The creation of human induced pluripotent stem cells (hiPSCs) has provided an unprecedented opportunity to study tissue morphogenesis and organ development through 'organogenesis-in-a-dish'. Current ...approaches to cardiac organoid engineering rely on either direct cardiac differentiation from embryoid bodies (EBs) or generation of aligned cardiac tissues from predifferentiated cardiomyocytes from monolayer hiPSCs. To experimentally model early cardiac organogenesis in vitro, our protocol combines biomaterials-based cell patterning with stem cell organoid engineering. 3D cardiac microchambers are created from 2D hiPSC colonies; these microchambers approximate an early-development heart with distinct spatial organization and self-assembly. With proper training in photolithography microfabrication, maintenance of human pluripotent stem cells, and cardiac differentiation, a graduate student with guidance will likely be able to carry out this experimental protocol, which requires ∼3 weeks. We envisage that this in vitro model of human early heart development could serve as an embryotoxicity screening assay in drug discovery, regulation, and prescription for healthy fetal development. We anticipate that, when applied to hiPSC lines derived from patients with inherited diseases, this protocol can be used to study the disease mechanisms of cardiac malformations at an early stage of embryogenesis.
Cardiovascular disease is the leading cause of death worldwide. Achieving the next phase of potential treatment strategies and better prognostic tools will require a concerted effort from ...interdisciplinary fields. Biomaterials-based cardiac tissue models are revolutionizing the area of preclinical research and translational applications. The goal of in vitro cardiac tissue modeling is to create physiological functional models of the human myocardium, which is a difficult task due to the complex structure and function of the human heart. This review describes the advances made in area of in vitro cardiac models using biomaterials and bioinspired platforms. The field has progressed extensively in the past decade, and we envision its applications in the areas of drug screening, disease modeling, and precision medicine.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Although adhesive interactions between cells and nanostructured interfaces have been studied extensively, there is a paucity of data on how nanostructured interfaces repel cells by directing cell ...migration and cell-colony organization. Here, by using multiphoton ablation lithography to pattern surfaces with nanoscale craters of various aspect ratios and pitches, we show that the surfaces altered the cells' focal-adhesion size and distribution, thus affecting cell morphology, migration and ultimately localization. We also show that nanocrater pitch can disrupt the formation of mature focal adhesions to favour the migration of cells towards higher-pitched regions, which present increased planar area for the formation of stable focal adhesions. Moreover, by designing surfaces with variable pitch but constant nanocrater dimensions, we were able to create circular and striped cellular patterns. Our surface-patterning approach, which does not involve chemical treatments and can be applied to various materials, represents a simple method to control cell behaviour on surfaces.
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IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, UL, UM, UPUK
Human organ-on-a-chip systems for drug screening have evolved as feasible alternatives to animal models, which are unreliable, expensive, and at times erroneous. While chips featuring single organs ...can be of great use for both pharmaceutical testing and basic organ-level studies, the huge potential of the organ-on-a-chip technology is revealed by connecting multiple organs on one chip to create a single integrated system for sophisticated fundamental biological studies and devising therapies for disease. Furthermore, since most organ-on-a-chip systems require special protocols with organ-specific media for the differentiation and maturation of the tissues, multi-organ systems will need to be temporally customizable and flexible in terms of the time point of connection of the individual organ units. We present a customizable Lego®-like plug & play system, μOrgano, which enables initial individual culture of single organ-on-a-chip systems and subsequent connection to create integrated multi-organ microphysiological systems. As a proof of concept, the μOrgano system was used to connect multiple heart chips in series with excellent cell viability and spontaneously physiological beat rates.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Drug discovery and development are hampered by high failure rates attributed to the reliance on non-human animal models employed during safety and efficacy testing. A fundamental problem in this ...inefficient process is that non-human animal models cannot adequately represent human biology. Thus, there is an urgent need for high-content in vitro systems that can better predict drug-induced toxicity. Systems that predict cardiotoxicity are of uppermost significance, as approximately one third of safety-based pharmaceutical withdrawals are due to cardiotoxicty. Here, we present a cardiac microphysiological system (MPS) with the attributes required for an ideal in vitro system to predict cardiotoxicity: i) cells with a human genetic background; ii) physiologically relevant tissue structure (e.g. aligned cells); iii) computationally predictable perfusion mimicking human vasculature; and, iv) multiple modes of analysis (e.g. biological, electrophysiological, and physiological). Our MPS is able to keep human induced pluripotent stem cell derived cardiac tissue viable and functional over multiple weeks. Pharmacological studies using the cardiac MPS show half maximal inhibitory/effective concentration values (IC₅₀/EC₅₀) that are more consistent with the data on tissue scale references compared to cellular scale studies. We anticipate the widespread adoption of MPSs for drug screening and disease modeling.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Organ-on-a-chip systems possess a promising future as drug screening assays and as testbeds for disease modeling in the context of both single-organ systems and multi-organ-chips. Although it ...comprises approximately one fourth of the body weight of a healthy human, an organ frequently overlooked in this context is white adipose tissue (WAT). WAT-on-a-chip systems are required to create safety profiles of a large number of drugs due to their interactions with adipose tissue and other organs
via
paracrine signals, fatty acid release, and drug levels through sequestration. We report a WAT-on-a-chip system with a footprint of less than 1 mm
2
consisting of a separate media channel and WAT chamber connected
via
small micropores. Analogous to the
in vivo
blood circulation, convective transport is thereby confined to the vasculature-like structures and the tissues protected from shear stresses. Numerical and analytical modeling revealed that the flow rates in the WAT chambers are less than 1/100 of the input flow rate. Using optimized injection parameters, we were able to inject pre-adipocytes, which subsequently formed adipose tissue featuring fully functional lipid metabolism. The physiologically relevant microfluidic environment of the WAT-chip supported long term culture of the functional adipose tissue for more than two weeks. Due to its physiological, highly controlled, and computationally predictable character, the system has the potential to be a powerful tool for the study of adipose tissue associated diseases such as obesity and type 2 diabetes.
Organs-on-a-chip possess a promising future as drug screening assays and testbeds for disease modeling in the context of both single-organ systems and multi-organ-chips.
Cell therapy offers much promise for the treatment of ischemic diseases by augmenting tissue vasculogenesis. Matrix-assisted cell transplantation (MACT) has been proposed as a solution to enhance ...cell survival and integration with host tissue following transplantation. By designing semi synthetic matrices (sECM) with the correct physical and biochemical signals, encapsulated cells are directed towards a more angiogenic phenotype. In this review, we describe the choice of cells suitable for pro-angiogenic therapies, the properties that should be considered when designing sECM for transplantation and their relative importance. Pre-clinical models where MACT has been successfully applied to promote angiogenesis are reviewed to show the great potential of this strategy to treat ischemic conditions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The activation of brown/beige adipose tissue (BAT) metabolism and the induction of uncoupling protein 1 (UCP1) expression are essential for BAT-based strategies to improve metabolic homeostasis. ...Here, we demonstrate that BAT utilizes actomyosin machinery to generate tensional responses following adrenergic stimulation, similar to muscle tissues. The activation of actomyosin mechanics is critical for the acute induction of oxidative metabolism and uncoupled respiration in UCP1+ adipocytes. Moreover, we show that actomyosin-mediated elasticity regulates the thermogenic capacity of adipocytes via the mechanosensitive transcriptional co-activators YAP and TAZ, which are indispensable for normal BAT function. These biomechanical signaling mechanisms may inform future strategies to promote the expansion and activation of brown/beige adipocytes.
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•BAT adrenergic stimulation induces an actomyosin-based mechanical response•Modulation of actomyosin responses alters oxidative metabolism in adipocytes•Thermogenic gene expression in adipocytes is in part regulated by YAP/TAZ
Tharp et al. show that brown adipocytes engage tensional actomyosin machinery, similar to muscle tissues, following adrenergic stimulation to mediate the thermogenic program and normal BAT function. These effects are mechanistically mediated through the YAP/TAZ pathway and, on a broad level, highlight the importance of cellular mechanics for cell metabolism.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Stem cell-derived β cells offer an alternative to primary islets for biomedical discoveries as well as a potential surrogate for islet transplantation. The expense and challenge of obtaining and ...maintaining functional stem cell-derived β cells calls for a need to develop better high-content and high-throughput culture systems. Microphysiological systems (MPS) are promising high-content
in vitro
platforms, but scaling for high-throughput screening and discoveries remain a challenge. Traditionally, simultaneous multiplexing of liquid handling and cell loading poses a challenge in the design of high-throughput MPS. Furthermore, although MPS for islet β culture/testing have been developed, studies on multi-day culture of stem-cell derived β cells in MPS have been limited. We present a scalable, multiplexed islet β MPS device that incorporates microfluidic gradient generators to parallelize fluid handling for culture and test conditions. We demonstrated the viability and functionality of the stem cell-derived enriched β clusters (eBCs) for a week, as assessed by the ∼2 fold insulin release by the clusters to glucose challenge. To show the scalable multiplexing for drug testing, we demonstrated the loss of stimulation index after long-term exposure to logarithmic concentration range of glybenclamide. The MPS cultured eBCs also confirmed a glycolytic bottleneck as inferred by insulin secretion responses to metabolites methyl succinate and glyceric acid. Thus, we present an innovative culture platform for eBCs with a balance of high-content and high-throughput characteristics.
Multiplexed microphysiological system as a high-content, higher throughput device for stem cell-derived β cell culture and drug screening.
Human induced pluripotent stem cell (hiPSC) derived angiogenesis models present a unique opportunity for patient-specific platforms to study the complex process of angiogenesis and the endothelial ...cell response to biomaterial and biophysical changes in a defined microenvironment. We present a refined method for differentiating hiPSCs into a CD31 + endothelial cell population (hiPSC-ECs) using a single basal medium from pluripotency to the final stage of differentiation. This protocol produces endothelial cells that are functionally competent in assays following purification. Subsequently, an in vitro angiogenesis model was developed by encapsulating the hiPSC-ECs into a tunable, growth factor sequestering hyaluronic acid (HyA) matrix where they formed stable, capillary-like networks that responded to environmental stimuli. Perfusion of the networks was demonstrated using fluorescent beads in a microfluidic device designed to study angiogenesis. The combination of hiPSC-ECs, bioinspired hydrogel, and the microfluidic platform creates a unique testbed for rapidly assessing the performance of angiogenic biomaterials.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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