Cardiovascular Research (2021) 117, 2742–2754 INVITED REVIEW doi:10.1093/cvr/cvab088 Organ-on-a-chip technology: a novel approach to investigate cardiovascular diseases Valentina Paloschi 1,2, Maria Sabater-Lleal 3,4, Heleen Middelkamp5, Aisen Vivas 5,6, Sofia Johansson 7, Andries van der Meer 6†, Maria Tenje 7† , and Downloaded from https://academic.oup.com/cardiovascres/article/117/14/2742/6174689 by guest on 27 January 2023 Lars Maegdefessel 1,2,8*† 1 Department for Vascular and Endovascular Surgery, Technical University Munich, Klinikum Rechts der Isar, Munich, Germany; 2German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance, Berlin, Germany; 3Research Institute of Hospital de la Santa Creu i Sant Pau, IIB Sant Pau, Genomics of Complex Diseases Group, Barcelona, Spain; 4 Cardiovascular Medicine Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden; 5BIOS/Lab on a Chip, University of Twente, Enschede, The Netherlands; 6Applied Stem Cell Technologies, University of Twente, Enschede, The Netherlands; 7Department of Materials Science and Engineering, Science for Life Laboratory, Uppsala University, Uppsala, Sweden; and 8Molecular Vascular Medicine Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden Received 8 September 2020; editorial decision 21 February 2021; accepted 13 March 2021; online publish-ahead-of-print 17 March 2021 Abstract The development of organs-on-chip (OoC) has revolutionized in vitro cell-culture experiments by allowing a better mimicry of human physiology and pathophysiology that has consequently led researchers to gain more meaningful insights into disease mechanisms. Several models of hearts-on-chips and vessels-on-chips have been demonstrated to recapitulate fundamental aspects of the human cardiovascular system in the recent past. These 2D and 3D systems include synchronized beating cardiomyocytes in hearts-on-chips and vessels-on-chips with layer-based structures and the inclusion of physiological and pathological shear stress conditions. The opportunities to discover novel targets and to perform drug testing with chip-based platforms have substantially enhanced, thanks to the utilization of patient-derived cells and precise control of their microenvironment. These organ models will provide an important asset for future approaches to personalized cardiovascular medicine and improved patient care. However, certain technical and biological challenges remain, making the global utilization of OoCs to tackle unanswered questions in cardiovascular science still rather challenging. This review article aims to introduce and summarize published work on hearts- and vessels-on chips but also to provide an outlook and perspective on how these advanced in vitro systems can be used to tailor disease models with patient-specific characteristics. 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 Keywords Organs-on-chips • Cell culture • Cardiovascular • Heart • Personalized medicine .................................................................................................................................................................................................... .. .. interplay of numerous cell subtypes, including endothelial cells (ECs) 1. Introduction .. (becoming dysfunctional), leukocytes and macrophages (triggering .. Cardiovascular diseases (CVDs) are a group of disorders affecting the .. inflammation), and smooth muscle cells (which dedifferentiate or heart and the vasculature that represent the number one cause of .. undergo apoptosis).3,4 .. mortality globally.1 Only in Europe, CVDs causes over 4 million deaths .. Unstable atherosclerotic plaques can rupture, which results in arterial each year,2 accounting for 47% of all deaths in Europe. The most .. thrombosis. Thrombosis, the formation of blood clots, prevents blood .. common underlying pathology in CVDs is atherosclerosis, which causes .. flow, and triggers life-threatening clinical conditions in the arterial sys- an ischaemia in the heart and in peripheral arteries. Atherosclerosis is de- .. tem, such as myocardial infarction (MI) and ischaemic forms of stroke .. fined as a chronic disease of the vasculature, whose architecture is slowly .. (IS). Although MI and IS are usually acute events resulting from chronic remodelled over time. This disease and remodelling process involves the .. atherosclerotic processes affecting coronary and carotid arteries, † These authors contributed equally to this study. * Corresponding author. Tel: þ49 894 140 3490, E-mail: lars.maegdefessel@tum.de C The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology. V This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Organs-on-chips for cardiovascular research 2743 .. respectively, venous thromboembolism (VTE) is mainly caused by hae- .. whereas OoCs are typically multi-structural engineered systems for mostatic or coagulation abnormalities.5 .. on-chip cell cultures. .. Another severe result of tissue ischaemia is heart failure (HF), which is .. In the following pages, we introduce the general concept and scientific most commonly associated with coronary artery disease (CAD).6 Other .. potential of OoC systems in CVD research. We further discuss the .. complications of ischaemia include arrhythmias caused by discontinued .. opportunities and challenges of utilizing OoCs in preclinical drug testing oxygen supply to the cardiac conduction system. In particular long-term .. and target discovery. .. complications after an acute MI that triggers pathological myocardial .. remodelling remain unsolved and are a major cause for high re-hospitali- .. .. zation rates.7 Hallmark features of this cardiac remodelling process8 are .. 2. Methods excessive deposition of extracellular matrix (ECM) leading to cardiac .. .. OoCs are micro-engineered in vitro models that recapitulate aspects of hu- fibrosis, chamber dilation (dilated cardiomyopathy), and cardiomyocyte .. hypertrophy. Apart from ischaemia being the key inducer leading to HF, .. man physiology and pathology. They can be used in drug discovery as well as .. Downloaded from https://academic.oup.com/cardiovascres/article/117/14/2742/6174689 by guest on 27 January 2023 for efficacy and toxicology testing.21 OoCs are defined as microfluidic cell- several non-cardiac therapies can cause adverse reactions that induce a .. culture devices that contain continuously perfused chambers being inhabited similar disease phenotype.9 In particular, anti-cancer treatment strategies .. .. by living human cells arranged in a three-dimensional organization that pre- (radiation as well as chemotherapies) are particularly known for their .. serves and mimics the tissue geometry.22,23 The high level of control, enabling cardiotoxic potential. Here, the new field of cardio-oncology aims at .. .. customised cell-culture environment in OoCs, is illustrated in Figure 1. This improving our understanding of molecular and clinical alterations that .. includes custom ECM topology, the integration of sensors and actuators for cancer therapies generate in the cardiovascular system.10 .. monitoring and electrical/mechanical stimuli, control of microfluidic channel .. Our knowledge about etiopathogenetic mechanisms in CVD has .. dimensions, and temporal and spatial flow profiles for pulsatile flow and dramatically benefited from advances in ‘-omics’ technologies. A typical .. chemical stimuli. Moreover, the precise microfluidic flow control enables an .. pipeline to tackle unanswered disease research questions exploits disci- .. optimal growing environment (influx of nutrients and efflux of cell-waste) as .. well as the circulation of drugs, signalling molecules, or immune cells. The fab- plines such as genomics and transcriptomics to identify novel targets in .. .. rication methods used to realize OoC systems were originally developed for human cohorts as well as in vivo models to validate these findings. .. the microelectronics industry, providing methods to define microfluidic chan- Genome-wide association studies (GWAS) to investigate CAD,11 .. .. nels and compartments with dimensions from a few micrometres (mm) to VTE,12,13 IS,14 and HF15 have evaluated hundreds to thousands of individ- .. several millimetres (mm), thus matching the dimensions of real arteries, veins, uals and led to the identification of multiple genetic loci associated with .. and functional units of organs. the respective disease. With the availability of new technologies and the .. .. In order to fabricate vessel models, it is imperative to consider the geome- combination of genomic data utilizing publicly available expression data- .. try as this affects the flow profile, wall shear stress, culture area, and the total sets, the translation from genomic loci to the discovery of causal genes is .. number of cells used in the specific model. The material most commonly .. starting to become a reality. Functional assessments using in vitro modula- .. used for proof-of-concept models is poly(dimethylsiloxane) (PDMS), a poly- tion in cultured cells or in vivo animal models are considered irreplace- .. mer developed in the late 1990s 24 to fabricate microfluidic channels. PDMS .. able to identify the actual genes or variants that are relevant for causing .. has several advantages for miniaturized cell cultures, such as simple fabrica- the associations while trying to validate biological relevance. However, .. tion, gas permeability, and optical transparency. Using PDMS, supportive .. the translation of discoveries across species remains challenging: the .. microfluidic channels can be moulded off a master that has the reverse topo- poor sequence conservation of most non-coding genes (unlike protein- .. graphical features. By removing the PDMS from the mould and bonding the .. structure onto a glass slide, a sealed channel structure can be created coding genes) substantially limits the experimental studies of exiting can- .. didates, such as long non-coding RNAs or circular RNAs.16 Moreover, .. (Figure 2). Inlet and outlet holes for connecting tubing for perfusion can easily .. be punched into the PDMS. the human circulatory system, including its mechanical, electrical, .. biochemical, and cellular complexity, is hard to mimic. Human in vitro .. In order to form vessel mimics, the microfluidic channels can then be .. coated with human ECs, forming artificial intima layers of vessels in which models that consist of single types of cells cultured under static condi- .. blood, plasma, or other cells of interest (e.g. monocytes and platelets) can be tions on a plastic surface in two dimensions poorly represent our physio- .. .. perfused.25 Specific culture media with different added stimuli or drugs can logical constitution.17 These simplistic models lack the three-dimensional .. be added to the system, and real-time observation of the system under a mi- .. complexity of the tissue, the effect of flow, the cell–cell interaction be- .. croscope can be used to evaluate the effect of certain stimuli. At the same tween blood cells and the endothelium as well as the involvement of the .. time, variations in flow and shear stress can provide more information than .. ECM that characterizes vascular tissue in vivo. .. static cultivation in well plates. To study disease onset, systems with higher In recent years, organs-on-chips (OoCs) have emerged as powerful .. complexity, including 3D lumen-chips that incorporate the media layer .. populated by SMCs, are required. A recent example of a perfusable artery- new tools to fill the translational gap from animal models to human dis- .. ease, with a particular potential to even replace animal testing in the fu- .. on-chip was published by Cho and Park,26 where SMCs and human umbilical .. vein ECs (HUVECs) were co-cultured in a PDMS channel. In an attempt to ture.18 OoC technology will improve the modelling of organs or organ .. systems for healthcare research while immensely impacting the precision .. induce the proper morphology and orientation of SMCs, wrinkles were .. formed on the circular PDMS channel surface during the moulding as contact medicine approach.18 OoCs comprise systems integrating either 2D cell .. .. guidance, and the HUVECs were aligned by medial perfusion. cultures on permeable membranes or cells cultured in 3D hydrogel scaf- .. One major disadvantage of using PDMS is that the material is porous, folds. In this current review article, we are referring to OoCs as defined .. thus absorbing especially hydrophobic compounds, which can lead to false- by the EU project ORCHID.19 Within the scale of novel physiologically .. .. negative read-outs from drug-screening studies.27 Also, the polymer is not relevant in vitro models, organoids need to be mentioned at this point. .. inert, meaning that silicon will leach from the structure into the cell culture They will however not be covered in greater detail in this present re- .. .. environment.28 Developments of more inert yet biocompatible materials are view. Organoids are self-organized three-dimensional tissue cultures de- .. therefore currently a major focus within the field.29 riving from stem cells. They differ from OoCs especially in biological .. In an effort to make the vascular structure more biomimetic, one can de- .. complexity, displaying multi-cellular self-assembled constructs,20 . sign the system to include multiple microfluidic compartments where some 2744 V. Paloschi et al. Downloaded from https://academic.oup.com/cardiovascres/article/117/14/2742/6174689 by guest on 27 January 2023 Figure 1 Fabricating heart-on-chip and vessel-on-chip models using micromachining allows for integration of several advanced features. .. can be filled with a biomimetic cell-culture scaffold to recreate the physiolog- .. Günther et al.42 They used a microfluidic platform for immobilizing small ar- ical environment of the vasculature.30–33 Alternatively, one can mould the .. teries obtained ex vivo from mice and long-term culturing under physiological complete device in a biological material such as collagen.34,35 Cells can fur- .. conditions (37 C, 45 mmHg transmural pressure). Live imaging allowed .. ther be introduced into the biomimetic scaffolds. This set-up allows investiga- .. them to determine the arteries’ inner and outer diameter in real time while tion of the tissue-blood interface in a controlled environment that cannot be .. assessing the effects of heterogeneous environmental changes on the micro- .. monitored in animals or patients. It can also be utilized to study the interac- .. vascular structure and function. tion with adjacent cells on disease sub-phenotypes, such as endothelial per- .. Cardiac models are often realized using the multichannel approach described meability, communication with blood cells, or platelet aggregation. Another .. .. above, as it provides the possibility to compartmentalize the different domains approach to fabricate vessel models in a biomimetic material is to utilize the .. of the cardiovascular system. This unprecedented modularity has led to the de- method of viscous fingering,36 thus forming co-centred channels instead of .. velopment of different heart-on-chip models that have focused on specific car- .. adjacent ones. The approach also has the advantage of generating circular .. diovascular subdomains of great interest to cardiovascular researchers. The vessel structures, which cannot be obtained via conventional PDMS mould- .. heart models can be fabricated to include multiple channels that are aligned in .. ing. The method has been used to study cell–cell interactions in 600 mm .. parallel either along the horizontal axis 43–45 or the vertical axis.46–48 The choice wide37 and 250 mm38 diameter vessel models. Although very interesting from .. of layout is often related to the assays used, where horizontally structured chan- a biological perspective, there are several technical challenges with integrat- .. nels, for example, allow for easy optical access of the separate compartments. .. ing biomimetic scaffolds into OoC systems, which have been discussed in .. There are also several reports of 2D-based cardiac models,46,47 for exam- more detail elsewhere.39 .. ple developed with an electrophysiological focus due to the increased sim- .. One option to exploit the angiogenic potential of the cells themselves is .. plicity of integrating planar electrodes with cell cultures. These systems are to increase the biological relevance of the vasculature models even further. .. often fabricated in more robust materials, such as silicon- or glass-coated, .. In such systems, vascular cells are seeded in the biomimetic scaffold and ex- .. with fibronectin, that enhance cellular adhesion. posed to mechanical stimuli via slow perfusion40 or a chemical gradient of .. Three-dimensional models, on the other hand, are more commonly found growth factors.41 This results in a vascular bed formation after 2–3 weeks, in- .. in models that mimic multi-organ systems or three-dimensional aspects that .. cluding both larger macro-vessels and dense capillary microvascular net- .. 2D models fail to recapitulate, such as the force of contraction measure- works. Taking a completely different approach to investigating small and .. ments and maturation of the co-culture micro-tissues. Such models include .. large artery diseases, one can utilize the advantages of microfluidics by devel- .. mechanically moveable parts, such as suspended membranes49,50 or cantile- oping advanced ex vivo culture platforms. They can be used if a higher com- .. vers structures51 that expose the encapsulated cardiac cells to mechanical .. stimulation during the culture. plexity is needed to investigate, for example, cell–cell interactions and their .. relevance for the onset of a certain disease. An initial attempt to investigate .. A fabrication method that is rapidly gaining attention, both for realizing structural changes occurring within the vessel wall was presented by .. vessels and heart constructs, is ‘bioprinting’, a type of additive manufacturing Organs-on-chips for cardiovascular research 2745 Downloaded from https://academic.oup.com/cardiovascres/article/117/14/2742/6174689 by guest on 27 January 2023 Figure 2 Schematic drawing showing the six basic steps of PDMS moulding to form a microfluidic channel that can be used in organs-on-chip. 1—A master is prepared having the inverse topography of the final channel structures, 2—PDMS pre-polymer is poured onto the master and polymerised upon heat treatment, 3—The moulded channels are released from the master, 4—Holes for connecting tubing for media perfusion are prepared in the PDMS by punching, 5—The PDMS surface is activated for bonding via plasma treatment, 6—The microfluidic channels are sealed by bonding the PMDS slab onto a glass microscope slide which may include patterned electrodes. or ‘3D printing’. In bio-printing, hydrogel cell suspensions are directly ex- ... a-actinin, connexin,43 and cardiac troponin-T.60 In addition to external elec- .. truded onto a substrate, building up the final biological structures layer-by- .. trical stimulation, maturation schemes that rely on external mechanical43,61 layer. Advantage of bioprinting is that the final device can include a high level .. and biochemical62 cues—or a combination of the aforementioned50,57— of topographical complexity and that multiple cell and material combinations .. have also been presented. .. can be realized. Bio-printed models of vascular networks52 have been .. The main use for microfabricated integrated electrodes in heart-on-chip reported, and recently, proof-of-principle of a drug toxicity screening heart- .. systems is, however, for on-chip read-out of electrophysiology. Here, the ion .. on-chip model using bioprinted cardiac cells was demonstrated.53 Although .. currents of the cells are measured extracellularly as changes in the field po- bioprinting is a rapidly developing technology, the challenge with interfacing .. tential. The extracellular field potential is closely linked to the QT interval .. through the corrected field potential duration.63 Multiple localized measure- bioprinted heart models with vascular models for controlled media perfusion .. still remains. .. ments of single cells enable assessment of the synchronization of beating cells .. and wavefront propagation. As demonstrated in open-format cell cultures, .. 2.1 Integrated electrodes for cellular .. cells can be cultured on top of high-density electrodes, so-called microelec- .. trode arrays (MEAs) for read-out of cellular electrophysiology.64–66 stimulation and read-out .. Customized MEAs can fit in virtually any microfluidic system and have been The inclusion of electrodes in cell-culture systems assist in producing highly .. .. reported for several OoC systems.46,54,67 controlled environments and allow for continuous read-out of parameters .. Normally, very close contact between the cells and the MEA is desired for essential to identifying cell behaviour. Various materials can be used for the .. .. optimal resolution and signal strength, resulting in cell culture on hard flat fabrication of the electrodes, including bio-friendly metals such as gold44 and .. surfaces and 2D cell models. To make the culture environment more biomi- platinum,46,54 and organic conducting materials, such as carbon55 or poly .. .. metic, Kujala et al.64 cultured cardiomyocytes on a 100-mm-thick micro- (3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT: PSS).56 .. grooved gelatin layer attached to the MEA and showed that the electrophysi- For the realization of reliable heart-on-chip models, it is very important to .. ology still could be mapped, although no longer with single-cell resolution. .. support the development of cells with a mature electrophysiological conduc- .. Other approaches that address the issue of 2D culture on hard surfaces are tion system, such as synchronized beating. Although some cultures of cardio- .. non-contact MEA measurements as explored by Sharf et al.68 and patterning .. myocytes show spontaneous synchronized beating already after a few days of MEAs on a soft PDMS substrate as demonstrated by Gaio et al.57 of culture even without external interactions,44,54,57 an effective method to ... .. Electrical sensors can also be integrated to monitor cell contraction. Quin induce synchronised beating in immature cultures is to assist the synchroniza- .. et al.65 have demonstrated this in an open-top structure, where interdigitated tion of the beating by using external electrical stimulation58,59 (Figure 2). .. electrodes were pattered for cell-contraction measurements in combination .. Typically, the cells are exposed to trains of electrical pulses similar to the .. with MEAs to monitor beating. Although highly correlated in normally func- electrical signalling of native cardiomyocytes. Often, this is achieved via large .. tioning cardiac tissue, contraction and electrophysiology are two different .. external electrodes, but more user-friendly custom systems have been devel- .. mechanisms that are important to follow in heart-on-chip models. oped using integrated electrodes on-chip.44,60 It has been shown that matura- .. Alternative ways to map cell contraction is to integrate mechanical sensors tion of cells may be improved by pacing the cells as proved by expression of .. into the heart model,54,66 or to utilize an external optical read-out with 2746 V. Paloschi et al. Downloaded from https://academic.oup.com/cardiovascres/article/117/14/2742/6174689 by guest on 27 January 2023 Figure 3 Heart-on-chip devices can recapitulate cardiac functions in vitro and integrate sensing units to monitor the cells in culture, e.g. action potential. Examples of cardiovascular diseases can be found in these devices, such as ischaemia and cardiac fibrosis. Integrated electrodes and mechanical actuation allow to monitor and stimulate the cells in culture, better recapitulating the cardiac microenvironment. .. computational motion tracking,62 or mapping the transient intracellular cal- .. systems (Figure 3). Heart-on-chip models aim to recapitulate the intricate cium signal using cells modified to include GCaMP6.44 .. conditions of the microenvironment that cells would experience in the heart. .. Nonetheless, the ability to include 3D cell cultures in these devices enables Integrated electrodes are also interesting for vessel models, as they can be .. used to assess the barrier integrity of the cultured cells via trans-endothelial .. an increase in their biological complexity. Control over the microenviron- electrical resistance (TEER) measurements. Most commonly, this method .. ment and the cultured cell types permits the recreation of relevant aspects .. works by integrating electrodes on either side of a porous membrane on .. of a specific disease (Table 1). Among the different reported heart-on-a-chip which the ECs are cultured. As the cells form tight junctions, it becomes in- .. platforms, models to address cardiac ischaemia, cardiac fibrosis, and cardio- .. toxicity can be found in the literature (Figure 3), whereas many other exiting creasingly more difficult for any electrical current to flow through the cell .. layer, and the measured electrical resistance increases. It is possible to com- .. approaches have been developed and summarized in further review .. articles.62,72–77 bine electrophysiological measurements and TEER as demonstrated by Maoz .. et al.,46 using a dual-channel, endothelialized heart-on-chip model. Further, .. 3D tubular vessels may incorporate TEER sensing capability by insertion of .. 2.2.1 Ischaemia .. electrodes inside the vessel and in a surrounding hydrogel matrix.69 .. The abrupt disruption of blood flow in ischaemia leads to the accumulation It may be noted that most models with integrated electrical sensing capa- .. of metabolic by-products while reducing the oxygen supply to the tissue. .. bilities are two-dimensional, which is explained by the well-established tech- .. This locally affects the contraction of cardiomyocytes. Liu et al.67 were able nology to form electrodes on hard 2D surfaces. However, electrodes can .. to replicate the hypoxic microenvironment and follow the action potential .. also interface more in vivo-like cardiac microtissue as in the case of Weng .. changes over time in the cell culture using patterned electrodes in a heart- et al.44 An increase in the number of publications on this topic is expected. .. on-a-chip device. The combination of the microenvironment cues, along Further, the hydrogel scaffold may be topologically patterned on top of the .. with the insights gained from the electrophysiology of the cells depicts the .. electrodes,64 or the electrodes themselves can be structured into 3D for- .. solid control that can be attained with these devices. mats.60,70 Alternatively, conducting and biocompatible scaffolds can be pre- .. .. pared, thus enabling electrical read-out via the porous scaffold itself.71 .. 2.2.2 Cardiac fibrosis .. In the scope of cardiac fibrosis, Kong et al.49 were able to recreate the in- .. 2.2 Heart-on-chip disease models .. creased ECM stiffness using a photopolymerizable hydrogel while including Heart-on-a-chip models developed so far have focused on establishing biomi- .. the mechanical load similar to the stimulus that cardiac fibroblasts would ex- .. metic, functional aspects of the heart, focusing in particular on the co-culture .. perience under pro-fibrotic conditions. The cyclic mechanical loading, along of multiple cell types, for example, cardiomyocytes and cardiac fibroblasts as .. with the exposure to a biochemical stimulus like transforming growth factor well as on the electromechanical stimulation of the cells cultured on these .. b (TGFb) can even more closely mimic the fibrotic microenvironment of the Organs-on-chips for cardiovascular research 2747 Table 1 Summary of the heart-on-chip platforms. Aspects of human cardiac physiology and disease in organs-on-chips .............................................................................................................................................................................................................................. 43,48,61,62,64,135 Cardiac physiology Defined 3D tissue organization 51,54,130,135 Force of contraction 43,46,57,62,64,65,67,135 Electrophysiology 44,46,62,135 Cardiac-vascular interactions 54,130,135 Body-on-chip approach 61 Cardiac disease and toxicity Hypertrophy 47,49 Arrhythmia 67 Ischaemia Downloaded from https://academic.oup.com/cardiovascres/article/117/14/2742/6174689 by guest on 27 January 2023 47,49 Fibrosis (e.g. fibroblast proliferation, collagen deposition, and valve calcification) 46,135 Inflammation 43,44,46,51,54,61,62,64,65,130,135 Cardiotoxicity & Pharmacology The selection criteria employed in this table were that the devices used in the study could be considered a heart-on-a-chip device, i.e. a microfluidic device where the microenviron- ment of the cells or tissue in culture can be controlled and/or be stimulated mechanically and/or electrically. Platforms where constructs are cultured in well-plates or make use of spheroid technology were not considered due to the lack of their microfluidic character, which is seen as a requirement for organs-on-chips. .. heart. There have also been non-microfluidic devices that make use of 3D .. electrophysiology, metabolism, and morphology, to name a few. Unveiling cardiac tissue models to mimic hallmarks of cardiac fibrosis using human .. what mechanisms underlie iPSC-derived cardiomyocytes maturity is an active .. induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) along .. field of research and is reviewed elsewhere.81 The models created with these with fibroblasts. Mastikhina et al.78 reported a model where expression of .. cells naturally inherit their limitations. There are examples of initiatives that collagen and brain natriuretic protein were upregulated when tissues .. aim to characterize iPSC-CMs and their response to pharmacological agents .. were exposed to TGF-b, a pro-fibrotic agent, and downregulated when .. with a known response as an attempt to benchmark different cell sources, subsequently exposed to an anti-fibrotic drug. Wang et al.35 used cardiac .. such as the CiPA initiative82 and the Pulse CRACK-IT83 project. In the future .. fibroblast overpopulation of the tissues to mimic a fibrotic scenario, thus .. development of heart-on-chip models, the drugs used for benchmarking in avoiding the pleiotropic effects of TGF-b. These two methods could be .. the mentioned initiatives can be used as a reference for new devices. With .. combined into microfluidic devices in additional adjacent compartments .. the aim of generating a model where the response of cardiac tissues exposed where, ECs can be integrated, and more complex, integrative models .. to libraries of compounds could determine the drug used, Lee et al.84 used a be made. .. .. machine-learning model for the drug-response analysis. This clearly .. demonstrates that heart-on-chip development is a multidisciplinary field, .. 2.2.3 Cardiotoxicity .. where pharmacologists, engineers, biologists, regulators, and clinicians, Interestingly, the vast majority of heart-on-chip devices reported in literature .. among others, play a key role in the development of these models and their .. state cardiotoxicity as their primary goal. Although cardiotoxicity encom- .. respective validation. passes a myriad of aetiologies, two common trends can be found in these .. devices: (i) an investigation of the toxic effects that specific drugs have on car- .. .. 2.3 Studying atherosclerosis using chip-based diac cells and (ii) the toxic effect produced by the co-cultured cells from .. other organs (e.g. liver). The highly controlled environment of the heart-on- .. systems .. Modelling the different stages of the advancement of atherosclerosis is con- chip devices makes it a highly appealing platform for drug toxicity studies, as .. evidenced by a large number of devices with this as its motivation of the .. sidered crucial in the development of vascular OoCs (summarized in Table 2 .. and Figure 4). Qiu et al.25 developed a 3D microvasculature-on-chip that is design. .. The highly controlled microenvironment in heart-on-chip models makes .. able to display physiological endothelial barrier function for several weeks, them very useful for modelling diseases. There is plenty of room to study dis- .. therefore allowing to study how chronic endothelial dysfunction develops. .. To model atherosclerosis progression, several devices have been created ease mechanisms that cannot be dissected in common platforms such as cell .. culture and animal models. However, one advantage of animal models com- .. with occlusion or stenosis of the lumen, which recreates the higher shear .. region commonly found in developing atherosclerotic plaques.38 pared with heart-on-a-chip models is the cross-talk between different organs, .. which are typically involved in the chronic diseases that lead to CVD.79 A ma- .. Westein et al.,85 Tovar-Lopez et al.,86 and Costa et al.87 have provided .. excellent examples of how to investigate atherosclerosis using chip-based jor challenge in developing heart-on-a-chip disease models would be the diffi- .. culty of integrating multi-organ co-cultures for longer periods of time, .. systems. These three models differ regarding their biological and technical typically around 1 week–7 days—after tissue formation. Nonetheless, .. complexity. The chips developed by Westein et al. and Tovar-Lopez et al. .. aspects of complex chronic diseases can still be modelled with OoCs, e.g., .. consist of a square channel in which an artificial atherosclerotic plaque is al- cardiac fibrosis, as mentioned above. .. ready embedded (Figure 4). In order to study 3D vessel geometry in a more .. Naturally, the aforementioned models make use of cardiac cells, a scarce .. physiologic way, Costa et al.87 created different 3D vascular structures by resource since primary cardiomyocytes proliferate poorly in vitro. With the .. using 3D-printed anatomical models based on observations generated .. advent of human iPSC-CMs, this major cell source bottleneck is gradually be- .. by computed tomography angiography. They were, therefore, able to ing overcome. However, hurdles still remain to improve their immaturity, .. closely mimic architectures found in both healthy and stenotic blood vessels. .. A disadvantage of these models is the lack of the ECM, as PDMS is respon- which ultimately affects the pharmacological response, with oncotherapy .. effects80 and the biomimetic aspects of the OoCs that employ human iPSC- .. sible for creating the lumen that directly surrounds the cells. If the occlusion CMs. Cardiac cell maturity can be defined in several aspects such as .. mechanism and pathophysiology of the development of atherosclerosis are 2748 V. Paloschi et al. Table 2 Summary of vessel-on-chip platforms. Aspects of human vascular physiology and disease in organs-on-chips .............................................................................................................................................................................................................................. 37,38,87,93,97,139,140 Vascular physiology Perfusable 3D blood vessels with defined geometries 31,141 Perfusable microvasculature with self-organized geometries 31,34 Angiogenesis 31,34,37,139 Endothelial-mural interactions 34,85,87,94,98,99 Blood perfusion 34,85,87,93,94,97–99 Vascular disease modelling Thrombosis 31,34,37,85,93,94,97,98,139 Inflammation (e.g. permeability and adhesion molecules) 31,34 Immune cell recruitment Downloaded from https://academic.oup.com/cardiovascres/article/117/14/2742/6174689 by guest on 27 January 2023 Vessel-on-chip devices included in this table consist of different types of perfusable blood vessels modelling human vascular physiology as well as pathophysiology. Figure 4 Vessels-on-chips devices are useful tools to study pathological mechanisms occurring within the vessel wall in early and later stages of athero- sclerosis. Preliminary research can be performed in easier fabricated straight PDMS channels, whereas for more extensive and complicated research questions, a more elaborate model can be used by producing a 3D lumen in a hydrogel. .. to be studied, a more flexible model in which monocytes and macrophages .. technology has therefore been used extensively to create new in vitro models can be included would be highly appreciated. .. to study thrombotic diseases.90–94 .. Although previous work has been published studying thrombosis on .. 2.4 Mimicking thrombosis on-a-chip .. microfluidic devices lacking ECs (reviewed in Westein et al.91 and Zhu Thrombosis is a complex process influenced by genetic and environmental .. et al.92), we here review OoC models of thrombosis typically consisting of a .. factors, involving three main components: abnormalities in the vessel wall .. tubular or rectangular mould that is covered by components of the ECM (endothelium), abnormalities in components of the blood (coagulation pro- .. (typically fibrin or collagen). ECs form a monolayer, therefore recreating en- .. teins and platelets), or abnormalities in fluid dynamics (turbulence flow and .. dothelial geometry and function. In normal healthy conditions, the endothe- shear stress).88,89 Unlike static in vitro cell-culture models, vessels-on-a-chip .. lium has anticoagulant and anti-inflammatory properties, therefore allowing can mimic the effect of flow and its interaction with the vessel wall while .. blood flow through the lumen and preventing platelet activation and fibrin .. overcoming inter-species differences of animal models. Microfluidic . clot formation.95 Upon endothelial damage or activation, coagulation and Organs-on-chips for cardiovascular research 2749 platelet aggregation are triggered to promote the formation of a thrombus.96 .. to promote blood clots. These studies serve as interesting examples for the .. Besides endothelial damage, several stimuli and the effect of blood flow are .. potential of vessels-on-chips to evaluate thrombotic side effects that would known to activate the endothelium. .. otherwise be missed in prior animal or static cell culture studies. They are .. Several examples of OoC have been published that study the different .. further proof of how OoCs can enhance drug safety during the process of triggers of clot formation through direct monitoring of platelet aggregation .. developing novel treatment strategies. .. under a microscope. In this direction, Zheng et al.34 engineered microvascu- .. lar networks by seeding HUVECs intro microfluidic circuits coated with col- .. 2.5 Future importance of organs-on-a-chip in .. lagen and demonstrated that upon perfusion with human blood, rolling and .. personalized cardiovascular medicine adhesion of platelets occurred only at sites of damage—or in stimulated en- .. Animal and genetic studies have implicated specific genes conferring in- dothelium where long fibres of von Willebrand factor (VWF) covered the .. .. creased susceptibility to many human diseases. Together with other emerg- surface of the activated endothelium. These structures had not been .. ing techniques (e.g. genome editing), OoCs have the potential to significantly reported in previous planar cultures or mouse models of thrombosis. .. .. enhance our understanding of how certain genes and epigenetic regulators Similarly, a recent study by Brouns et al.94 explored the anticoagulant effect .. are capable of influencing our personal CVD risk. Downloaded from https://academic.oup.com/cardiovascres/article/117/14/2742/6174689 by guest on 27 January 2023 of intact endothelium (created by a microfluidic model coated with .. HUVECs) through natural anticoagulants while comparing with artificially .. The novel genome editing CRISPR/Cas9 technology enables the introduc- .. tion of targeted mutations or specific gene knock-outs104 into human cells. damaged endothelium covering a highly thrombogenic surface created with .. This methodology has allowed the generation of stable cell lines carrying the collagen and tissue factor. .. .. desired genetic mutation/knock-out while eliminating the effect of inter-indi- One of the key advantages of OoCs is their great capacity to tightly con- .. vidual variations due to the genetic background. trol vessel geometry and flow, which has contributed significantly to the un- .. .. Although primary cells from specific individuals can be difficult to obtain derstanding of flow-based changes on platelet activation and the risk of .. and cannot be cultured indefinitely, stable cell lines with the desired genomic thrombosis. To better understand the contribution of atherosclerosis and .. background can be generated from human iPSCs. Human iPSCs are obtained vessel stenosis to thrombosis, stenotic vessels perfused with whole human .. .. from a somatic cell and can be differentiated into all cell types.110,111 They blood have been developed. The results indicate that platelets aggregate at .. can therefore be derived from accessible adult tissues of any patient, such as the outlet zone of constriction, therefore concluding that the shear rate is .. .. the blood or the skin. There are current protocols available for the genera- crucial for platelet adhesion and aggregation.85,87,90,97 .. tion of cardiomyocytes,112 defined atrial and ventricular cardiomyocyte sub- Although these examples clearly contribute to advancing research and un- .. .. types,113 ECs as well as pericytes and vascular SMCs.114,115 An important derstanding of the coagulation process, most of these models are not practi- .. limitation of iPSC-CMs relates to their immaturity. In fact, they share more cal to enhance clinical diagnosis. Several commercial devices are emerging .. similarities with foetal than adult human CMs. Although human iPSC-CMs with the idea to provide simple tools that can be used for clinical testing. .. .. express high levels of cardiac-specific genes and display a striated pattern for Towards this aim, Mannino et al.97 proposed a commercial simple endothe- .. a-actinin and myosin light chain similar to the adult ventricular myocardium, lial-coated cylindrical microchannel to test the effect of local vascular geome- .. .. their shape is typically roundish (and not elongated), whereas their cell body tries on blood cell-endothelium interactions. One year later, Jain et al.98 .. is smaller.111 In addition, the intrinsic immaturity of iPSC-CMs is reflected by demonstrated that a microfluidic device coated with ECs could be fixed and .. electrophysiological impairments,111 which constitute a valid criticism when .. still retain their ability to modulate haemostasis under flow. The device was .. employing these cells for the investigation of arrhythmias. Various methods able to detect differences in patients taking antiplatelet medication and there- .. to enhance maturity are constantly developed and refined, including expo- fore providing a new tool for clinical laboratories for coagulation testing in .. .. sure to electrical stimulation, application of mechanical strain, and culturing more robust and practical diagnostic assays. .. human iPSC-CMs in three-dimensional tissue configuration.112 A great Mathur et al.99 created a model for thrombo-inflammation where they first .. advantage of the iPSC differentiation protocols is that they allow for patient- .. isolated blood outgrowth endothelial cells (BOEC) from healthy subjects .. specific iPSC-derived systems that can be conveniently edited to test the and then introduced these cells to microfluidic channels. These cells were .. effect of specific disease-related mutations.113,114 The aforementioned .. then stimulated with TNFa and exposed to re-calcified human whole blood .. gene-editing can be used to restore the effect of specific genes or mutations to compare the inflammatory response of these cells to models using .. while exploring genotypic effects on specific phenotypes. HUVECs. Due to the easy isolation method of these BOECs, and a different .. .. The opportunity to build OoC with disease-relevant cells can minimize response of healthy and unhealthy patients, this model could be used as a .. the current challenges of most genomic studies. For example, vessel- and tool in personalized medicine approaches for certain pathologies, in which .. heart-on-chip designs could be used to test whether genetic associations .. thrombo-inflammation is an essential contributor to disease exacerbation. .. emerging from recent CAD, VTE, HF, or IS GWAS will increase the disease Sepsis is a disease often coinciding with thrombus formation in smaller .. risk through an effect in the cardiovascular system. At the same time, blood .. vessels and arteries, which can lead to ischaemic events of the heart and sub- .. from individuals with a known genetic background can be perfused into sequent HF.100 Many of the models described above can be used to model .. different microchannels to study interactions between blood cells and the thrombotic sepsis in a microfluidic chip. In sepsis, high levels of inflammation .. endothelium. Overall, the advanced possibilities of OoCs using novel avail- .. in blood vessels are observed and accompanied by an elevation of TNF-a, .. able technologies offer great potential to finally elucidate the effect of genetic interleukin-1, and interferons. These factors activate the endothelium to a .. factors on CVD phenotypes, therefore enabling us to more rapidly and effi- .. pro-thrombotic state and strongly induce thrombus formation.101,102 After .. ciently move towards the era of personalized medicine and pharmacoge- incubation with these factors, or other known contributors to sepsis, a blood .. nomics. OoCs have already demonstrated their potential to support clinical .. perfusion assay can be performed to better understand more mechanisms .. trials of certain drug candidates.120 The opportunity to create patient-specific involved in sepsis-associated thrombosis. .. organ-on-chips could therefore pave the way for the development of precise Finally, clinical applicability has also been demonstrated by using vessels- .. and individualized therapies. .. on-chips to predict thrombotic side effects in drug candidates prior to human .. It is essential to mention that the majority of advances in personalized clinical trials. For example, Tsai and collaborators used a vessel-on-chip de- .. medicine stem from organoids-based research. In this context, Atchinson .. vice to perfuse blood samples from patients with sickle cell disease and test .. et al.116 have developed micro-engineered blood vessels based on SMCs de- the effect of certain drugs in microvascular occlusion and thrombosis.93 .. rived from human iPSC of Hutchinson–Gilford progeria, a rare accelerating Another study by Barrile et al.103 used a vascular channel coated with ECs .. aging disorder that causes early onset of atherosclerosis. The work .. and perfused whole human blood to study the potential for different drugs . highlighted the possibility of the in vitro system to recapitulate the key features 2750 V. Paloschi et al. of the disease and how to utilize it as a patient-specific drug testing device. .. .. complex, multi-layered, and interconnected tissue architectures remain More recently, self-assembled 3D blood vessel organoids have been gener- .. impossible with the current engineering approaches utilised to build ated from human iPSCs. Blood vessel organoids are made of endothelial net- .. .. OoCs. However, the possibility to combine biological self-assembly ca- works and pericytes that are genetically identical and can recapitulate the .. pabilities of organoids with the controllable assembly of microfabricated formation of a vascular lumen and basement membrane deposition.117 .. However, it is not yet possible to mimic blood flow perfusion in these models .. OoCs represents an attractive advancement of both technologies. This .. has been demonstrated by a novel microphysiological model of the hu- and therefore to obtain functional parameters, such as permeability or .. immune-cell adhesion/extravasation under defined in vitro conditions. This, as .. man retina derived from hiPSCs incorporated in a two-channel chip sep- .. arated by a thin porous membrane mimicking the endothelial barrier and aforementioned, is one of the major advantages of OoC systems. .. Moreover, several studies showing the integration of gene-editing technol- .. enabling the exchange of nutrients and metabolites.136 .. ogy and organoids culture systems have been published.118,119 An example .. An important challenge for cardiovascular OoC platforms is the ability was presented in a cystic fibrosis organoid model, where mutations in CFTR .. to reproduce the chronic aspect underlying the progression of CVDs. gene were repaired in intestinal stem cells by CRISPR/Cas9 system.120 .. Animal models in this respect are still somewhat irreplaceable; however, .. Downloaded from https://academic.oup.com/cardiovascres/article/117/14/2742/6174689 by guest on 27 January 2023 Genome editing in iPSC-derived organoids have also been proved useful for .. investigators can benefit from OoCs to study a specific mechanism and drug testing,121 or to study the effect of virus infections.122,123 Very recent .. .. subsequently refine the targets to be evaluated in further animal models. work in organoids made from iPSCs have shown that SARS-CoV-2 can infect .. The field of OoC is growing, and an increasing number of academic engineered human blood vessel organoids and leak out into the blood- .. stream124 and that the infection could be inhibited by human recombinant .. groups and companies are becoming active in the development of OoC .. systems.19 All these OoC systems have different layouts and interfaces, soluble angiotensin-converting enzyme 2 (ACE2). .. Few examples of personalized medicine accompanied by OoCs have been .. which prevents them from being easily connected, interchanged, or .. compared. This lack of common standards for OoC systems is consid- published in the CVD field.125,126 Wang et al.127 created a heart-on-a-chip .. using genetically engineered iPSC to proof that contractile deficiencies in car- .. ered to be one of the major challenges in their future development and diomyocytes associated with Barth syndrome were caused by a mutation in .. implementation.137 Initiatives are currently emerging in the field to ad- .. tafazzin gene, thus elucidating the biological mechanism and providing poten- .. dress this challenge, for example, the development of a Translational tial therapeutic targets to treat the disease. Another interesting approach is .. Organ-on-Chip Platform, which aims to be an ‘open’ platform based on .. the integration of clinical data with the fabrication of OoC devices was used .. standards that are defined and supported by the stakeholders (develop- by Costa et al.,87 as described in detail in the atherosclerosis section above. .. ers, manufacturers, users) in the field (https://top.hdmt.technology/). Here, CTA data of a coronary artery formed the basis to construct personal- .. .. Standardization and harmonization are not only of importance for the ized chips with the measured grade of stenosis. This method allowed the .. .. technical aspects of an OoC but also for the cells and tissues that are in- researchers to reproduce in vitro the unique (personalized) flow profile of .. tegrated into the chips. Human cells are highly variable in terms of individual patients and to observe the formation of thrombi dynamically. .. Similarly, blood vessel-on-chips can be perfused with human blood from indi- .. growth, stability, and function, particularly when primary tissues or stem viduals treated with different anticoagulant therapies to assess drug response .. cells are used as a source. Mainly, users from the industry consider ac- .. of specific patients in a personalized manner.128 .. cess to human cell material, including patient material, to be a major chal- .. lenge.138 In the field of stem-cell technology, efforts are made to address .. .. this issue by setting up open databases of available human stem-cell lines, 3. Conclusion and outlook .. including their characteristics, sources, and restrictions to use (e.g. .. .. https://hpscreg.eu/). OoCs have advanced the drug development process by stimulating .. Overall, the use of OoC is rapidly moving from basic science to trans- scientists to increase the level of complexity of better mimicking human .. .. lational research to validate results from genomic studies and provide biology and physiology on a more systemic level. Further, the possibility .. better models for drug testing, paving the way for personalized medicine. to integrate more than one organ in the same model is an important and .. .. The combination of organ-on-chips with gene editing and iPSC use for on-going effort in the field.129–131 Microfluidic devices that contain the .. function of different organs have been connected with each other via .. better control of genetic background is becoming an innovative, attrac- .. tive alternative for the functional study of the cardiovascular system. vessels-on-chips to obtain the so-called ‘body-on-a-chip’. This concept .. captures the potential efficacy and toxicity of a drug in different .. .. organs.129,132 An important advancement in the validation of multi- .. Acknowledgements .. organs-on-chip usage in drug screening has been the integration of sen- .. We thank Susan Peacock (Uppsala University) for manuscript editing sors to ensure continuous assessment of the microenvironment parame- .. and support with the literature review. .. ters (pH, oxygen, and temperature).133 Kamei et al.134 have developed .. an integrated system composed by a healthy heart-on-chip connected to .. Conflict of interest: none declared. .. a liver cancer-on-chip and recapitulated the cardiotoxic effect of the .. anti-cancer drug doxorubicin. The side effect of the doxorubicin treat- .. .. Funding ment on cardiac cells was due to toxic metabolites produced in the liver .. cancer cells. .. MS-L is supported by a Miguel Servet contract from the ISCIII Spanish Health .. Institute (CP17/00142) and co-financed by the European Social Fund. MT Especially relevant for cardiovascular research are models that include .. and SJ have received funding from the European Research Council (ERC) un- both vasculature and the heart. This can be achieved either by building a .. .. der the European Union’s Horizon 2020 research and innovation programme microfabricated vascular network that cardiac tissue can be shaped .. (grant agreement No 757444) and the Knut and Alice Wallenberg around135 or by generating vascularized cardiac microtissues via co-cul- .. .. Foundation (grant number WAF 2016.0112). HM and AvdM acknowledge ture with ECs. The latter approach shows a higher biological relevance .. financial support from the Dutch Research Council (NWO) under the but comes with the challenge to interface the microtissue constructs .. Gravitation Grant ‘NOCI’ Program (Grant No. 024.003.001). AvdM .. with microfluidic circuits for controlled perfusion. To recapitulate . acknowledges funding received from the Dutch Cardiovascular Alliance Organs-on-chips for cardiovascular research 2751 (CVON-2018-29) and the European Research Council (ERC) under the .. discovery rate implicate new loci for coronary artery disease. Nat Genet 2017;49: .. Advanced Grant ‘VESCEL’ Program (Grant no. 669768). Research in LM’s .. 1385–1391. laboratories is supported by the European Research Council (ERC Starting .. 12. Lindström S, Wang L, Smith EN, Gordon W, Vlieg AH, de Andrade M, Brody JA, .. Pattee JW, Haessler J, Brumpton BM, Chasman DI, Suchon P, Chen M-H, Turman Grant no. 679777), a DZHK Junior Research Group (JRG_LM_MRI), the .. C, Germain M, Wiggins KL, MacDonald J, Braekkan SK, Armasu SM, Pankratz N, SFB1123 and TRR267 of the German Research Council (DFG), the National .. Jackson RD, Nielsen JB, Giulianini F, Puurunen MK, Ibrahim M, Heckbert SR, .. 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