Standardizing designed and emergent quantitative features in microphysiological systems.

Microphysiological systems (MPSs) are cellular models that replicate aspects of organ and tissue functions in vitro. In contrast with conventional cell cultures, MPSs often provide physiological mechanical cues to cells, include fluid flow and can be interlinked (hence, they are often referred to as microfluidic tissue chips or organs-on-chips). Here, by means of examples of MPSs of the vascular system, intestine, brain and heart, we advocate for the development of standards that allow for comparisons of quantitative physiological features in MPSs and humans. Such standards should ensure that the in vivo relevance and predictive value of MPSs can be properly assessed as fit-for-purpose in specific applications, such as the assessment of drug toxicity, the identification of therapeutics or the understanding of human physiology or disease. Specifically, we distinguish designed features, which can be controlled via the design of the MPS, from emergent features, which describe cellular function, and propose methods for improving MPSs with readouts and sensors for the quantitative monitoring of complex physiology towards enabling wider end-user adoption and regulatory acceptance.

Vascularized hiPSC-derived 3D cardiac microtissue on chip.

Functional vasculature is essential for delivering nutrients, oxygen, and cells to the heart and removing waste products. Here, we developed an in vitro vascularized human cardiac microtissue (MT) model based on human induced pluripotent stem cells (hiPSCs) in a microfluidic organ-on-chip by coculturing hiPSC-derived, pre-vascularized, cardiac MTs with vascular cells within a fibrin hydrogel. We showed that vascular networks spontaneously formed in and around these MTs and were lumenized and interconnected through anastomosis. Anastomosis was fluid flow dependent: continuous perfusion increased vessel density and thus enhanced the formation of the hybrid vessels. Vascularization further improved endothelial cell (EC)-cardiomyocyte communication via EC-derived paracrine factors, such as nitric oxide, and resulted in an enhanced inflammatory response. The platform sets the stage for studies on how organ-specific EC barriers respond to drugs or inflammatory stimuli.

Software Tool for Automatic Quantification of Sarcomere Length and Organization in Fixed and Live 2D and 3D Muscle Cell Cultures In Vitro.

Sarcomeres are the structural units of the contractile apparatus in cardiac and skeletal muscle cells. Changes in sarcomere characteristics are indicative of changes in the sarcomeric proteins and function during development and disease. Assessment of sarcomere length, alignment, and organization provides insight into disease and drug responses in striated muscle cells and models, ranging from cardiomyocytes and skeletal muscle cells derived from human pluripotent stem cells to adult muscle cells isolated from animals or humans. However, quantification of sarcomere length is typically time consuming and prone to user-specific selection bias. Automated analysis pipelines exist but these often require either specialized software or programming experience. In addition, these pipelines are often designed for only one type of cell model in vitro. Here, we present an easy-to-implement protocol and software tool for automated sarcomere length and organization quantification in a variety of striated muscle in vitro models: Two dimensional (2D) cardiomyocytes, three dimensional (3D) cardiac microtissues, isolated adult cardiomyocytes, and 3D tissue engineered skeletal muscles. Based on an existing mathematical algorithm, this image analysis software (SotaTool) automatically detects the direction in which the sarcomere organization is highest over the entire image and outputs the length and organization of sarcomeres. We also analyzed videos of live cells during contraction, thereby allowing measurement of contraction parameters like fractional shortening, contraction time, relaxation time, and beating frequency. In this protocol, we give a step-by-step guide on how to prepare, image, and automatically quantify sarcomere and contraction characteristics in different types of in vitro models and we provide basic validation and discussion of the limitations of the software tool. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Staining and analyzing static hiPSC-CMs with SotaTool Alternate Protocol: Sample preparation, acquisition, and quantification of fractional shortening in live reporter hiPSC lines Support Protocol 1: Finding the image resolution Support Protocol 2: Advanced analysis settings Support Protocol 3: Finding sarcomere length in non-aligned cells.

Advanced In Vitro Modeling to Study the Paradox of Mechanically Induced Cardiac Fibrosis.

In heart failure, cardiac fibrosis is the result of an adverse remodeling process. Collagen is continuously synthesized in the myocardium in an ongoing attempt of the heart to repair itself. The resulting collagen depositions act counterproductively, causing diastolic dysfunction and disturbing electrical conduction. Efforts to treat cardiac fibrosis specifically have not been successful and the molecular etiology is only partially understood. The differentiation of quiescent cardiac fibroblasts to extracellular matrix-depositing myofibroblasts is a hallmark of cardiac fibrosis and a key aspect of the adverse remodeling process. This conversion is induced by a complex interplay of biochemical signals and mechanical stimuli. Tissue-engineered 3D models to study cardiac fibroblast behavior in vitro indicate that cyclic strain can activate a myofibroblast phenotype. This raises the question how fibroblast quiescence is maintained in the healthy myocardium, despite continuous stimulation of ultimately profibrotic mechanotransductive pathways. In this review, we will discuss the convergence of biochemical and mechanical differentiation signals of myofibroblasts, and hypothesize how these affect this paradoxical quiescence. Impact statement Mechanotransduction pathways of cardiac fibroblasts seem to ultimately be profibrotic in nature, but in healthy human myocardium, cardiac fibroblasts remain quiescent, despite continuous mechanical stimulation. We propose three hypotheses that could explain this paradoxical state of affairs. Furthermore, we provide suggestions for future research, which should lead to a better understanding of fibroblast quiescence and activation, and ultimately to new strategies for the prevention and treatment of cardiac fibrosis and heart failure.

Engineered models of the human heart: Directions and challenges.

Human heart (patho)physiology is now widely studied using human pluripotent stem cells, but the immaturity of derivative cardiomyocytes has largely limited disease modeling to conditions associated with mutations in cardiac ion channel genes. Recent advances in tissue engineering and organoids have, however, created new opportunities to study diseases beyond "channelopathies." These synthetic cardiac structures allow quantitative measurement of contraction, force, and other biophysical parameters in three-dimensional configurations, in which the cardiomyocytes in addition become more mature. Multiple cardiac-relevant cell types are also often combined to form organized cardiac tissue mimetic constructs, where cell-cell, cell-extracellular matrix, and paracrine interactions can be mimicked. In this review, we provide an overview of some of the most promising technologies being implemented specifically in personalized heart-on-a-chip models and explore their applications, drawbacks, and potential for future development.