Growth Responses in Isolated Elastic, Muscular and Resistance-Sized Arterial Segments of the Rat
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To evaluate whether intravascular phenomena contribute to local differences in growth responses of the arterial wall, we evaluated responses to organoid culture in a broad variety of arterial preparations. Arterial segments were isolated from adult, normotensive rats, sympathectomized, denuded from endothelium, and suspended in medium supplemented with serum. As judged from the nuclear incorporation of the thymidine analogue 5-bromo-2’-deoxyuridine (BrdUrd), this induced a transient stimulation of DNA synthesis in only a fraction of the arterial smooth muscle cells in all types of arteries. This intramedial DNA synthesis was more marked in renal arteries than in carotid arteries or aortae and was least pronounced in main pulmonary, femoral, and superior mesenteric artery and in mesenteric resistance-sized arteries. Organoid culture of isolated arteries did not increase the cross-sectional area of the media or the number of medial cells. It rather resulted in proliferation of smooth-muscle-like cells outside the media. In addition, smooth-muscle-like cells migrated out of the isolated arterial segments during culture. The rate of proliferation of these isolated cells did not differ between large arteries of different anatomical origin. However, isolated cells derived from mesenteric resistance arteries proliferated at a rate that was 4 times slower than that of large artery cells. The presence of endothelium significantly reduced medial DNA synthesis in carotid and renal artery segments, but not in mesenteric resistance-sized preparations. These data indicate that growth responses of the arterial wall differ quantitatively with the anatomical location and branching order of the vascular segment. In addition to the regional heterogeneity of endothelial effects on mitogenic responses of arterial smooth muscle, this seems to be due to regional differences in the susceptibility of arterial smooth muscle to exogenous growth factors. In this respect, we speculate that subsets of growth-resistant and growth-prone arterial smooth muscle cells could be heterogeneously distributed over the arterial tree.Keywords:
Mesenteric arteries
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ABSTRACT Over the last decades, organoids have been established from the majority of tissue resident stem and iPS cells. They hold great promise for our understanding of mammalian organ development, but also for the study of disease or even personalized medicine. In recent years, several reports hinted at intraculture organoid variability, but a systematic analysis of such a heterogeneity has not been performed before. Here, we used RNA-seq of individual organoids to address this question. Importantly, we find that batch-to-batch variation is very low, even when prepared by different researchers. On the other hand, there is organoid-to-organoid variability within a culture. Using differential gene expression, we did not identify specific pathways that drive this variability, pointing towards possible effects of the microenvironment within the culture condition. Taken together, our study provides a framework for organoid researchers to properly consider experimental design.
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Culturing and passaging of iPSC derived intestinal organoids derived using STEMDIFF intestinal organoid kit. We usually use organoids after 5 passages once consistent growth has been established and until 15th passage.
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This protocol describes the passaging of organoid cultures. It has been developed by the organoid derivation team within the Cellular Generation and Phenotyping Group at the Wellcome Sanger Institute. The team has extensive experience passaging and expanding organoid models. The method described has mainly been used for the passaging cancer organoids with successful propagation of organoids derived from colon, pancreas and oesophageal tumours.
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Abstract Three-dimensional (3D) organoid culture holds great promises in cancer precision medicine. However, Matrigel and stem cell-stimulating supplements are necessary for culturing 3D organoid cells. It costs a lot of money and consumes more time and effort compared with 2D cultured cells. Therefore, the establishment of cheaper and Matrigel-free organoid culture that can maintain the characteristics of a part of 3D organoids is demanded. In the previous study, we established a dog bladder cancer (BC) 3D organoid culture system by using their urine samples. Here, we successfully isolated cells named “2.5D organoid” from multiple strains of dog BC 3D organoids using 2.5 organoid media. The cell proliferation speed of 2.5D organoids was faster than parental 3D organoid cells. The expression pattern of stem cell markers was close to 3D organoids. Injection of 2.5D organoid cells into immunodeficient mice formed tumors and showed the histopathological characteristics of urothelial carcinoma similar to the injection of dog BC 3D organoids. The 2.5D organoids had a similar sensitivity profile for anti-cancer drug treatment to their parental 3D organoids. These data suggest that our established 2.5D organoid culture method might become a reasonable and useful tool instead of 3D organoids in dog BC research and therapy.
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Organoids are three-dimensional structures formed by self-organizing growth of cells in vitro, which own many structures and functions similar with those of corresponding in vivo organs. Although the organoid culture technologies are rapidly developed and the original cells are abundant, the organoid cultured by current technologies are rather different with the real organs, which limits their application. The major challenges of organoid cultures are the immature tissue structure and restricted growth, both of which are caused by poor functional vasculature. Therefore, how to develop the vascularization of organoids has become an urgent problem. We presently reviewed the progresses on the original cells of organoids and the current methods to develop organoids vascularization, which provide clues to solve the above-mentioned problems.类器官是在体外通过细胞自组织生长而形成的三维结构，它具有许多与相应在体器官相类似的结构和功能。类器官培养技术发展迅速，起源细胞类型丰富，但是现有技术培养的类器官和真正的器官还具有很大差别，限制了类器官的广泛应用。限制类器官的主要问题有组织结构不成熟和生长受限，而缺少功能性血管系统则是根本原因，如何在类器官模型中发展血管系统已成为亟待解决的问题。本文主要综述了类器官的起源细胞以及类器官血管化的研究进展，为后续研究提供了线索。.
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Here, we describe a protocol to generate organoids from human thyroid cancer cells. Starting from the same patient-derived cells, we establish both organoids and primary lines. The organoid medium is supplemented with conditioned medium obtained from the primary cell line. This modification enables culture of the organoid lines for up to 10 months. Even after long-term culture, the organoids retain the genetic and phenotypic characteristics of their tissue of origin.
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Organoids, three-dimensional in vitro tissue cultures derived from pluripotent (embryonic or induced) or adult stem cells, are promising models for the study of human processes and structures, disease onset and preclinical drug development. An increasing amount of omics data has been generated for organoid studies. Here, we introduce OrganoidDB (http://www.inbirg.com/organoid_db/), a comprehensive resource for the multi-perspective exploration of the transcriptomes of organoids. The current release of OrganoidDB includes curated bulk and single-cell transcriptome profiles of 16 218 organoid samples from both human and mouse. Other types of samples, such as primary tissue and cell line samples, are also integrated to enable comparisons with organoids. OrganoidDB enables queries of gene expression under different modes, e.g. across different organoid types, between different organoids from different sources or protocols, between organoids and other sample types, across different development stages, and via correlation analysis. Datasets and organoid samples can also be browsed for detailed information, including organoid information, differentially expressed genes, enriched pathways and single-cell clustering. OrganoidDB will facilitate a better understanding of organoids and help improve organoid culture protocols to yield organoids that are highly similar to living organs in terms of composition, architecture and function.
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This SOP defines the procedure for thawing a frozen cryovial of organoids into 1 well of a 6 well plate for further culture. It has been developed by the organoid derivation team within the Cellular Generation and Phenotyping Group at the Wellcome Sanger Institute. The team has extensive experience passaging and expanding organoid models. The method described has mainly been used for cancer organoids with successful propagation of organoids derived from colon, pancreas and oesophageal tumours.
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