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Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration
Abstract
In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.
... The ability to explant and expand various stem cells in vitro has been instrumental to study the biology of several regenerative tissues. In the stomach, early studies reported the successful explantation of mouse and human gastric epithelial cells, although these cultures could not be maintained long term [24][25][26][27][28] . Seminal follow-up studies demonstrated that embedding gastric glands in Matrigel facilitates the indefinite expansion of epithelial cells as three-dimensional (3D) organoids, which require Lgr5-expressing gland base-like cells to maintain the culture 13,15,[29][30][31][32] . ...
The glandular stomach is composed of two regenerative compartments termed corpus and antrum, and our understanding of the transcriptional networks that maintain these tissues is incomplete. Here we show that cell types with equivalent functional roles in the corpus and antrum share similar transcriptional states including the poorly characterized stem cells of the isthmus region. To further study the isthmus, we developed a monolayer two-dimensional (2D) culture system that is continually maintained by Wnt-responsive isthmus-like cells capable of differentiating into several gastric cell types. Importantly, 2D cultures can be converted into conventional three-dimensional organoids, modelling the plasticity of gastric epithelial cells in vivo. Finally, we utilized the 2D culture system to show that Sox2 is both necessary and sufficient to generate enterochromaffin cells. Together, our data provide important insights into gastric homeostasis, establish a tractable culture system to capture isthmus cells and uncover a role for Sox2 in enterochromaffin cells.
... In vitro experimentation using organoids and induced pluripotent stem cell models offers opportunities in the study of the microbiome, particularly in the human genetic context (Fig. 3). Organoids can now be generated from many organs and from different species, including humans (99) and mice (100). Human adult lung stem cells can generate lung organoids (99), and human blastocysts can be modeled to investigate toxin exposure and viral susceptibility during embryogenesis (101). ...
Interactions between the developing microbiome and maturing immune system in early life are critical for establishment of a homeostasis beneficial to both host and commensals. The lung harbors a diverse community of microbes associated with health and local or systemic disease. We discuss how early life colonization and community changes correlate with immune development and health and disease throughout infancy, childhood, and adult life. We highlight key advances in microbiology, immunology, and computational biology that allow investigation of the functional relevance of interactions between the respiratory microbiome and host immune system, which may unlock the potential for microbiome-based therapeutics.
... Drug discovery programs are beginning to exploit recent advances in patient-derived organoid cultures which have been shown to better model the heterogeneity observed within and between patients [23]. Approaches to screen the impact of various drugs on different organoid models to explore patient heterogeneity have primarily cultured organoids in 2D monolayers [29], [30], on the surface of hydrogel layers with dilute hydrogel in the surrounding media (the "slurry" method) [17], or within 3D hydrogel plugs [31]. Only the last of these approaches enables true 3D culture, in which cells are embedded within ECM. ...
Complex 3D bioengineered tumour models provide the opportunity to better capture the heterogeneity of patient tumours. Patient-derived organoids are emerging as a useful tool to study tumour heterogeneity and variation in patient responses. Organoid cultures typically require a 3D microenvironment that can be manufactured easily to facilitate screening. Here we set out to create a high-throughput, "off-the-shelf" platform which permits the generation of organoid- containing microtissues for standard phenotypic bioassays and image-based readings. To achieve this, we developed the Scaffold-supported Platform for Organoid-based Tissues (SPOT) platform. SPOT is a 3D gel-embedded in vitro platform that can be produced in a 96- or 384-well plate format and enables the generation of flat, thin and dimensionally-defined microgels. SPOT has high potential for adoption due to its reproducible manufacturing methodology, compatibility with existing instrumentation, and reduced within-sample and between-sample variation, which can pose challenges to both data analysis and interpretation. Using SPOT we generate cultures from patient derived pancreatic ductal adenocarcinoma organoids and assess the cellular response to standard-of-care chemotherapeutic compounds, demonstrating our platform's usability for drug screening. We envision 96/384-SPOT will provide a useful tool to assess drug sensitivity of patient-derived organoids and easily integrate into the drug discovery pipeline.
... This has been best exemplified in the context of intestinal cancers. Gastric organoids derived from murine and human tissue and co-cultured with CD8+ CTLs and DCs have been employed to elucidate important mechanistic insights into PD-L1 regulation in gastric tumor progression [134][135][136]. Such a system also allowed for convenient introduction of microbes such as Helicobacter pylori (H. ...
Immunosuppression in cancer has emerged as a major hurdle to immunotherapy efforts. Immunosuppression can arise from oncogene-induced signaling within the tumor as well as from tumor-associated immune cells. Understanding various mechanisms by which the tumor can undermine and evade therapy is critical in improving current cancer immunotherapies. While mouse models have allowed for the characterization of key immune cell types and their role in tumor development, extrapolating these mechanisms to patients has been challenging. There is need for better models to unravel the effects of genetic alterations inherent in tumor cells and immune cells isolated from tumors on tumor growth and to investigate the feasibility of immunotherapy. Three-dimensional (3D) organoid model systems have developed rapidly over the past few years and allow for incorporation of components of the tumor microenvironment such as immune cells and the stroma. This bears great promise for derivation of patient-specific models in a dish for understanding and determining the impact on personalized immunotherapy. In this review, we will highlight the significance of current experimental models employed in the study of tumor immunosuppression and evaluate current tumor organoid-immune cell co-culture systems and their potential impact in shedding light on cancer immunosuppression.
... electrical resistance assays to measure functional permeability, while maintaining the unique characteristics of ex vivo organoid cultures, such as molecular identity to the original tissue and presence of a number of different epithelial cell types, such as parietal, chief and mucous neck surface mucosal cells from the stomach (Fig. 4). 47 High-throughput microscopy can be performed in 2D-organoid-derived monolayer cultures, which would be difficult to perform in the 3D equivalent. ...
Cancer comprises a collection of highly proliferative and heterogeneous cells growing within an adaptive and evolving tumour microenvironment. Cancer survival rates have significantly improved following decades of cancer research. However, many experimental and preclinical studies do not translate to the bedside, reflecting the challenges of modelling the complexities and multicellular basis of human disease. Organoids are novel, complex, three-dimensional ex vivo tissue cultures that are derived from embryonic stem cells, induced pluripotent stem cells or tissue-resident progenitor cells, and represent a near-physiological model for studying cancer. Organoids develop by self-organisation, and can accurately represent the diverse genetic, cellular and pathophysiological hallmarks of cancer. In addition, co-culture methods and the ability to genetically manipulate these organoids have widened their utility in cancer research. Organoids thus offer a new and exciting platform for studying cancer and directing personalised therapies. This review aims to highlight how organoids are shaping the future of cancer research.
... However, the process of microinjection can become time consuming and labor intensive when the question of the epithelial response to H. pylori is the focus. Thus, we use a modified method described by Schlaermann et al. (2016), and our group to generate gastric epithelial monolayers derived from 3-dimensional tissue-derived gastric organoids (Teal, Bertaux-Skeirik, et al., 2018;Teal, Steele, et al., 2018). This methodology is optimized to establish monolayers from organoids and ideal for wound healing assays and epithelial/bacterial co-culture experiments. ...
... However, the process of microinjection can become time consuming and labor intensive when the question of the epithelial response to H. pylori is the focus. Thus, we use a modified method described by Schlaermann et al. (2016), and our group to generate gastric epithelial monolayers derived from 3-dimensional tissue-derived gastric organoids (Teal, Bertaux-Skeirik, et al., 2018;Teal, Steele, et al., 2018). This methodology is optimized to establish monolayers from organoids and ideal for wound healing assays and epithelial/bacterial co-culture experiments. ...
While the incidence of gastric cancer in the United States is relatively low due to the diagnosis and treatment of the major risk factor Helicobacter pylori (H. pylori), 5-year patient survival is only approximately 29%. Even after H. pylori infection has been eradicated there is still a risk of developing gastric cancer. Gastric cancer is the final clinical outcome that is often initiated by a sustained inflammatory response and altered epithelial cell differentiation and metaplasia in response to H. pylori infection. Identifying the early epithelial responses to H. pylori infection is important in advancing our understanding of the events that shape a conducive environment for the progression of gastric cancer. Thus, we developed an human gastric tissue-derived organoid-based approach to identify the initiating molecular events that trigger gastric cancer development in response to bacterial infection.
Preparation of kidney tissue single-cell suspensions is the basis of single-cell sequencing, flow cytometry and primary cell culture, but it is difficult to prepare high quality whole kidney single-cell suspensions because of the complex structure of the kidney. We explored a technique called stepwise enzymatic digestion (StE) method for preparing a single-cell suspension of rat whole kidney tissue which contained three main steps. The first step is to cut the kidney into a homogenate. The second step is the digestion of renal tubules using Multi Tissue Dissociation Kit 2 and the last step is the digestion of glomeruli using type IV collagenase. We also compared it with two previous techniques, mechanical grinding method and simple enzymatic digestion method. The StE method had the advantages of high intrinsic glomerular cells and immune cells harvest rate, high singlets rate and high cell viability compared with the other two techniques. In conclusion, the StE method is feasible, highly efficient, and worthy of further research and development.
Complex 3D bioengineered tumour models provide the opportunity to better capture the heterogeneity of patient tumours. Patient-derived organoids are emerging as a useful tool to study tumour heterogeneity and variation in patient responses. Organoid cultures typically require a 3D microenvironment that can be manufactured easily to facilitate screening. Here we set out to create a high-throughput, “off-the-shelf” platform which permits the generation of organoid-containing engineered microtissues for standard phenotypic bioassays and image-based readings. To achieve this, we developed the Scaffold-supported Platform for Organoid-based Tissues (SPOT) platform. SPOT is a 3D gel-embedded in vitro platform that can be produced in a 96- or 384-well plate format and enables the generation of flat, thin and dimensionally-defined microgels. SPOT has high potential for adoption due to its reproducible manufacturing methodology, compatibility with existing instrumentation, and reduced within-sample and between-sample variation, which can pose challenges to both data analysis and interpretation. Using SPOT, we generate cultures from patient derived pancreatic ductal adenocarcinoma organoids and assess the cellular response to standard-of-care chemotherapeutic compounds, demonstrating our platform's usability for drug screening. We envision 96/384-SPOT will provide a useful tool to assess drug sensitivity of patient-derived organoids and easily integrate into the drug discovery pipeline.
Gastric cancer is the fifth most commonly diagnosed cancer in the world. With over one million diagnosed cases per year, it is the third leading cause of cancer-related deaths worldwide. Since the establishment of cell culture in the early 1900s along with the generation of the first cell lines to study gastric cancer and disease, the breadth of knowledge and resources has grown substantially. A key element of the evolution of modeling for gastric carcinogenesis, in particular, has been the advent of genetic engineering and organoid culture. Genetic engineering has allowed the study of specific genes and “humanizing” model systems for preclinical use, while organoid culture provides a physiologically similar in vitro cell culture of patient-derived tissue. This chapter describes the generation and use of commonly used models in vitro and in vivo to study gastric disease and recent developments in methods and technology.
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