3D Cell Culture: Techniques For and Beyond Organoid Applications.

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.

Static 3D Osteoblast Cell Culture on 3D Printed Titanium Scaffolds.

In vitro cell cultures are a very useful tool for the validation of biomaterial cytocompatibility, especially for bone tissue engineering scaffolds and bone implants. In this chapter, a protocol for a static three-dimensional osteoblast cell culture on titanium scaffolds and subsequent analysis of osteogenic capacity is presented. The protocol is explained for additively manufactured titanium scaffolds, but it can be extrapolated to other scaffolds with similar size and structure, while differing in composition or manufactured technology. Additionally, the protocol can be used for culture of other adherent cell types beyond osteoblast cells such as mesenchymal stem cells.

Differentiation of Fibroblasts to Adipocytes in 3D for a Co-culture with Mammary Organoids and Immunohistological Analysis.

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.

Fibroblast-Epithelium Co-culture Methods Using Epithelial Organoids and Cell Line-Derived Spheroids.

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.

Organotypic 3D Cell Culture of the Embryonic Lacrimal Gland.

Ectodermal organ development, including lacrimal gland, is characterized by an interaction between an epithelium and a mesenchyme. Murine lacrimal gland is a good model to study non-stereotypical branching morphogenesis. In vitro cultures allow the study of morphogenesis events with easy access to high-resolution imaging. Particularly, embryonic lacrimal gland organotypic 3D cell cultures enable the follow-up of branching morphogenesis thanks to the analysis of territories organization by immunohistochemistry. In this chapter, we describe a method to culture primary epithelial fragments together with primary mesenchymal cells, isolated from embryonic day 17 lacrimal glands.

Fibroblast-induced mammary epithelial branching depends on fibroblast contractility.

Epithelial branching morphogenesis is an essential process in living organisms, through which organ-specific epithelial shapes are created. Interactions between epithelial cells and their stromal microenvironment instruct branching morphogenesis but remain incompletely understood. Here, we employed fibroblast-organoid or fibroblast-spheroid co-culture systems and time-lapse imaging to reveal that physical contact between fibroblasts and epithelial cells and fibroblast contractility are required to induce mammary epithelial branching. Pharmacological inhibition of ROCK or non-muscle myosin II, or fibroblast-specific knock-out of Myh9 abrogate fibroblast-induced epithelial branching. The process of fibroblast-induced branching requires epithelial proliferation and is associated with distinctive epithelial patterning of yes associated protein (YAP) activity along organoid branches, which is dependent on fibroblast contractility. Moreover, we provide evidence for the in vivo existence of contractile fibroblasts specifically surrounding terminal end buds (TEBs) of pubertal murine mammary glands, advocating for an important role of fibroblast contractility in branching in vivo. Together, we identify fibroblast contractility as a novel stromal factor driving mammary epithelial morphogenesis. Our study contributes to comprehensive understanding of overlapping but divergent employment of mechanically active fibroblasts in developmental versus tumorigenic programs.

Imagine beyond: recent breakthroughs and next challenges in mammary gland biology and breast cancer research.

On 8 December 2022 the organizing committee of the European Network for Breast Development and Cancer labs (ENBDC) held its fifth annual Think Tank meeting in Amsterdam, the Netherlands. Here, we embraced the opportunity to look back to identify the most prominent breakthroughs of the past ten years and to reflect on the main challenges that lie ahead for our field in the years to come. The outcomes of these discussions are presented in this position paper, in the hope that it will serve as a summary of the current state of affairs in mammary gland biology and breast cancer research for early career researchers and other newcomers in the field, and as inspiration for scientists and clinicians to move the field forward.

Segmentation and Tracking of Mammary Epithelial Organoids in Brightfield Microscopy.

We present an automated and deep-learning-based workflow to quantitatively analyze the spatiotemporal development of mammary epithelial organoids in two-dimensional time-lapse (2D+t) sequences acquired using a brightfield microscope at high resolution. It involves a convolutional neural network (U-Net), purposely trained using computer-generated bioimage data created by a conditional generative adversarial network (pix2pixHD), to infer semantic segmentation, adaptive morphological filtering to identify organoid instances, and a shape-similarity-constrained, instance-segmentation-correcting tracking procedure to reliably cherry-pick the organoid instances of interest in time. By validating it using real 2D+t sequences of mouse mammary epithelial organoids of morphologically different phenotypes, we clearly demonstrate that the workflow achieves reliable segmentation and tracking performance, providing a reproducible and laborless alternative to manual analyses of the acquired bioimage data.

Expandable Lung Epithelium Differentiated from Human Embryonic Stem Cells.

BACKGROUND: The progenitors to lung airway epithelium that are capable of long-term propagation may represent an attractive source of cells for cell-based therapies, disease modeling, toxicity testing, and others. Principally, there are two main options for obtaining lung epithelial progenitors: (i) direct isolation of endogenous progenitors from human lungs and (ii) in vitro differentiation from some other cell type. The prime candidates for the second approach are pluripotent stem cells, which may provide autologous and/or allogeneic cell resource in clinically relevant quality and quantity. METHODS: By exploiting the differentiation potential of human embryonic stem cells (hESC), here we derived expandable lung epithelium (ELEP) and established culture conditions for their long-term propagation (more than 6 months) in a monolayer culture without a need of 3D culture conditions and/or cell sorting steps, which minimizes potential variability of the outcome. RESULTS: These hESC-derived ELEP express NK2 Homeobox 1 (NKX2.1), a marker of early lung epithelial lineage, display properties of cells in early stages of surfactant production and are able to differentiate to cells exhibitting molecular and morphological characteristics of both respiratory epithelium of airway and alveolar regions. CONCLUSION: Expandable lung epithelium thus offer a stable, convenient, easily scalable and high-yielding cell source for applications in biomedicine.

Single Organoids Droplet-Based Staining Method for High-End 3D Imaging of Mammary Organoids.

In the last decade, organoids became a tremendously popular technique in developmental and cancer biology for their high pathophysiological relevance to in vivo models with the advantage of easier manipulation, real-time observation, potential for high-throughput studies, and reduced ethical issues. Among other fundamental biological questions, mammary organoids have helped to reveal mechanisms of mammary epithelial morphogenesis, mammary stem cell potential, regulation of lineage specification, mechanisms of breast cancer invasion or resistance to therapy, and their regulation by stromal microenvironment. To exploit the potential of organoid technology to the fullest, together with optimal organoid culture protocols, visualization of organoid architecture and composition in high resolution in three dimensions (3D) is required. Whole-mount imaging of immunolabeled organoids enables preservation of the 3D cellular context, but conventional confocal microscopy of organoid cultures struggles with the large organoid sample size and relatively long distance from the objective to the organoid due to the 3D extracellular matrix (ECM) that surrounds the organoid. We have overcome these issues by physical separation of single organoids with their immediate stroma from the bulk ECM. Here we provide a detail protocol for the procedure, which entails single organoid collection and droplet-based staining and clearing to allow visualization of organoids in the greatest detail.

An Organotypic Assay to Study Epithelial-Fibroblast Interactions in Human Breast.

Epithelial-stromal interactions play an essential role in regulation of mammary gland development, homeostasis, and tumorigenesis. Fibroblasts constitute a substantial proportion of mammary gland stromal cells in human breast and have been recognized for their paracrine signaling and extracellular matrix production and remodeling roles during normal breast development as well as in breast cancer. However, our current knowledge on human breast fibroblast functions is incomplete. Here we provide a detailed protocol for an organotypic human breast assay to facilitate research in the roles of human breast fibroblasts in mammary epithelial morphogenesis and early tumorigenesis.

A Robust Mammary Organoid System to Model Lactation and Involution-like Processes.

The mammary gland is a highly dynamic tissue that changes throughout reproductive life, including growth during puberty and repetitive cycles of pregnancy and involution. Mammary gland tumors represent the most common cancer diagnosed in women worldwide. Studying the regulatory mechanisms of mammary gland development is essential for understanding how dysregulation can lead to breast cancer initiation and progression. Three-dimensional (3D) mammary organoids offer many exciting possibilities for the study of tissue development and breast cancer. In the present protocol derived from Sumbal et al., we describe a straightforward 3D organoid system for the study of lactation and involution ex vivo. We use primary and passaged mouse mammary organoids stimulated with fibroblast growth factor 2 (FGF2) and prolactin to model the three cycles of mouse mammary gland lactation and involution processes. This 3D organoid model represents a valuable tool to study late postnatal mammary gland development and breast cancer, in particular postpartum-associated breast cancer. Graphic abstract: Mammary gland organoid isolation and culture procedures.

Fibroblasts: The grey eminence of mammary gland development.

The essential role of mammary gland stroma in the regulation of mammary epithelial development, function, and cancer has long been recognized. Only recently, though, the functions of individual stromal cell populations have begun to become more clarified. Mammary fibroblasts have emerged as master regulators and modulators of epithelial cell behavior through paracrine signaling, extracellular matrix production and remodeling, and through regulation of other stromal cell types. In this review article, we summarize the crucial studies that helped to untangle the roles of fibroblasts in mammary gland development. Furthermore, we discuss the origin, heterogeneity, and plasticity of mammary fibroblasts during mammary development and cancer progression.

Mammary Organoids and 3D Cell Cultures: Old Dogs with New Tricks.

3D cell culture methods have been an integral part of and an essential tool for mammary gland and breast cancer research for half a century. In fact, mammary gland researchers, who discovered and deciphered the instructive role of extracellular matrix (ECM) in mammary epithelial cell functional differentiation and morphogenesis, were the pioneers of the 3D cell culture techniques, including organoid cultures. The last decade has brought a tremendous increase in the 3D cell culture techniques, including modifications and innovations of the existing techniques, novel biomaterials and matrices, new technological approaches, and increase in 3D culture complexity, accompanied by several redefinitions of the terms "3D cell culture" and "organoid". In this review, we provide an overview of the 3D cell culture and organoid techniques used in mammary gland biology and breast cancer research. We discuss their advantages, shortcomings and current challenges, highlight the recent progress in reconstructing the complex mammary gland microenvironment in vitro and ex vivo, and identify the missing 3D cell cultures, urgently needed to aid our understanding of mammary gland development, function, physiology, and disease, including breast cancer.

3D Cell Culture Models Demonstrate a Role for FGF and WNT Signaling in Regulation of Lung Epithelial Cell Fate and Morphogenesis.

FGF signaling plays an essential role in lung development, homeostasis, and regeneration. We employed mouse 3D cell culture models and imaging to study ex vivo the role of FGF ligands and the interplay of FGF signaling with epithelial growth factor (EGF) and WNT signaling pathways in lung epithelial morphogenesis and differentiation. In non-adherent conditions, FGF signaling promoted formation of lungospheres from lung epithelial stem/progenitor cells (LSPCs). Ultrastructural and immunohistochemical analyses showed that LSPCs produced more differentiated lung cell progeny. In a 3D extracellular matrix, FGF2, FGF7, FGF9, and FGF10 promoted lung organoid formation. FGF9 showed reduced capacity to promote lung organoid formation, suggesting that FGF9 has a reduced ability to sustain LSPC survival and/or initial divisions. FGF7 and FGF10 produced bigger organoids and induced organoid branching with higher frequency than FGF2 or FGF9. Higher FGF concentration and/or the use of FGF2 with increased stability and affinity to FGF receptors both increased lung organoid and lungosphere formation efficiency, respectively, suggesting that the level of FGF signaling is a crucial driver of LSPC survival and differentiation, and also lung epithelial morphogenesis. EGF signaling played a supportive but non-essential role in FGF-induced lung organoid formation. Analysis of tissue architecture and cell type composition confirmed that the lung organoids contained alveolar-like regions with cells expressing alveolar type I and type II cell markers, as well as airway-like structures with club cells and ciliated cells. FGF ligands showed differences in promoting distinct lung epithelial cell types. FGF9 was a potent inducer of more proximal cell types, including ciliated and basal cells. FGF7 and FGF10 directed the differentiation toward distal lung lineages. WNT signaling enhanced the efficiency of lung organoid formation, but in the absence of FGF10 signaling, the organoids displayed limited branching and less differentiated phenotype. In summary, we present lung 3D cell culture models as useful tools to study the role and interplay of signaling pathways in postnatal lung development and homeostasis, and we reveal distinct roles for FGF ligands in regulation of mouse lung morphogenesis and differentiation ex vivo.

Primary Mammary Organoid Model of Lactation and Involution.

Mammary gland development occurs mainly after birth and is composed of three successive stages: puberty, pregnancy and lactation, and involution. These developmental stages are associated with major tissue remodeling, including extensive changes in mammary epithelium, as well as surrounding stroma. Three-dimensional (3D) mammary organoid culture has become an important tool in mammary gland biology and enabled invaluable discoveries on pubertal mammary branching morphogenesis and breast cancer. However, a suitable 3D organoid model recapitulating key aspects of lactation and involution has been missing. Here, we describe a robust and straightforward mouse mammary organoid system modeling lactation and involution-like process, which can be applied to study mechanisms of physiological mammary gland lactation and involution as well as pregnancy-associated breast cancer.

Unraveling the Breast: Advances in Mammary Biology and Cancer Methods.

The field of mammary gland biology and breast cancer research encompasses a wide range of topics and scientific questions, which span domains of molecular, cell and developmental biology, cancer research, and veterinary and human medicine, with interdisciplinary overlaps to non-biological domains. Accordingly, mammary gland and breast cancer researchers employ a wide range of molecular biology methods, in vitro techniques, in vivo approaches as well as in silico analyses. The list of techniques is ever-expanding; together with the refinement of established, staple techniques in the field, new technologies keep emerging thanks to technological advances and scientific creativity. This issue of the Journal of Mammary Gland Biology and Neoplasia represents a compilation of original articles and reviews focused on methods used in mammary gland biology and breast cancer research.