Hypothermic and cryogenic preservation of cardiac tissue-engineered constructs.

Cardiac tissue engineering (cTE) has already advanced towards the first clinical trials, investigating safety and feasibility of cTE construct transplantation in failing hearts. However, the lack of well-established preservation methods poses a hindrance to further scalability, commercialization, and transportation, thereby reducing their clinical implementation. In this study, hypothermic preservation (4 °C) and two methods for cryopreservation (i.e., a slow and fast cooling approach to -196 °C and -150 °C, respectively) were investigated as potential solutions to extend the cTE construct implantation window. The cTE model used consisted of human induced pluripotent stem cell-derived cardiomyocytes and human cardiac fibroblasts embedded in a natural-derived hydrogel and supported by a polymeric melt electrowritten hexagonal scaffold. Constructs, composed of cardiomyocytes of different maturity, were preserved for three days, using several commercially available preservation protocols and solutions. Cardiomyocyte viability, function (beat rate and calcium handling), and metabolic activity were investigated after rewarming. Our observations show that cardiomyocytes' age did not influence post-rewarming viability, however, it influenced construct function. Hypothermic preservation with HypoThermosol® ensured cardiomyocyte viability and function. Furthermore, fast freezing outperformed slow freezing, but both viability and function were severely reduced after rewarming. In conclusion, whereas long-term preservation remains a challenge, hypothermic preservation with HypoThermosol® represents a promising solution for cTE construct short-term preservation and potential transportation, aiding in off-the-shelf availability, ultimately increasing their clinical applicability.

Convergence of melt electrowriting and extrusion-based bioprinting for vascular patterning of a myocardial construct.

To progress cardiac tissue engineering strategies closer to the clinic, thicker constructs are required to meet the functional need following a cardiac event. Consequently, pre-vascularization of these constructs needs to be investigated to ensure survival and optimal performance of implantable engineered heart tissue. The aim of this research is to investigate the potential of combining extrusion-based bioprinting (EBB) and melt electrowriting for the fabrication of a myocardial construct with a precisely patterned pre-vascular pathway. Gelatin methacryloyl (GelMA) was investigated as a base hydrogel for the respective myocardial and vascular bioinks with collagen, Matrigel and fibrinogen as interpenetrating polymers to support myocardial functionality. Subsequently, extrusion-based printability and viability were investigated to determine the optimal processing parameters for printing into melt electrowritten meshes. Finally, an anatomically inspired vascular pathway was implemented in a dual EBB set-up into melt electrowritten meshes, creating a patterned pre-vascularized myocardial construct. It was determined that a blend of 5% GelMA and 0.8 mg·ml-1collagen with a low crosslinked density was optimal for myocardial cellular arrangement and alignment within the constructs. For the vascular fraction, the optimized formulation consisted of 5% GelMA, 0.8 mg·ml-1collagen and 1 mg·ml-1fibrinogen with a higher crosslinked density, which led to enhanced vascular cell connectivity. Printability assessment confirmed that the optimized bioinks could effectively fill the microfiber mesh while supporting cell viability (∼70%). Finally, the two bioinks were applied using a dual EBB system for the fabrication of a pre-vascular pathway with the shape of a left anterior descending artery within a myocardial construct, whereby the distinct cell populations could be visualized in their respective patterns up to D14. This research investigated the first step towards developing a thick engineered cardiac tissue construct in which a pre-vascularization pathway is fabricated within a myocardial construct.

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Small molecule-mediated rapid maturation of human induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) do not display all hallmarks of mature primary cardiomyocytes, especially the ability to use fatty acids (FA) as an energy source, containing high mitochondrial mass, presenting binucleation and increased DNA content per nuclei (polyploidism), and synchronized electrical conduction. This immaturity represents a bottleneck to their application in (1) disease modelling-as most cardiac (genetic) diseases have a middle-age onset-and (2) clinically relevant models, where integration and functional coupling are key. So far, several methods have been reported to enhance iPSC-CM maturation; however, these protocols are laborious, costly, and not easily scalable. Therefore, we developed a simple, low-cost, and rapid protocol to promote cardiomyocyte maturation using two small molecule activators of the peroxisome proliferator-activated receptor ß/δ and gamma coactivator 1-alpha (PPAR/PGC-1α) pathway: asiatic acid (AA) and GW501516 (GW). METHODS AND RESULTS: Monolayers of iPSC-CMs were incubated with AA or GW every other day for ten days resulting in increased expression of FA metabolism-related genes and markers for mitochondrial activity. AA-treated iPSC-CMs responsiveness to the mitochondrial respiratory chain inhibitors increased and exhibited higher flexibility in substrate utilization. Additionally, structural maturity improved after treatment as demonstrated by an increase in mRNA expression of sarcomeric-related genes and higher nuclear polyploidy in AA-treated samples. Furthermore, treatment led to increased ion channel gene expression and protein levels. CONCLUSIONS: Collectively, we developed a fast, easy, and economical method to induce iPSC-CMs maturation via PPAR/PGC-1α activation. Treatment with AA or GW led to increased metabolic, structural, functional, and electrophysiological maturation, evaluated using a multiparametric quality assessment.

Cardiac circadian rhythms in time and space: The future is in 4D.

The circadian clock synchronizes the body into 24-h cycles, thereby anticipating variations in tissue-specific diurnal tasks, such as response to increased cardiac metabolic demand during the active period of the day. As a result, blood pressure, heart rate, cardiac output, and occurrence of fatal cardiovascular events fluctuate in a diurnal manner. The heart contains different cell types that make up and reside in an environment of biochemical, mechanical, and topographical signaling. Cardiac architecture is essential for proper heart development as well as for maintenance of cell homeostasis and tissue repair. In this review, we describe the possibilities of studying circadian rhythmicity in the heart by using advanced in vitro systems that mimic the native cardiac 3D microenvironment which can be tuned in time and space. Harnessing the knowledge that originates from those in vitro models could significantly improve innovative cardiac modeling and regenerative strategies.

A Roadmap to Cardiac Tissue-Engineered Construct Preservation: Insights from Cells, Tissues, and Organs.

Worldwide, over 26 million patients suffer from heart failure (HF). One strategy aspiring to prevent or even to reverse HF is based on the transplantation of cardiac tissue-engineered (cTE) constructs. These patient-specific constructs aim to closely resemble the native myocardium and, upon implantation on the diseased tissue, support and restore cardiac function, thereby preventing the development of HF. However, cTE constructs off-the-shelf availability in the clinical arena critically depends on the development of efficient preservation methodologies. Short- and long-term preservation of cTE constructs would enable transportation and direct availability. Herein, currently available methods, from normothermic- to hypothermic- to cryopreservation, for the preservation of cardiomyocytes, whole-heart, and regenerative materials are reviewed. A theoretical foundation and recommendations for future research on developing cTE construct specific preservation methods are provided. Current research suggests that vitrification can be a promising procedure to ensure long-term cryopreservation of cTE constructs, despite the need of high doses of cytotoxic cryoprotective agents. Instead, short-term cTE construct preservation can be achieved at normothermic or hypothermic temperatures by administration of protective additives. With further tuning of these promising methods, it is anticipated that cTE construct therapy can be brought one step closer to the patient.

Fiber Scaffold Patterning for Mending Hearts: 3D Organization Bringing the Next Step.

Heart failure (HF) is a leading cause of death worldwide. The most common conditions that lead to HF are coronary artery disease, myocardial infarction, valve disorders, high blood pressure, and cardiomyopathy. Due to the limited regenerative capacity of the heart, the only curative therapy currently available is heart transplantation. Therefore, there is a great need for the development of novel regenerative strategies to repair the injured myocardium, replace damaged valves, and treat occluded coronary arteries. Recent advances in manufacturing technologies have resulted in the precise fabrication of 3D fiber scaffolds with high architectural control that can support and guide new tissue growth, opening exciting new avenues for repair of the human heart. This review discusses the recent advancements in the novel research field of fiber patterning manufacturing technologies for cardiac tissue engineering (cTE) and to what extent these technologies could meet the requirements of the highly organized and structured cardiac tissues. Additionally, future directions of these novel fiber patterning technologies, designs, and applicability to advance cTE are presented.

Engineering of cell-laden gelatin-based microgels for cell delivery and immobilization in regenerative therapies.

Cell-based therapies often face the challenge of low cell retention and viability upon transplantation. Hence, biomaterials, which can immobilize transplanted cells, while at the same time support cell viability, are essential for successful clinical application. Noteworthy, biomaterials in the micrometer range such as microcapsules or microspheres have the advantage of a minimally invasive introduction into tissue.Hence, we established an approach to generate gelatin-based cell carriers in the form of microspherical hydrogels. Fibroblasts were microencapsulated in glycidylmethacrylate (GMA)-functionalized gelatin by photopolymerization. While the degree of GMA-functionalization was kept constant, the hydrogel cross-linking density was adjusted by varying the time of irradiation or the average gelatin-chain length.Stable microspheres were synthesized from 10 wt% GMA-gelatin solutions for all irradiation periods tested (0.5 -2 min). Evaluation of cell viability revealed that microgels with the same weight content of biopolymer but with decreased cross-linking densities and thus decreased storage and E modulus, resulted in best cell support. Noteworthy, encapsulated cells partially migrated out of the microspheres and attached to the spherical surface.10 wt% GMA-gelatin-based hydrogels with E moduli comparable to the native cellular niche proved to be a promising biomaterial suitable for the production of cell-laden microspheres and shall be evaluated further for biomedical application.

Secretome released from hydrogel-embedded adipose mesenchymal stem cells protects against the Parkinson's disease related toxin 6-hydroxydopamine.

Neurodegenerative diseases, as Parkinson's disease (PD), involve irreversible neural cell damage and impairment. In PD, there is a selective degeneration of the dopaminergic neurons leading to motor symptoms. A common finding in PD neurodegeneration is the increase of reactive oxygen species (ROS), leading to oxidative stress. To date there are only interventions to relieve PD symptoms, however progress has been made in the development of therapies that target the immune system or use its components as therapeutic agents; among these, mesenchymal stem cells (MSCs), which are able to express neuroprotective factors as cytokines, chemokines and angiogenic molecules, collectively named secretome, that accumulate in MSC culture medium. However, lasting cell-free administration of secretome in vitro or in vivo is challenging. We used the conditioned media from rat adipose tissue-derived MSCs (RAA-MSCs) to check for neuroprotective activity towards pro-oxidizing agents such as hydrogen peroxide (H2O2) or the dopaminergic selective toxin 6-hydroxydopamine (6-OHDA) that is commonly used to model PD neurodegeneration. When neuroblastoma SH-SY5Y cells were pre-conditioned with 100% RAA-MSC media, then treated with H2O2 and 6-OHDA, mortality and ROS generation were reduced. We implemented the controlled release of RAA-MSC secretome from injectable biodegradable hydrogels that offer a possible in situ implant with mini-invasive techniques. The hydrogels were composed of type I bovine collagen (COLL) and low-molecular-weight hyaluronic acid (LMWHA) or COLL and polyethylene glycol (PEG). Hydrogels were suitable for RAA-MSC embedding up to 48h and secretome from these RAA-MSCs was active and counteracted 6-OHDA toxicity, with upregulation of the antioxidant enzyme sirtuin 3 (SIRT3). These results support a biomaterials-based approach for controlled delivery of MSC-produced neuroprotective factors in a PD-relevant experimental context.