Cardiovascular diseases (CVD) are the leading cause of mortality and substantially contribute to healthcare and economic burdens worldwide (1). Despite enormous funding investments and implementation of various drug development strategies, ninety percent of new drugs fails in clinical trials (2). Cardiovascular disease and cancer have the lowest success rates in discovering new drug candidates, largely due to adverse effects of the therapeutic agents that result in clinical and subclinical cardiotoxicity (3–6). Because the elucidation of disease pathogenesis and high-throughput screening are of paramount importance in the development of new drugs, various 2D and 3D cell model systems have been published over the past several decades (7–9). In the traditional setting, cells are routinely cultured in 2D format for the purposes of disease research and drug screening, but 2D models inherently lack the structural complexity of their in vivo counterparts (10–12).Therefore, intensive research efforts have been made to develop next-generation culture methods that mimic important features of native tissues, such as the cellular and acellular microenvironment and cell-cell interactions when performing 3D cell culture experiments in vitro. The rapidly evolving fields of organoid engineering and biological model systems are continually providing new insights into basic experimental biology, human disease mechanisms and drug response efficacy research and are driving new therapeutic innovations (13, 14). Cardiac organoids are organized structures that self-assemble into miniaturized models composed of progenitor cells, cardiomyocytes, endothelial cells, and fibroblasts in a three-dimensional microenvironment that mimic the in vivo native organs (15, 16). Unlike traditional 2D myocardial culture systems, cardiac organoids recapitulate the human-specific aspects of heart histogenesis, physiology and developmental trajectory. Therefore, cardiac organoids have grown in popularity because they provide a unique opportunity to model cardiac diseases drug screening, and toxicity testing (17, 18). They are considered as powerful gateway to understand cardiac functions not only from a basic science perspective but also for the development of personalized therapies (19, 20). Towards that end, a wide range of cardiac organoid models have been reported over the past few years, applying a variety of experimental strategies (21, 22). Nonetheless, progress in the study of cardiac organoids is much slower than that of its other counterparts including brain, liver, kidney, and intestinal models (20–25). This represents an opportunity for active research related to the biofabrication techniques for the engineering of different models of cardioids for translational regenerative medicine applications (23–28).

In this glance article, we begin with history of the development of organoid technology and the use of organ-specific biomaterials derived from decellularized hearts in culture for cardiac organoid research. We then summarized their applications as in vitro model systems and drug screening tools. Despite the tremendous potentials of organoids models, it is essential to understand the existing hurdles and limitations of organoid technology. Therefore, we highlighted the current challenges and respective advantages of cardiac organoid research for basic and translational applications.

The initial efforts to generate organs in vitro began with pioneering dissociation-reaggregation experiments. In these early studies, Henry Van Peters Wilson showed that sponge cells, when mechanically separated, have the ability to come together again and self-organize, ultimately forming a complete organism (29). Several decades after this initial experiment, multiple research teams conducted dissociation-reaggregation studies, successfully creating various types of organs from separated cells of the amphibian pronephros and chicken embryos (30, 31). However, the initial observation of in vitro tissue-like colonies formation occurred through the co-culture of keratinocytes and 3T3 fibroblasts (32). An important milestone in this journey was the development of the differential adhesion hypothesis, which was prompted by the observation that cells, upon mechanical dissociation and subsequent aggregation, could autonomously reorganize into the original tissue structures from which they were derived. This phenomenon underscored the inherent capacity of cells for spatial organization and tissue reconstruction. Advancements in stem cell biology further elucidated the potential of stem cells to differentiate and organize into organ-like structures in vitro, as evidenced by the formation of teratomas and embryoid bodies. These entities demonstrated the ability of differentiated cells to form arrangements mimicking various tissue types. From the end of the 19th century (1998) to the beginning of the 20th century (2006), intensive research on stem cell technology, especially human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), triggered a wave of studies on the mechanisms and fate of stem cells in different culture environments (33, 34). In 2009, Hans Clever and team found that individual intestinal stem cells expressing leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) stem cells produce crypt-villus structures in vitro, independent of mesenchymal niche (i.e., stroma), marking the creation of the first organoids. Their findings showed that LGR5+ stem cells are involved in rapid regeneration of intestinal tissue and can self-organize the intestinal crypt-villus units from a single stem cell without requiring a surrounding cell niche (35). Since then, a variety of organoid models have been reported by different research groups, including intestinal, brain, heart, kidney, and liver, among others (35–39). These studies laid the groundwork for numerous other studies on organoids across various systems, such as the mesendoderm (including organs like the stomach, liver, pancreas, lung, and kidney) and neuroectoderm (such as the brain and retina), employing either adult stem cells (ASCs) or pluripotent stem cells (40).

The primary basis for the development of organoid culture relies on the ability of homogeneous cells to self-organize and mimic the key features of the source tissue. The key steps in self-organization rely heavily on a series of highly regulated signaling pathways that play an important role in the self-organization and cell differentiation processes in the developmental cascade of pattern formation through a variety of morphogenetic rearrangements. The traditional method of organoid culture, expansion and development involves the enzymatic digestion of tissue fragments and derivation of stem cells, progenitor cells, followed by culturing them in a 3D microenvironment using growth factor-conditioned medium (15). A variety of morphogens are used to culture of cardiac organoids, including Wnt-3a epidermal growth factor (EGF), fibroblast growth factor (FGF), R-spondin, gastrin, noggin, Human Bone Morphogenetic Protein 4, Human Activin-A, Insulin, B-27 supplement, L-ascorbic acid 2 phosphate, Aprotinin, Palmitic acid. The methods for creating in vitro cardiac organoids using stem cells, progenitor cells, cardiomyocytes (CMs), endothelial cells (ECs), and cardiac fibroblasts, fall into two categories: scaffold-based and scaffold-free techniques (Figure 1). Scaffold-based methods utilize biomaterials like hydrogels or decellularized bioscaffolds, whereas scaffold-free techniques typically involve promoting the spherical aggregation of cultured cells in an anti-adhesive setting, in addition to some newly emerged techniques, used to facilitate cardiac organoid formation, including microarray technology, 3D bioprinted models, and scaffolds based on electrospun fiber mats. Scaffold-free 3D cultures are created by the self-assembly of cells, while scaffold-based cultures use hydrogels or other scaffolds to support tissue replicas. In either case, the cells are organized into a three-dimensional structure, which is essential for preserving their morphology, phenotype, and polarity. Scaffold-free systems can create artificial 3D heart tissues that maintain mechanical integrity without the need for external support (41). This paper will focus solely on the development of cardiac models using scaffold formation methods with decellularized materials. To understand the basic concepts and operating principles of biomaterials and bioengineering strategies suitable for organoids applications, the readers can refer to more specialized reviews (42, 43).

A large portion of organoids have been grown in Matrigel, a complex and poorly defined biomaterial produced from the secretions of Engelbreth-Holm-Swarm mouse sarcoma cells (44). Although affordable and versatile, Matrigel is highly complex; a proteomic study reveals that it comprises more than 1,800 distinct proteins (45). It is challenging to determine the signals required for organoid construction and function due to Matrigel's vague nature, which is made more challenging by lot-to-lot variances in Matrigel (46, 47). Additionally, Matrigel based culture environment may lack some of the constituents required for the production of healthy organoid models. For example, gut organoids generated and maintained in Matrigel lack the distinctive villous structure of mammalian intestines, which may be caused by insufficient laminin-511 and associated components (48, 49). Also, it is becoming increasingly evident that the permeabilities and mechanical characteristics of 3D environments can significantly impact the development of cells (50), organoids (51), tissues, and organs (52, 53). The mechanical behaviors-like pore size, elasticity, stress relaxation moduli, and creep compliances (54, 55) -cannot be readily distinguished from the biochemical signal outputs in the Matrigel-based culture environments. Moreover, Matrigel samples have diverse mechanical characteristics; certain parts of these hydrogels are been known to exhibit elastic moduli and stiffness related properties that are considerably higher than the average elastic modulus of the sample (56, 57). Finally, due to possible immunogenicity, Matrigel's origins in mouse cells make it unsuitable for use in clinical transplantation of humans (58). With all these shortcomings, there is growing demand to create Matrigel-free culture techniques for the growth and maintenance of organoids. Extracellular matrix (ECM) proteins function as an adhesive substrate, a signaling cue source, and a growth factor sequestration mechanism during organ development (59).

Organoid technology has recently brought a dramatic shift to biomedical research by creating 3D models that replicate cellular heterogeneity, structure, and functions of tissues (40). In recent years, organoids have been used widely in disease modeling and drug discovery in several organs, such as the brain (79), the kidney (39), the liver (80, 81), and the intestine (82). However, due to the heart's complex structure and vascularization, the progress of developing cardiac organoids has been slower than that of other organs. Regardless of these limitations, human cardiac organoids are widely considered as a novel model system for studying cardiac diseases (Table 1), drug screening and cardiotoxicity testing (84–91) (Tables 1, 2).

Advancements in cardiac organoids that allow appropriate structural and functional elements of the normal heart are imperative. These include the formation of chambers, myocardium thickness, and contractibility. One recent advancement in cardiac chamber formation using organoids includes the hHO model by Lewis-Israeli et al., which involves self-organization into multiple miniature chambers via BMP4 and Activin A (37). Additionally, Lin et al. successfully established a “human-heart-in-a-jar”, demonstrating an electrically and mechanically functional miniature ventricle (83). Although these characteristics provide an obvious advantage for studying diseases and drug interactions over using simpler 2D or 3D models, they lack essential features including left-right symmetry, conductance, and valves (91). Bioreactors have been suggested in the literature to improve the maturity of cardiac organoids (92). They work by improving media circulation, which leads to elevated uptake of nutrients, and providing mechanical stretch stimuli, which promote organoid maturation (93, 94).

Lastly, poor vascularization of cardiac organoids has been observed in several models, with some even developing necrosis from limited perfusion (24). Organ-chip systems have addressed this drawback, allowing 3D microchannel scaffolds supporting millimeter-thick cardiac tissue that is optimally perfused (24, 95). A limitation of these systems is the inability to replicate the 3D configuration and multicellular composition of normal cardiac tissue. Indeed, a multicellular composition, incorporating endothelial cells and fibroblasts, enables enhanced disease modeling and cardiomyocyte maturation (87). A potential solution to this has been observed with a Heart-on-Chip (HoC) model described by Cofiño-Fabres et al., involving the addition of hiPSC-derived cardiomyocytes with other cells typically found in cardiac tissue (96). Another alternative to improve vascularization involves the development of hiPSC-derived self-assembling vascular spheres, followed by encasing with hiPSC-derived cardiomyocytes (97).

In summary, while cardiac organoids represent a significant advancement in heart disease modeling and therapeutic research, challenges remain in fully replicating the complexity of the heart's structure and function. The absence of a cardiac conduction system, functional vascularization, and the lack of standardized fabrication methods are key obstacles that hinder the development of more accurate and reliable models. However, with ongoing advancements in 3D modeling techniques, such as Heart-on-Chip systems, these limitations are gradually being addressed. As the field progresses, cardiac organoids hold great promise for improving disease modeling, drug screening, and personalized medicine, paving the way for more effective treatments and better understanding of cardiovascular diseases. Continued research into fabrication methods, tissue engineering, and functional integration will be essential for realizing the full potential of cardiac organoids in both clinical and preclinical applications.