3D organoid
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The collection on Methods and Models in Mammary Gland Biology and Breast Cancer Research showcases recent advances in tools and models that enhance our understanding of mammary gland development and breast cancer. This collection includes sixteen articles, collectively addressing approaches to investigate key aspects of mammary gland biology and tumorigenesis, including hormonal signaling, tissue architecture, tumor microenvironment, and species-specific mammary development. The issue highlights innovations such as optimized progesterone receptor reporters, improved menopause models, and 3D-printed mammary epithelial structures. It also features advancements in organoid-based studies, in situ labeling of epithelial proliferation in large animals, preclinical models for breast cancer prevention, and high-resolution imaging techniques. Methodologies for studying macrophage-cancer cell interactions and lysosomal function are provided as step-by-step protocols. Additionally, review articles provide insights into diverse mammalian organoid systems, rat mammary tumor models, and strategies for modeling breast cancer metastasis. Together, these contributions advance mammary gland research by refining experimental approaches, expanding model diversity, and fostering translational applications in breast cancer.
- MeSH
- lidé MeSH
- mléčné žlázy lidské * patologie růst a vývoj fyziologie MeSH
- mléčné žlázy zvířat * patologie růst a vývoj fyziologie MeSH
- modely nemocí na zvířatech MeSH
- nádorové mikroprostředí fyziologie MeSH
- nádory prsu * patologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- úvodní články MeSH
- úvodníky MeSH
BACKGROUND: The choroid plexus (ChP) is the secretory epithelial structure located in the brain ventricles. Choroid plexus tumors (CPTs) are rare neoplasms predominantly occurring in young patients with intensified malignancy in children. CPT treatment is hindered by insufficient knowledge of tumor pathology and the limited availability of valid models. METHODS: Genomic and transcriptomic data from CPT patients were analyzed to identify the putative pathological pathway. Cellular and molecular techniques were employed to validate bioinformatic results in CPT patient samples. Pharmacologic inhibition of Wnt/β-catenin signaling was assessed in CPT cells. Cell-based assays of ChP cell lines were performed following CRISPR-Cas9-derived knockout and overexpression of Wnt/β-catenin pathway genes. A 3D CPT model was generated through CRISPR-Cas9-derived knockout of APC. RESULTS: We discovered that Wnt/β-catenin signaling is activated in human CPTs, likely as a consequence of large-scale chromosomal instability events of the CPT genomes. We demonstrated that CPT-derived cells depend on autocrine Wnt/β-catenin signaling for survival. Constitutive Wnt/β-catenin pathway activation, either through knockout of the negative regulator APC or overexpression of the ligand WNT3A, induced tumorigenic properties in ChP 2D in vitro models. Increased activation of the Wnt/β-catenin pathway in ChP organoids, through treatment with a potent GSK3β inhibitor, reduced the differentiation of mature ChP epithelial cells. Remarkably, the depletion of APC was sufficient to induce the oncogenic transformation of ChP organoids. CONCLUSIONS: Our research identifies Wnt/β-catenin signaling as a critical driver of CPT tumorigenesis and provides the first 3D in vitro model for future pathological and therapeutic studies of CPT.
- MeSH
- beta-katenin metabolismus genetika MeSH
- karcinogeneze metabolismus MeSH
- lidé MeSH
- nádorové buňky kultivované MeSH
- nádory plexus chorioideus * patologie metabolismus genetika MeSH
- plexus chorioideus metabolismus patologie MeSH
- proliferace buněk MeSH
- regulace genové exprese u nádorů MeSH
- signální dráha Wnt * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Súhrn: Ľudská placenta predstavuje životne dôležitú bariéru medzi matkou a vyvíjajúcim sa plodom počas tehotenstva. Porucha včasného vývoja placenty je spojená so závažnými poruchami tehotenstva. Napriek jej komplexnému vývoju stále nie sú úplne objasnené rôzne molekulárne procesy riadiace vývoj placenty a špecializáciu buniek trofoblastu. Jednou z hlavných prekážok je nedostatok vhodných bunkových modelových systémov. Tradičné dvojrozmerné (2D) bunkové kultúry nedokážu imitovať podmienky in vivo a nezachytávajú zložité medzibunkové interakcie nevyhnutné na štúdium vývoja placenty. Avšak trojrozmerné (3D) modely organoidov, odvodené z kmeňových buniek, ktoré replikujú prirodzenú organizáciu a architektúru buniek výrazne zlepšili naše chápanie správania sa trofoblastov a ich medicínskych aplikácií. Organoidy s relevantnými fenotypmi poskytujú cennú platformu na modelovanie fyziológie a patológie placenty, vrátane modelovania porúch placenty. Sú veľkým prísľubom pre personalizovanú medicínu, zlepšenie diagnostiky a hodnotenia účinnosti a bezpečnosti farmaceutických liečiv. Tento článok poskytuje stručný prehľad trofoblastových kmeňových buniek, invázie trofoblastu a rozvíjajúcej sa úlohy organoidov v gynekológii.
The human placenta serves as a vital barrier between the mother and the developing fetus during pregnancy. A defect in the early development of the placenta is associated with severe pregnancy disorders. Despite its complex development, various molecular processes control placental development, and the specialization of trophoblast cells is still not fully understood. One primary obstacle is the lack of suitable cell model systems. Traditional two-dimensional (2D) cell cultures fail to mimic in vivo conditions and do not capture the intricate intercellular interactions vital for studying placental development. However, three-dimensional (3D) organoid models derived from stem cells that replicate natural cell organization and architecture have greatly improved our understanding of trophoblast behavior and its medicinal applications. Organoids with relevant phenotypes provide a valuable platform to model both placental physiology and pathology, including the modeling of placental disorders. They hold great promise for personalized medicine, improved diagnostics, and the evaluation of pharmaceutical drug efficacy and safety. This article provides a concise overview of trophoblast stem cells, trophoblast invasion, and the evolving role of organoids in gynecology.
- MeSH
- kmenové buňky fyziologie MeSH
- komplikace těhotenství MeSH
- lidé MeSH
- organoidy fyziologie MeSH
- placenta * cytologie patologie MeSH
- těhotenství MeSH
- trofoblasty fyziologie MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- přehledy MeSH
Mammary epithelial ducts, the main functional compartment of the mammary gland, are embedded in an adipocyte-rich stroma, which is essential for proper mammary gland development, function, and tissue homeostasis. Moreover, the adipocyte compartment has an important role in cancer progression. To better understand cell-to-cell interactions and the role of the adipocytes in the mammary gland, development of proper in vitro models which realistically mimic in vivo conditions has been essential. In this chapter, we describe a simple and effective method for generating mammary gland adipocytes from mammary fibroblasts and their subsequent co-culture with mammary epithelial organoids to further investigate the role of adipocytes in epithelial development and morphogenesis.
- MeSH
- epitelové buňky * MeSH
- fibroblasty MeSH
- kokultivační techniky MeSH
- mléčné žlázy zvířat * MeSH
- organoidy MeSH
- tukové buňky MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Fibroblasts are an integral cell type of mammary gland stroma, which plays crucial roles in development, homeostasis, and tumorigenesis of mammary epithelium. Fibroblasts produce and remodel extracellular matrix proteins and secrete a plethora of paracrine signals, which instruct both epithelial and other stromal cells of the mammary gland through mechanisms, which have not been fully understood. To enable deciphering of the intricate fibroblast-epithelial interactions, we developed several 3D co-culture methods. In this chapter, we describe methods for establishment of various types of embedded 3D co-cultures of mammary fibroblasts with mammary epithelial organoids, mammary tumor organoids, or breast cancer spheroids to investigate the role of fibroblasts in mammary epithelial development, morphogenesis, and tumorigenesis. The co-culture types include dispersed, aggregated, and transwell cultures.
In the rapidly evolving landscape of cell biology and biomedical research, three-dimensional (3D) cell culture has contributed not only to the diversification of experimental tools available but also to their improvement toward greater physiological relevance. 3D cell culture has emerged as a revolutionary technique that bridges the long-standing gap between traditional two-dimensional (2D) cell culture and the complex microenvironments found in living organisms. By providing conditions for establishing critical features of in vivo environment, such as cell-cell and cell-extracellular matrix interactions, 3D cell culture enables proper tissue-like architecture and differentiated function of cells. Since the early days of 3D cell culture in the 1970s, the field has witnessed remarkable progress, with groundbreaking discoveries, novel methodologies, and transformative applications. One particular 3D cell culture technique has caught the attention of many scientists and has experienced an unprecedented boom and enthusiastic application in both basic and translational research over the past decade - the organoid technology. This book chapter provides an introduction to the fundamental concepts of 3D cell culture including organoids, an overview of 3D cell culture techniques, and an overview of methodological- and protocol-oriented chapters in the book 3D Cell Culture.
Organoids are 3-dimensional (3D) self-assembled structures capable of replicating the microanatomy and physiology of the epithelial components of their organ of origin. Adult stem cell (ASC) derived organoids from the liver have previously been shown to differentiate into primarily mature cholangiocytes, and their partial differentiation into functional hepatocytes can be promoted using specific media compositions. While full morphological differentiation of mature hepatocytes from ASCs has not yet been reported for any species, the functional differentiation can be approximated using various media compositions. Six differentiation media formulations from published studies on hepatic organoids were used for the differentiation protocol. Target species for these protocols were humans, mice, cats, and dogs, and encompassed various combinations and concentrations of four major hepatocyte media components: Bone morphogenetic protein 7 (BMP7), Fibroblast Growth Factor 19 (FGF19), Dexamethasone (Dex), and Gamma-Secretase Inhibitor IX (DAPT). Additionally, removing R-spondin from basic organoid media has previously been shown to drive the differentiation of ASC into mature hepatocytes. Differentiation media (N = 20) were designed to encompass combinations of the four major hepatocyte media components. The preferred differentiation of ASC-derived organoids from liver tissue into mature hepatocytes over cholangiocytes was confirmed by albumin production in the culture supernatant. Out of the twenty media compositions tested, six media resulted in the production of the highest amounts of albumin in the supernatant of the organoids. The cell lines cultured using these six media were further characterized via histological staining, transmission electron microscopy, RNA in situ hybridization, analysis of gene expression patterns, immunofluorescence, and label-free proteomics. The results indicate that preferential hepatocyte maturation from canine ADC-derived organoids from liver tissue is mainly driven by Dexamethasone and DAPT components. FGF19 did not enhance organoid differentiation but improved cell culture survival. Furthermore, we confirm that removing R-spondin from the media is crucial for establishing mature hepatic organoid cultures.
- Publikační typ
- časopisecké články MeSH
... Mesenchymal stem cells in medicine 184 -- 12.3.2 Pluripotent stem cells in medicine 186 -- 12.4 Organoids ... ... 190 -- 12.5 From cells and organoids to organs: the journey from 2D to 3D tissue culture 191 -- 12.6 ...
First edition 227 stran : ilustrace ; 30 cm
- Konspekt
- Lékařské vědy. Lékařství
- Učební osnovy. Vyučovací předměty. Učebnice
- NLK Obory
- biologie
- NLK Publikační typ
- učebnice vysokých škol
Organoids are complex multicellular structures that stem cells self-organize in three-dimensional (3D) cultures into anatomical structures and functional units similar to those seen in the organs from which they originate. This review describes the construction of thyroid organoids and the research progress that has occurred in models of thyroid-related disease. As a novel tool for modeling in a 3D multicellular environment, organoids help provide some useful references for the study of the pathogenesis of thyroid disease.
- MeSH
- kmenové buňky MeSH
- organoidy * MeSH
- štítná žláza * MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Three-dimensional (3D) cell cultures are to date the gold standard in biomedical research fields due to their enhanced biological functions compared to conventional two-dimensional (2D) cultures. 3D cell spheroids, as well as organoids, are better suited to replicate tissue functions, which enables their use both as in vitro models for basic research and toxicology, as well as building blocks used in tissue/organ biofabrication approaches. Culturing 3D spheroids from bone-derived cells is an emerging technology for both disease modelling and drug screening applications. Bone tissue models are mainly limited by the implementation of sophisticated devices and procedures that can foster a tissue-specific 3D cell microenvironment along with a dynamic cultivation regime. In this study, we consequently developed, optimized and characterized an advanced perfused microfluidic platform to improve the reliability of 3D bone cell cultivation and to enhance aspects of bone tissue maturation in vitro. Moreover, biomechanical stimulation generated by fluid flow inside the arrayed chamber, was used to mimic a more dynamic cell environment emulating a highly vascularized bone we expected to improve the osteogenic 3D microenvironment in the developed multifunctional spheroid-array platform. The optimized 3D cell culture protocols in our murine bone-on-a-chip spheroid model exhibited increased mineralization and viability compared to static conditions. As a proof-of-concept, we successfully confirmed on the beneficial effects of a dynamic culture environment on osteogenesis and used our platform for analysis of bone-derived spheroids produced from primary human pre-osteoblasts. To conclude, the newly developed system represents a powerful tool for studying human bone patho/physiology in vitro under more relevant and dynamic culture conditions converging the advantages of microfluidic platforms with multi-spheroid array technologies.
- Publikační typ
- časopisecké články MeSH
