC-derived engineered cardiac spheroids

Deep phenotyping of testosterone-prompted fibrosis in arrhythmogenic right ventricular cardiomyopathy using iPSC-derived engineered cardiac spheroids

Development and application of 3D cardiac tissues derived from human pluripotent stem cells.

Development and application of 3D cardiac tissues derived from human pluripotent stem cells.

Conductive Microfibers Improve Stem Cell-Derived Cardiac Spheroid Maturation.

Conventional two-dimensional (2D) cardiomyocyte differentiation protocols create cells with limited maturity, which impairs their predictive capacity and has driven interest in three-dimensional (3D) engineered cardiac tissue models of varying maturity and scalability. Cardiac spheroids are attractive high-throughput models that have demonstrated improved functional and transcriptional maturity over conventional 2D differentiations. However, these 3D models still tend to have limited contractile and electrical maturity compared to highly engineered cardiac tissues; hence, we incorporated a library of conductive polymer microfibers in cardiac spheroids to determine if fiber properties could accelerate maturation. Conductive microfibers improved contractility parameters of cardiac spheroids over time versus nonconductive fibers, specifically, when they were short, for example, 5 μm, and when there was moderate fiber mass per spheroid, for example, 20 μg. Spheroids with optimal conductive microfiber length and concentration developed a thicker ring-like perimeter and a less compacted cavity, improving their contractile work compared to control cardiac spheroids. Functional improvements correlated with increased expression of contractility and calcium handling-related cardiac proteins, as well as improved calcium handling abilities and drug response. Taken together, these data suggest that conductive microfibers can improve cardiac spheroid performance to improve cardiac disease modeling.

Highly Stretchable 3D Microelectrode Array for Noninvasive Functional Evaluation of Cardiac Spheroids and Midbrain Organoids.

Organoids are 3D biological models that recapitulate the complex structures and functions of human organs. Despite the rapid growth in the generation of organoids, in vitro assay tools are still limited to 2D forms. Thus, a comprehensive and continuous functional evaluation of the electrogenic organoids remains a challenge. Here, a highly stretchable 3D multielectrode array (sMEA) with protruding microelectrodes is presented for functional evaluation of electrogenic organoids. The optimized serpentine structures with bridge structures cover the surface of the organoids conformally even in immersion. The protruding microelectrodes form a stable contact with the organoids and allow electrophysiological recordings with high signal-to-noise ratio (SNR). sMEAs are fabricated in wafer-scale for repeatable, scalable, and mass production and packed into an easy-to-use, user-friendly, and robust microwell for fast dissemination of technology. The versatility of sMEA is validated by measuring electrophysiological signals from cardiac spheroids and midbrain organoids with a wide range of sizes from 500 to 1500µm. Also, electrophysiological signals recorded with high SNR enable functional evaluation of the effects of drugs. The proposed sMEA with high SNR and user-friendly interface could be the key player in high-throughput drug screening, 3D spatiotemporal mapping of electrogenic organoids, and standardization of protocols for quality assessment.

Sericin improves alginate-gelatin hydrogels’ mechanical properties, porosity, durability and viability of fibroblast in cardiac spheroids

Biofabrication of cardiac patches is a challenging strategy proposed as an alternative to transplantation for end-stage heart failure patients. The optimization of the bioink used for this aim can be limited by costs, properties and biocompatibility of its building blocks. Lately, sericin has emerged within a wide range of natural proteins thanks to its bioadhesive and biocompatibility potential. In this study, we assessed for the first time the effects of adding silk sericin on alginate-gelatin hydrogels, proposed for cardiac applications. To this aim, we first biofabricated sericin-containing hydrogels with increasing protein concentrations. Thus, we characterized hydrogels’ mechanical behaviour, porosity and structure through rheology, Brillouin microspectroscopy and SEM. Then, we bioprinted the formulated hydrogels and evaluated their effects on human cardiac spheroids (CSs) in vitro. Our mechanical characterisation demonstrated that adding sericin significantly enhanced the elasticity and the viscosity of alginate-gelatin hydrogels. Sericin also modified hydrogels’ swelling behaviour and their pore size, increasing by 20%, 62%, and 92% in Ser1%, Ser2% and Ser3%, respectively. Although Ser1% did not exhibit significant effects on CSs, Ser2% and Ser3% enhanced cardiac cell viability for up to 14 days compared to the sericin-free hydrogel by acting on the fibroblast population. Sericin-based bioinks showed better printability and durability with +33% and +28% intact patches after 28 days of culture at 37°C compared to alginate-gelatin. Altogether our results validated the use of sericin as a promising component for the optimization of bioink intended for cardiac applications.

3D in vitro modelling of post-partum cardiovascular health reveals unique characteristics and signatures following hypertensive disorders in pregnancy

BackgroundHypertensive disorders of pregnancy (HDP) affect 2–8% of pregnancies and are associated postpartum with increased cardiovascular disease (CVD) risk, although mechanisms are poorly understood.MethodsHuman induced pluripotent stem cells (iPSC)-derived cardiomyocytes, cardiac fibroblasts and coronary artery endothelial cells were cocultured to form cardiac spheroids (CSs) in collagen type-1 hydrogels containing 10% patient plasma collected five years postpartum [n = 5 per group: normotensive control, gestational hypertension (GH) and preeclampsia (PE)]. Plasma-treated CSs were assessed for cell viability and contractile function and subjected to immunofluorescence staining and imaging. A quantitative proteomic analysis of plasma samples was conducted (controls n = 21; GH n = 5; PE n = 12).ResultsContraction frequency (CF) was increased in PE-treated CSs (CF: 45.5 ± 3.4 contractions/minute, p < 0.001) and GH-treated CSs (CF: 45.7 ± 4.0 contractions/minute, p < 0.001), compared to controls (CF = 21.8 ± 2.6 contractions/min). Only PE-treated CSs presented significantly increased fractional shortening (FS) % (9.95 ± 1.8%, p < 0.05) compared to controls (3.7 ± 1.1%). GH-treated CSs showed a reduction in cell viability (p < 0.05) and an increase in α-SMA expression (p < 0.05). Proteomics analyses identified twenty differentially abundant proteins, with hemoglobin A2 being the only protein perturbed in both GH and PE versus control plasma (p < 0.05).ConclusionsThe innovative patient-relevant CS platforms led to the discovery of biomarkers/targets linked to cell death signaling and cardiac remodeling in GH-induced CVD and vascular/endothelial cell dysfunction in PE-induced CVD.

Chlorella-enriched hydrogels protect against myocardial damage and reactive oxygen species production in an in vitro ischemia/reperfusion model using cardiac spheroids

Microalgae have emerged as promising photosynthetic microorganisms for biofabricating advanced tissue constructs, with improved oxygenation and reduced reactive oxygen species (ROS) production. However, their use in the engineering of human tissues has been limited due to their intrinsic growth requirements, which are not compatible with human cells. In this study, we first formulated alginate-gelatin (AlgGel) hydrogels with increasing densities ofChlorella vulgaris. Then, we characterised their mechanical properties and pore size. Finally, we evaluated their effects on cardiac spheroid (CS) pathophysiological response under control and ischemia/reperfusion (I/R) conditions. Our results showed that the addition ofChlorelladid not affect AlgGel mechanical properties, while the mean pore size significantly decreased by 35% in the presence of the 107cells ml-1microalgae density. Under normoxic conditions, the addition of 107Chlorellacells ml-1significantly reduced CS viability starting from 14 d in. No changes in pore size nor CS viability were measured for hydrogels containing 105and 106Chlorellacells ml-1. In our I/R model, allChlorella-enriched hydrogels reduced cardiac cell sensitivity to hypoxic conditions with a corresponding reduction in ROS production, as well as protected against I/R-induced reduction in cell viability. Altogether, our results support a promising use ofChlorella-enriched Alg-Gel hydrogels for cardiovascular tissue engineering.

Knock-down of FOXO3, GATA2, NFE2L2 and AHR promotes doxorubicin-induced cardiotoxicity in human cardiomyocytes

Knock-down of FOXO3, GATA2, NFE2L2 and AHR promotes doxorubicin-induced cardiotoxicity in human cardiomyocytes

The hanging‐heart chip: A portable microfluidic device for high‐throughput generation of contractile embryonic stem cell‐derived cardiac spheroids

AbstractStem cell‐derived cardiac spheroids are promising models for cardiac research and drug testing. However, generating contracting cardiac spheroids remains challenging because of the laborious experimental procedure. Here, we present a microfluidic hanging‐heart chip (HH‐chip) that uses a microchannel and flow‐driven system to facilitate cell loading and culture medium replacement operations to reduce the laborious manual handling involved in the generation of a large quantity of cardiac spheroids. The effectiveness of the HH‐chip was demonstrated by simultaneously forming 50 mouse embryonic stem cell‐derived embryonic bodies, which sequentially differentiated into 90% beating cardiac spheroids within 15 days of culture on the chip. A comparison of our HH‐chip method with traditional hanging‐drop and low‐attachment plate methods revealed that the HH‐chip could generate higher contracting proportions of cardiac spheroids with higher expression of cardiac markers. Additionally, we verified that the contraction frequencies of the cardiac spheroids generated from the HH‐chip were sensitive to cardiotoxic drugs. Overall, our results suggest that the microfluidic hanging drop chip‐based approach is a high‐throughput and highly efficient method for generating contracting mouse embryonic stem cell‐derived cardiac spheroids for cardiac toxicity and drug testing applications.

Embedding Biomimetic Vascular Networks via Coaxial Sacrificial Writing into Functional Tissue.

Printing human tissues and organs replete with biomimetic vascular networks is of growing interest. While it is possible to embed perfusable channels within acellular and densely cellular matrices, they do not currently possess the biomimetic architectures found in native vessels. Here, coaxialsacrificial writing into functional tissues (co-SWIFT) is developed, an embedded bioprinting method capable of generating hierarchically branching, multilayered vascular networks within both granular hydrogel and densely cellular matrices. Coaxial printheads are designed with an extended core-shell configuration to facilitate robust core-core and shell-shell interconnections between printed branching vessels during embedded bioprinting. Using optimized core-shell ink combinations, biomimetic vessels composed of a smooth muscle cell-laden shell that surrounds perfusable lumens are coaxially printed into granular matrices composed of: 1) transparent alginate microparticles, 2) sacrificial microparticle-laden collagen, or 3) cardiac spheroids derived from human induced pluripotent stem cells. Biomimetic blood vessels that exhibit good barrier function are produced by seeding these interconnected lumens with a confluent layer of endothelial cells. Importantly, it is found that co-SWIFT cardiac tissues mature under perfusion, beat synchronously, and exhibit a cardio-effective drug response in vitro. This advance opens new avenues for the scalable biomanufacturing of vascularized organ-specific tissues for drug testing, disease modeling, and therapeutic use.

New cardiac platform for the management of cardiovascular risk following preeclampsia using in vitro bioengineered cardiac spheroids and patient-derived stem cells

New cardiac platform for the management of cardiovascular risk following preeclampsia using in vitro bioengineered cardiac spheroids and patient-derived stem cells

Exploring the Dimensions of Pre-Clinical Research: 3D Cultures as an Investigative Model of Cardiac Fibrosis in Chagas Disease.

A three-dimensional (3D) cell culture can more precisely mimic tissues architecture and functionality, being a promising alternative model to study disease pathophysiology and drug screening. Chagas disease (CD) is a neglected parasitosis that affects 7 million people worldwide. Trypanosoma cruzi's (T. cruzi) mechanisms of invasion/persistence continue to be elucidated. Benznidazole (BZ) and Nifurtimox (NF) are trypanocidal drugs with few effects on the clinical manifestations of the chronic disease. Chronic Chagas cardiomyopathy (CCC) is the main manifestation of CD due to its frequency and severity. The development of fibrosis and hypertrophy in cardiac tissue can lead to heart failure and sudden death. Thus, there is an urgent need for novel therapeutic options. Our group has more than fifteen years of expertise using 3D primary cardiac cell cultures, being the first to reproduce fibrosis and hypertrophy induced by T. cruzi infection in vitro. These primary cardiac spheroids exhibit morphological and functional characteristics that are similar to heart tissue, making them an interesting model for studying CD cardiac fibrosis. Here, we aim to demonstrate that our primary cardiac spheroids are great preclinical models which can be used to develop new insights into CD cardiac fibrosis, presenting advances already achieved in the field, including disease modeling and drug screening.

Silk fibroin increases the elasticity of alginate-gelatin hydrogels and regulates cardiac cell contractile function in cardiac bioinks

Silk fibroin (SF) is a natural protein extracted from Bombyx mori silkworm thread. From its common use in the textile industry, it emerged as a biomaterial with promising biochemical and mechanical properties for applications in the field of tissue engineering and regenerative medicine. In this study, we evaluate for the first time the effects of SF on cardiac bioink formulations containing cardiac spheroids (CSs). First, we evaluate if the SF addition plays a role in the structural and elastic properties of hydrogels containing alginate (Alg) and gelatin (Gel). Then, we test the printability and durability of bioprinted SF-containing hydrogels. Finally, we evaluate whether the addition of SF controls cell viability and function of CSs in Alg–Gel hydrogels. Our findings show that the addition of 1% (w/v) SF to Alg–Gel hydrogels makes them more elastic without affecting cell viability. However, fractional shortening (FS%) of CSs in SF–Alg–Gel hydrogels increases without affecting their contraction frequency, suggesting an improvement in contractile function in the 3D cultures. Altogether, our findings support a promising pathway to bioengineer bioinks containing SF for cardiac applications, with the ability to control mechanical and cellular features in cardiac bioinks.

Human in vitro disease modeling of ATTR cardiac amyloidosis

Abstract Funding Acknowledgements Type of funding sources: Private company. Main funding source(s): Pfizer Alnylam Transthyretin amyloid cardiomyopathy (ATTR-CM) is a progressive disease with deposition of TTR amyloid fibrils in the myocardium and cardiac dysfunction with dismal prognosis. In ATTR-CM, either genetic mutations (variant; v) or ageing (wild-type; wt) leads to TTR instability. Late diagnosis and a lack of human disease models limit disease understanding and therapeutic discovery. This study aimed to model ATTR-CM in vitro by exposing various cardiac cell types and multicellular 3D cardiac spheroids to TTR fibrils, characterizing disease phenotypes and providing insights into the cellular mechanisms of disease progression. Human recombinant TTR (WT, V30M, V122I) and acid induced fibrils were generated and characterized. Seeding hiPSC-cardiomyocytes (CMs) and endothelial cells (ECs) on TTR fibrils resulted in cell viability reduction. Confocal microscopy revealed the extracellular attachment of TTR fibrils, leading to sarcomere disorganization, prolonged calcium transients and disrupted electromechanical coupling in hiPSC-CMs, and declined migration capacity of ECs showing aberrant cell morphology. Similarly, cardiac spheroids incorporating TTR fibrils resulted in decreased cell viability and prolonged calcium transients with lower amplitude. Our models show the discovery of early cellular ATTR-CM phenotypes, providing a novel approach to study the disease progression, potentially facilitating the identification of novel therapeutic targets and biomarkers.Cytotoxicity of TTR fibrils on hiPS-CMs

Investigations of cardiac fibrosis rheology by in vitro cardiac tissue modeling with 3D cellular spheroids

Investigations of cardiac fibrosis rheology by in vitro cardiac tissue modeling with 3D cellular spheroids

Regeneration of Nonhuman Primate Hearts With Human Induced Pluripotent Stem Cell-Derived Cardiac Spheroids.

The clinical application of human induced pluripotent stem cell-derived cardiomyocytes (CMs) for cardiac repair commenced with the epicardial delivery of engineered cardiac tissue; however, the feasibility of the direct delivery of human induced pluripotent stem cell-derived CMs into the cardiac muscle layer, which has reportedly induced electrical integration, is unclear because of concerns about poor engraftment of CMs and posttransplant arrhythmias. Thus, in this study, we prepared purified human induced pluripotent stem cell-derived cardiac spheroids (hiPSC-CSs) and investigated whether their direct injection could regenerate infarcted nonhuman primate hearts. We performed 2 separate experiments to explore the appropriate number of human induced pluripotent stem cell-derived CMs. In the first experiment, 10 cynomolgus monkeys were subjected to myocardial infarction 2 weeks before transplantation and were designated as recipients of hiPSC-CSs containing 2×107 CMs or the vehicle. The animals were euthanized 12 weeks after transplantation for histological analysis, and cardiac function and arrhythmia were monitored during the observational period. In the second study, we repeated the equivalent transplantation study using more CMs (6×107 CMs). Recipients of hiPSC-CSs containing 2×107 CMs showed limited CM grafts and transient increases in fractional shortening compared with those of the vehicle (fractional shortening at 4 weeks after transplantation: 26.2±2.1%; 19.3±1.8%; P<0.05), with a low incidence of posttransplant arrhythmia. Transplantation of increased dose of CMs resulted in significantly greater engraftment and long-term contractile benefits (fractional shortening at 12 weeks after transplantation: 22.5±1.0%; 16.6±1.1%; P<0.01, left ventricular ejection fraction at 12 weeks after transplantation: 49.0±1.4%; 36.3±2.9%; P<0.01). The incidence of posttransplant arrhythmia slightly increased in recipients of hiPSC-CSs containing 6×107 CMs. We demonstrated that direct injection of hiPSC-CSs restores the contractile functions of injured primate hearts with an acceptable risk of posttransplant arrhythmia. Although the mechanism for the functional benefits is not fully elucidated, these findings provide a strong rationale for conducting clinical trials using the equivalent CM products.

Vascularised cardiac spheroids-on-a-chip for testing the toxicity of therapeutics

Microfabricated organ-on-a-chips are rapidly becoming the gold standard for the testing of safety and efficacy of therapeutics. A broad range of designs has emerged, but recreating microvascularised tissue models remains difficult in many cases. This is particularly relevant to mimic the systemic delivery of therapeutics, to capture the complex multi-step processes associated with trans-endothelial transport or diffusion, uptake by targeted tissues and associated metabolic response. In this report, we describe the formation of microvascularised cardiac spheroids embedded in microfluidic chips. Different protocols used for embedding spheroids within vascularised multi-compartment microfluidic chips were investigated first to identify the importance of the spheroid processing, and co-culture with pericytes on the integration of the spheroid within the microvascular networks formed. The architecture of the resulting models, the expression of cardiac and endothelial markers and the perfusion of the system was then investigated. This confirmed the excellent stability of the vascular networks formed, as well as the persistent expression of cardiomyocyte markers such as cTNT and the assembly of striated F-actin, myosin and α-actinin cytoskeletal networks typically associated with contractility and beating. The ability to retain beating over prolonged periods of time was quantified, over 25 days, demonstrating not only perfusability but also functional performance of the tissue model. Finally, as a proof-of-concept of therapeutic testing, the toxicity of one therapeutic associated with cardiac disfunction was evaluated, identifying differences between direct in vitro testing on suspended spheroids and vascularised models.

Generation of Self-Induced Myocardial Ischemia in Large-Sized Cardiac Spheroids without Alteration of Environmental Conditions Recreates Fibrotic Remodeling and Tissue Stiffening Revealed by Constriction Assays.

A combination of human-induced pluripotent stem cells (hiPSCs) and 3D microtissue culture techniques allows the generation of models that recapitulate the cardiac microenvironment for preclinical research of new treatments. In particular, spheroids represent the simplest approach to culture cells in 3D and generate gradients of cellular access to the media, mimicking the effects of an ischemic event. However, previous models required incubation under low oxygen conditions or deprived nutrient media to recreate ischemia. Here, we describe the generation of large spheroids (i.e., larger than 500 μm diameter) that self-induce an ischemic core. Spheroids were generated by coculture of cardiomyocytes derived from hiPSCs (hiPSC-CMs) and primary human cardiac fibroblast (hCF). In the proper medium, cells formed aggregates that generated an ischemic core 2 days after seeding. Spheroids also showed spontaneous cellular reorganization after 10 days, with hiPSC-CMs located at the center and surrounded by hCFs. This led to an increase in microtissue stiffness, characterized by the implementation of a constriction assay. All in all, these phenomena are hints of the fibrotic tissue remodeling secondary to a cardiac ischemic event, thus demonstrating the suitability of these spheroids for the modeling of human cardiac ischemia and its potential application for new treatments and drug research.