Current limitations of organoid technology

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!!1. Lack of a vascular system The human body is dynamic; blood constantly flows through the blood vessels to exchange nutrients, oxygen, and waste ensuring cell survival. However, organoids lack a vascular system and are cultured through static methods. As the organoids keep growing, the cells in the center are unable to get enough nutrition and the exchange of waste becomes difficult. Other factors that affect the vascularization of organoids include cytokine concentration, the shape of the organoid, and flow rate. Currently, in vitro organoid vascularization is carried out by addition of vascular cells or genetic engineering. Organoids are small in size: They can only grow to around 1 millimeter in size. They cannot become larger because they rely on nutrients diffusing to their centre. Diffusion can only occur across a small distance. In contrast, organs have blood vessels that supply nutrients deep into the tissues. To make functional organs, they need to grow big enough, and for this, they need to have blood vessels. But advances are being made. Although still only mm in size, a heart organoid can be grown that resembles a foetal heart. It has all the major cardiac cell types, small heart chambers, and a vascular network. After several days of culture, they even start to beat. !!!Vascularizing organoids through co-culture with endothelial cells The vascularization of organoids, a critical aspect of creating physiologically relevant miniature organs, often involves the co-culture with endothelial cells. Endothelial cells are derived from mesoderm and are required for the formation of new blood vessels. This interaction mirrors the intricate interaction that occurs during embryonic development and early organogenesis, encouraging endothelial cells to self-assemble into capillary-like structures within the organoid. In the context of human brain organoids, co-culturing with vascular cells, specifically a monolayer of endothelial cells, has been employed to achieve organoid vascularization. In 2018, Pham et al., successfully vascularized 34-day-old human brain organoids by embedding them in Matrigel, enriched with endothelial cells derived from human iPSCs, along with bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor A (VEGF 165), and fibroblast growth factor 2 (FGF-2). After 34 days of co-culture, these brain organoids displayed a network of penetrating vessels expressing platelet endothelial cell adhesion molecule, a key endothelial marker. Perhaps the most common method for organoid vascularization involves co-culturing human organoids with human umbilical vascular endothelial cells (HUVECs). In brain organoids vascularized through HUVEC co-culture for over 200 days, Shi et al., found that vascularization initially manifests in the ventricular zone (VZ)-like region, which houses neural stem and progenitor cells (NSPCs), mirroring the developmental patterns of the human brain vasculature. Vascularized brain organoids exhibit more robust growth compared to their non-vascularized counterparts, suggesting that vascularization promotes the proliferation of NSPCs and/or the survival of neurons, even in an in vitro environment. !!!Three-dimensional bioprinting to achieve organoid vascularization Bioprinting technologies allow researchers to precisely position cells and biomaterials to create intricate structures, including vascular networks. By using bioinks composed of endothelial cells and supportive matrix materials, researchers can fabricate organoids with predefined vascular patterns. Bioprinting allows for fine control over the architecture of the vascular network. It can create highly customized organoids with intricate vasculature. Bioprinting technologies allow researchers to precisely position cells and biomaterials to create intricate structures, including vascular networks. By using bioinks composed of endothelial cells and supportive matrix materials, researchers can fabricate organoids with predefined vascular patterns. Bioprinting allows for fine control over the architecture of the vascular network. It can create highly customized organoids with intricate vasculature. Bioprinting has emerged as a transformative technology for creating large-scale tissue constructs with intricate vascular networks. Two primary methods, Filament Deposition Fabrication (FFF) and Fused Deposition Modeling (FDM), are frequently employed to achieve this goal. These techniques involve depositing cell-laden hydrogel filaments in a controlled manner to form specific patterns, offering immense potential for tissue engineering. Although bioprinting enables the creation of tissues with spatially organized cells and extracellular matrices, it faces challenges related to resolution. The printing resolution is limited, particularly in extrusion-based bioprinting, which has a resolution of about 100 µm, whereas the diameter of typical capillaries is approximately 10 µm. !!!Advancing organoids-on-a-chip for organoid vascularization Organoids-on-a-chip is a promising solution to the lack of complexity of living environments of 2D culture. This innovative technique involves the construction of organoids within microfluidic devices, with meticulous consideration of the anatomical structure and physiological characteristics of the target organ. While traditional microfluidic organoids lack blood circulation, vascularized organoids-on-a-chip devices offer several advantages for studying brain-related events. To create organoids-on-a-chip, researchers aim to replicate the 3D mechanical and biochemical environment of the target organ. This process involves leveraging the growth characteristics of cells and utilizing micro-processing technologies, such as soft lithography, to construct the necessary model for cell growth. These models are designed to closely resemble the structure and function of the organ they mimic for disease modeling, insights into pathological processes, pharmacology modeling and aiding drug testing and evaluation. When applied to brain organoids, vascularized organoid-on-a-chip devices provide several unique benefits. The devices simplify the evaluation of blood-brain barrier (BBB) permeability as well as detailed analyses of BBB function, including calcium imaging to monitor neuronal activity after drug delivery. !!2. Organoids and immune cells The immune system comprises immune organs, immune cells, and regulatory proteins that work together to protect the body from foreign invaders. Epithelial cells and immune cells interact to maintain the physiological balance in humans. It is difficult to establish an interacting and integrating model of organoids and immune cells to study disease models, therapy efficacies, and drug evaluation. Current culture technologies cannot meet in vivo simultaneous interactions of tumor and immune cells. Tumor organoids with an established immune system will help to study the disease in detail, produce clinically relevant results, and reduce the time between the laboratory and the hospital (3). !!3. Organoids culture medium Matrigel is widely used to cultivate organoids as it provides them with an extracellular matrix enabling the investigation of cell-matrix interactions. Matrigel is derived from the secretion of Engelbreth-Holm-Swarm mouse sarcoma cells and includes laminin, collagen, heparan sulfate proteoglycan, and entactin. Despite these advantages, Matrigel is extremely complex and contains more than 180 unique proteins. The impossibility of precisely defining its composition makes it difficult to fully understand its influence on organoids’ structure and function. Matrigel may also lack important components that are necessary for the cultivation of organoids in optimal conditions. Further, Matrigel’s animal origin limits its use in human clinical applications. Considering these disadvantages, there is a need to develop Matrigel-independent organoid culture methods (4,5). References # van den Berg, C. W., Ritsma, L., Avramut, M. C., Wiersma, L. E., van den Berg, B. M., Leuning, D. G., et al. (2018). Renal Subcapsular Transplantation of PSC-Derived Kidney Organoids Induces Neo-Vasculogenesis and Significant Glomerular and Tubular Maturation In Vivo. Stem Cell Rep. 10 (3), 751–765. doi:10.1016/j.stemcr.2018.01.041 # Homan KA, Gupta N, Kroll KT, Kolesky DB, Skylar-Scott M, Miyoshi T, Mau D, Valerius MT, Ferrante T, Bonventre JV, Lewis JA, Morizane R. Flow-enhanced vascularization and maturation of kidney organoids in vitro. Nat Methods. 2019 Mar;16(3):255-262. doi: 10.1038/s41592-019-0325-y. Epub 2019 Feb 11. PMID: 30742039; PMCID: PMC6488032. # Campinoti S, Gjinovci A, Ragazzini R, Zanieri L, Ariza-McNaughton L, Catucci M, Boeing S, Park JE, Hutchinson JC, Muñoz-Ruiz M, Manti PG, Vozza G, Villa CE, Phylactopoulos DE, Maurer C, Testa G, Stauss HJ, Teichmann SA, Sebire NJ, Hayday AC, Bonnet D, Bonfanti P. Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds. Nat Commun. 2020 Dec 11;11(1):6372. doi: 10.1038/s41467-020-20082-7. PMID: 33311516; PMCID: PMC7732825. # Andrews MG, Kriegstein AR. Challenges of Organoid Research. Annu Rev Neurosci. 2022 Jul 8;45:23-39. doi: 10.1146/annurev-neuro-111020-090812. Epub 2022 Jan 5. PMID: 34985918. # Kozlowski MT, Crook CJ, Ku HT. Towards organoid culture without Matrigel. Commun Biol. 2021 Dec 10;4(1):1387. doi: 10.1038/s42003-021-02910-8. PMID: 34893703; PMCID: PMC8664924 # PLoS Biol. 2020 Vascularized human cortical organoids (vOrganoids) model cortical development in vivo. Authors: Shi Y, Sun L, Wang M, et al. # Neuroreport. 2018 Generation of human vascularized brain organoids. Authors: Pham MT, Pollock KM, Rose MD, et al. # Small 16, 1905505. 2020, Bioprinting of Multiscaled Hepatic Lobules within a Highly Vascularized Construct. Authors: Kang, D. et al. # Biomolecules 11, 966. 2021, Engineering the Vasculature of Stem-Cell-Derived Liver Organoids. Authors: Zhang, X., Tang, L. & Yi, Q # Stem Cells, 39, 1017–1024 (2021). Vascularization of Human Brain Organoids. Authors: Matsui, T. K., Tsuru, Y., Hasegawa, K. & Kuwako, K. # Frontiers in Bioengineering and Biotechnology, 9, (2021). Review on the Vascularization of Organoids and Organoids-on-a-Chip. Authors: Zhao, X. et al. * Vascularized Tissue Organoids: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9951914/ * Vascularizing Organoids to Promote Long-Term Organogenesis on a Chip: https://www.mdpi.com/2674-1172/2/4/19 * Review on the Vascularization of Organoids and Organoids-on-a-Chip: https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2021.637048/full * Flow-enhanced vascularization and maturation of kidney organoids in vitro: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488032/ * Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells: https://www.nature.com/articles/s41598-019-52204-7 * Give Them Vasculature and Immune Cells: How to Fill the Gap of Organoids: https://karger.com/cto/article/212/5/369/836906/Give-Them-Vasculature-and-Immune-Cells-How-to-Fill {br} Keyword: growing organoid vasculature
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