The reproducibility of 3D cardiac models poses a challenge that must be addressed to ensure the full applicability to high- or mid-throughput screening platforms. To this end, rigorous standardization and optimization of iPSC and MSC differentiation protocols, and organoid or spheroid generation procedures, are essential. For full integration into drug discovery platforms, either during target discovery or pre-clinical toxicology screening, technologies must be developed to improve compatibility with automated handling, high-content imaging, and real-time assay platforms. In complex 3D cell culture models, parameters such as organoid size, complexity, and maturity are challenging to control and synchronize across long-term cultures.

An important trend supporting scale-up and improved reproducibility in organoid culture is the move to serum-free, chemically defined cell culture media to remove sources of inter- and intra-laboratory variability. In addition, the removal of animal-derived components and particularly growth factors or cytokines derived from mammalian cell culture, such as BMP-4, is an important step in scale-up to reduce cost whilst improving reproducibility. TGF beta family proteins such as BMP-4, are notoriously difficult to produce in a highly pure form from mammalian protein production platforms, such as CHO and NSO cells, this can lead to differences between suppliers and lots from the same supplier. Additionally, media development and refinement, including the correct selection of components and optimization of concentrations is necessary to ensure reproducible results in organoid cell culture.

For those cardiac organoid models that rely on Matrigel, there is a drive to replace this with synthetic hydrogels, or purified recombinant extracellular matrix components, such as vitronectin. This addresses both supply chain challenges that limit scale-up, remove animal-derived components with inherent variability from the cell culture system, and importantly improve compatibility with automated cell culture. Replacement of Matrigel (or similar matrices) with synthetic hydrogels is proving an industry-wide challenge with few widely adopted solutions to date.

Automation of organoid culture and functional bioassays are key areas being addressed in order to scale organoid cell culture systems, as they play a critical role in achieving consistent organoid cultures and high-quality data. Automating laboratory processes such as sample preparation, incubation and imaging can help increase the reproducibility of the culture system while decreasing labor costs. Significant advances are being made to deliver successful scale-up of organoid cultures, and there is growing interest in overcoming remaining barriers to fully-defined 3D-cell culture systems.

Advances in tissue engineering are providing opportunities to build more complex models of the human heart. As the complexity and size of organoid models increase, the need for a form of vascularisation to provide nutrients to these models is an area of active research. Several recent technological advances, including the use of microfluidics and micropatterning techniques, have allowed researchers to build more physiologically relevant complex models of heart tissues. This may improve the ability to reproduce complex aspects of cardiotoxicity and disease. Recently, micro vascularized cardiac organoids have been successfully generated, and particularly intriguing, is recent work on blood vessel organoids comprising self-organizing endothelial cells. The vascular lineage induction was achieved by modulating bone morphogenetic protein 4 signaling (BMP4), vascular endothelial growth factor A (VEGF 165), and fibroblast growth factor 2 (FGF-2) levels. Blood vessel capillary networks formed by the self-organization of endothelial cells and pericytes present a fascinating avenue for further development of organoid models. Although of intense basic research interest, these complex models may have limited application in high throughput studies necessary for efficient new drug discovery. A simple and reproducible blood vessel organoid model however may open opportunities for diabetes and cardiovascular target discovery.

Finally, an important limitation of current cardiac organoids is the cells often do not express markers associated

with maturity, unlike native tissue, meaning that they may not be able to accurately replicate the full range of cardiac functions. Growth factors and cytokines have been identified to play essential roles during cardiac development and are necessary for cardiac tissue regeneration processes, therefore have been explored as key components of cardiac stem cell and organoid culture focusing on the maturation of the cardiac organoids. Cytokines such as Activin A, bone morphogenic protein 4 (BMP4), and vascular epithelial growth factor (VEGF) have all been found to contribute to cardiac lineage commitment when used in cardiac stem cell culture systems. In particular, Activin A and BMP4 have been associated with higher levels of myocyte markers in cardiac organoid cultures, whereas VEGF has been found to stimulate cardiomyocyte proliferation. This indicates the importance of selective growth factor and cytokine use in cardiac stem cell and organoid culture systems and the potential to push cardiac organoids to form more adult-like cell types.

Recent developments in cardiac research have enabled scientists to create three-dimensional organoids with atrial tissue, to provide more accurate models for studying atrial maturation processes. By supplementing these atrial organoids with growth factors, such as BMP9, cell maturity, and maturation were partially increased. There are, however, still some challenges that need to be overcome before atrial organoids can offer reliable, functional cardiac tissues and organs. Further research into controlling atrial organoid maturation at the genetic level will promote progress in this field. Moreover, further study into specializing atrial organoids using various techniques could yield meaningful results that have the potential to revolutionize cardiac treatments in the future. Increasing the complexity of cell-cell interactions through introducing additional neuronal and endothelial cells, and exploring the contribution from the extracellular matrix, as part of cardiomyocyte-containing cardiac organoids is being explored to improve the maturity and clinical relevance of these systems.

Cardiac organoid models and related 3D models such as spheroids are powerful tools that are poised to start to contribute to the early detection of drug, environmental toxin, and pollutants cardiac toxicity, and have applications in regenerative medicine. At Qkine we specialize in animal-free, high-purity, highly reproducible growth factors for commercial manufacture of organoid cultures and research use. Our team are happy to discuss collaborative or supply chain enhancement projects to scale-up and improve reproducibility in your organoid models.

Please contact us at proteins@qkine.com, or visit our organoid resource page for more information.