Long-term live imaging and multiscale analysis identify heterogeneity and core principles of epithelial organoid morphogenesis (original) (raw)
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2020
Organoids are morphologically heterogeneous three-dimensional cell culture systems. To understand the cell organisation principles of their morphogenesis, we imaged hundreds of pancreas and liver organoids in parallel using light sheet and bright field microscopy for up to seven days. We quantified organoid behaviour at single-cell (microscale), individual-organoid (mesoscale), and entire-culture (macroscale) levels. At single-cell resolution, we monitored formation, monolayer polarisation and degeneration, and identified diverse behaviours, including lumen expansion and decline (size oscillation), migration, rotation and multi-organoid fusion. Detailed individual organoid quantifications lead to a mechanical 3D agent-based model. A derived scaling law and simulations support the hypotheses that size oscillations depend on organoid properties and cell division dynamics, which is confirmed by bright field macroscale analyses of entire cultures. Our multiscale analysis provides a syst...
Subpopulations of Organoid-Forming Cells Have Different Motility
Applied Sciences
Cancer stem cells from oropharyngeal squamous cell carcinoma (OPSCC) have the ability to self-renew and differentiate into heterogeneous three-dimensional structures carrying features of tumor cells. Here, we describe a simple and label-free method for generating tumor organoids, and imaging them using live digital holographic microscopy (DHM) on the basis of the phase shift caused by light passing through the cells. We show early events of cell aggregation during tumor-organoid formation, and display their heterogeneity in terms of optical parameters up to an optical volume of 105 µm3. Lastly, by sorting OPSCC epithelial cells, we demonstrate that CD44+ cells displayed greater motility and tumor-forming capacity than those of CD44− cells. These results were in line with previous reports highlighting increased invasive and tumorigenic potential in tumor cells expressing high levels of CD44. Our method provides insight into the formation of tumor organoids, and could be used to asses...
Scientific Reports
Pancreatic ductal adenocarcinoma (PDAC) reportedly progresses very rapidly through the initial carcinogenesis stages including DNA damage and disordered cell death. However, such oncogenic mechanisms are largely studied through observational diagnostic methods, partly because of a lack of live in vitro tumour imaging techniques. Here we demonstrate a simple live-tumour in vitro imaging technique using micro-patterned plates (micro/nanoplates) that allows dynamic visualisation of PDAC microtumours. When PDAC cells were cultured on a micro/nanoplate overnight, the cells self-organised into non-spheroidal microtumours that were anchored to the micro/nanoplate through cell-in-cell invasion. This self-organisation was only efficiently induced in small-diameter rough microislands. Using a time-lapse imaging system, we found that PDAC microtumours actively stretched to catch dead cell debris via filo/lamellipoedia and suction, suggesting that they have a sophisticated survival strategy (analogous to that of starving animals), which implies a context for the development of possible therapies for PDACs. The simple tumour imaging system visualises a potential of PDAC cells, in which the aggressive tumour dynamics reminds us of the need to review traditional PDAC pathogenesis. Despite accumulating evidence on the pathological features exhibited by cancer cells in various carcinomas, recent in vitro cancer cell studies have focused on the behavior of single cells in isolation. In contrast, in analyses conducted at the tumor tissue level, in vivo methodologies still largely depend on observational diagnostic methods such as histopathological analysis and in vivo imaging systems using fluorescent imaging probes. Consequently, the underlying pathophysiological tumor dynamics in tissue remains mostly unclear. Thus, in current cancer research, directly linking in vitro cell-based studies with in vivo tissue-based pathological studies could result in a huge gap in our understanding. Indeed, we have been confronted with numerous unexpected difficulties in clinical trials of molecular-targeted anticancer agents for which there should be near perfect evidence for targets from conventional evaluations undertaken both in vivo and in vitro. Recently, the importance of using three-dimensional (3D) cell culture systems has remarkably increased in the field of anticancer drug development 1-4 ; 3D cell culture systems are expected to provide more physiologically relevant information to the in vivo setting as compared with traditional two-dimensional culture systems 5-8. PDAC, which constitutes approximately 90% of pancreatic cancers, is still one of the most lethal malignant tumours 9. KRAS mutation is the initiating genetic event for pancreatic intraepithelial neoplasia (PanINs), premalignant lesions of PDACs 10. Recent work has shown that once PDACs become detectable, they progress from T1 stage to T4 stage in approximately 14 months 11. PDACs rapidly progress through highly frequent DNA damage and mitotic abnormalities through unknown catastrophic events 12,13. Generally, the epithelial-mesenchymal
Organoids: inception and utilization of 3D organ models
2020
Over the previous decade, one of the most exciting advancements in stem cell technology has been the development of organoid culture system. Organoids are new research tools created in-vitro, to form self-organizing 3-Dimensional structures that encompass some of the crucial characteristics of the represented organ. Organoids are grown from stem cells from an organ of interest. There are potentially as many types of organoids as there are different tissues and organs in a body. It is challenging for scientists to understand the underlying mechanism of biological processes with complex spatial cellular organization and tissue dynamics. Also, how they are disrupted in a disease is impossible to study in-vivo, but discovery of organoids is revolutionizing the fields of biology. Since success in these platforms will be restricted without the proficiency to alter the genomic content, genome engineering was also applied in recently discovered organoid cultures for correcting mutations. Th...
OrgDyn: feature- and model-based characterization of spatial and temporal organoid dynamics
Bioinformatics, 2020
Summary Organoid model systems recapitulate key features of mammalian tissues and enable high throughput experiments. However, the impact of these experiments may be limited by manual, non-standardized, static or qualitative phenotypic analysis. OrgDyn is an open-source and modular pipeline to quantify organoid shape dynamics using a combination of feature- and model-based approaches on time series of 2D organoid contour images. Our pipeline consists of (i) geometrical and signal processing feature extraction, (ii) dimensionality reduction to differentiate dynamical paths, (iii) time series clustering to identify coherent groups of organoids and (iv) dynamical modeling using point distribution models to explain temporal shape variation. OrgDyn can characterize, cluster and model differences among unique dynamical paths that define diverse final shapes, thus enabling quantitative analysis of the molecular basis of tissue development and disease. Availability and Implementation https:...
Automated high-speed 3D imaging of organoid cultures with multi-scale phenotypic quantification
Nature Methods
Current imaging approaches limit the ability to perform multiscale characterization of 3D organotypic cultures (organoids) in large numbers. Here, we present an automated multiscale 3D imaging platform synergizing high-density organoid cultures with 3D live single objective light-sheet imaging. It is composed of disposable microfabricated organoid culture chips embedding optical components and a custom laser beam steering unit coupled to a commercial inverted microscope. It streamlines organoid culture and high content 3D imaging on a single user-friendly instrument with minimal manipulations and unprecedented throughput of 300 organoids per hour in 3D. Collecting large number of 3D stacks allowed training deep learning-based algorithms to quantify the organoids morphogenetic organizations at multi-scales, ranging from the sub-cellular scale to the whole organoid level. We validated the versatility and robustness of our approach on intestine, hepatic, neuroectoderm organoids and oncospheres.
Journal of Mathematical Biology, 2009
Collective phenomena in multi-cellular assemblies can be approached on different levels of complexity. Here, we discuss a number of mathematical models which consider the dynamics of each individual cell, so-called agent-based or individual-based models (IBMs). As a special feature, these models allow to account for intracellular decision processes which are triggered by biomechanical cell-cell or cell-matrix interactions. We discuss their impact on the growth and homeostasis of multi-cellular systems as simulated by lattice-free models. Our results demonstrate that cell polarisation subsequent to cell-cell contact formation can be a source of stability in epithelial monolayers. Stroma contact-dependent regulation of tumour cell proliferation and migration is shown to result in invasion dynamics in accordance with the migrating cancer stem cell hypothesis. However, we demonstrate that different regulation mechanisms can equally well comply with present experimental results. Thus, we suggest a panel of experimental studies for the in-depth validation of the model assumptions.
Organoids: a novel modality in disease modeling
Bio-Design and Manufacturing, 2021
Limitations of monolayer culture conditions have motivated scientists to explore new models that can recapitulate the architecture and function of human organs more accurately. Recent advances in the improvement of protocols have resulted in establishing three-dimensional (3D) organ-like architectures called 'organoids' that can display the characteristics of their corresponding real organs, including morphological features, functional activities, and personalized responses to specific pathogens. We discuss different organoid-based 3D models herein, which are classified based on their original germinal layer. Studies of organoids simulating the complexity of real tissues could provide novel platforms and opportunities for generating practical knowledge along with preclinical studies, including drug screening, toxicology, and molecular pathophysiology of diseases. This paper also outlines the key challenges, advantages, and prospects of current organoid systems.
Cancers, 2019
Primary liver cancer, consisting predominantly of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), remains one of the most lethal malignancies worldwide. This high malignancy is related to the complex and dynamic interactions between tumour cells, stromal cells and the extracellular environment. Novel in vitro models that can recapitulate the tumour are essential in increasing our understanding of liver cancer. Herein, primary liver cancer-derived organoids have opened up new avenues due to their patient-specificity, self-organizing ability and potential recapitulation of many of the tumour properties. Organoids are solely of epithelial origin, but incorporation into co-culture models can enable the investigation of the cellular component of the tumour microenvironment. However, the extracellular component also plays a vital role in cancer progression and representation is lacking within current in vitro models. In this review, organoid technology is discussed in the con...