CRISPR/Cas9-Mediated Genome Editing of Mouse Small Intestinal Organoids

2016 ◽  
pp. 3-11 ◽  
Author(s):  
Gerald Schwank ◽  
Hans Clevers
Keyword(s):  
Genome Editing ◽  
Intestinal Organoids ◽  
Small Intestinal

scholarly journals Porcine small intestinal organoids as a model to explore ETEC–host interactions in the gut

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Bjarne Vermeire ◽  
Liara M. Gonzalez ◽  
Robert J. J. Jansens ◽  
Eric Cox ◽  
Bert Devriendt
Keyword(s):  
Intestinal Epithelial ◽  
Gut Health ◽  
Functional Responses ◽  
Host Pathogen Interactions ◽  
Epithelial Surface ◽  
Host Interactions ◽  
Intestinal Organoids ◽  
Host Pathogen ◽  
Small Intestinal

AbstractSmall intestinal organoids, or enteroids, represent a valuable model to study host–pathogen interactions at the intestinal epithelial surface. Much research has been done on murine and human enteroids, however only a handful studies evaluated the development of enteroids in other species. Porcine enteroid cultures have been described, but little is known about their functional responses to specific pathogens or their associated virulence factors. Here, we report that porcine enteroids respond in a similar manner as in vivo gut tissues to enterotoxins derived from enterotoxigenic Escherichia coli, an enteric pathogen causing postweaning diarrhoea in piglets. Upon enterotoxin stimulation, these enteroids not only display a dysregulated electrolyte and water balance as shown by their swelling, but also secrete inflammation markers. Porcine enteroids grown as a 2D-monolayer supported the adhesion of an F4+ ETEC strain. Hence, these enteroids closely mimic in vivo intestinal epithelial responses to gut pathogens and are a promising model to study host–pathogen interactions in the pig gut. Insights obtained with this model might accelerate the design of veterinary therapeutics aimed at improving gut health.

Defined serum-free culture of human infant small intestinal organoids with predetermined doses of Wnt3a and R-spondin1 from surgical specimens

2021 ◽  
Author(s):  
Yuka Matsumoto ◽  
Hiroyuki Koga ◽  
Mirei Takahashi ◽  
Kazuto Suda ◽  
Takanori Ochi ◽  
...  
Keyword(s):  
Human Infant ◽  
Serum Free ◽  
Intestinal Organoids ◽  
Small Intestinal

scholarly journals Intestinal organoids expose heterogeneity in SARS-CoV-2 susceptibility

2021 ◽  
Author(s):  
Kyung Ku Jang ◽  
Maria E Kaczmarek ◽  
Simone Dallari ◽  
Ying-Han Chen ◽  
Jordan Axelrad ◽  
...  
Keyword(s):  
Viral Infections ◽  
Ex Vivo ◽  
Intestinal Barrier ◽  
Infectious Agents ◽  
Interferon Signature ◽  
Interindividual Differences ◽  
Intestinal Organoids ◽  
Small Intestinal ◽  
Substantial Heterogeneity ◽  
Primary Tissue

Organoids generated from primary human specimens facilitate investigation of the intestinal barrier by recreating the complex cellular composition of the epithelium. Although the significance remains unclear, intestinal organoid lines display heterogeneity in their growth and morphology. We hypothesized that organoids will also display variability in the degree to which they are susceptible to infectious agents. Using SARS-CoV-2 as a model, we found orders of magnitude differences in the amount of SARS-CoV-2 recovered from small intestinal and colonic organoids generated from different donors. SARS-CoV-2 burden did not correlate with demographic or clinical features associated with donors, but rather reflected the expression level of the virus receptor ACE2. Remarkably, organoid ACE2 transcript levels matched the amount of ACE2 detected in primary tissue from the same individual, indicating that certain properties of the intestinal epithelium are retained during ex vivo differentiation. Longitudinal transcriptomics of organoids identified a delayed yet robust interferon signature, the magnitude of which corresponded to the degree of SARS-CoV-2 infection. These results suggest that intestinal organoids display substantial heterogeneity in their ability to support viral infections and can potentially inform mechanisms behind interindividual differences in susceptibility to infectious disease.

33 Single-Cell Level Analysis Reveals Heterogeneous Expression of Stem-Cell Specific Genes in Human Small Intestinal Organoids

2016 ◽  
Vol 150 (4) ◽  
pp. S11
Author(s):  
Kohei Suzuki ◽  
Satoru Fujii ◽  
Ami Kawamoto ◽  
Fumiaki Ishibashi ◽  
Toru Nakata ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Single Cell Level ◽  
Cell Level ◽  
Intestinal Organoids ◽  
Heterogeneous Expression ◽  
Small Intestinal ◽  
Level Analysis

scholarly journals Cell isolation and Organoid Culture from Mouse Liver and Small Intestine

2021 ◽  
Author(s):  
Wenyi Chen ◽  
Qigu Yao ◽  
Ruo Wang ◽  
Yanping Xu ◽  
Jiong Yu ◽  
...  
Keyword(s):  
Small Intestine ◽  
Bile Ducts ◽  
Disease Modeling ◽  
Organoid Culture ◽  
Intrahepatic Bile Ducts ◽  
Tissue Organization ◽  
Intestinal Organoids ◽  
Gravity Settling ◽  
Small Intestinal

Abstract Background: Organoid culture enables disease modeling and drug screening in vitro. Organoids are from organs (e.g., brain, small intestine, kidney, lung, and liver). To facilitate the establishment of liver and small-intestinal organoids, we developed a protocol for collecting cholangiocytes and crypts and culturing organoids.Methods: Cholangiocytes were collected from intrahepatic bile ducts, gallbladder, and crypts from the small intestine using gravity settling and multi-step centrifugation methods, and embedded in Matrigel to grow into three-dimensional spheroids in suitable culture medium. Passaging, cryopreservation, and thawing were performed to assess organoid cell stability. RNA and DNA extraction, as well as immunostaining procedure were optimized. For preclinical modeling, the growth rate of cholangiocyte organoids (cho-orgs) was harmonized.Results: Large amount of Cholangiocytes and small intestine crypts were collected. Cholangiocytes developed into cyst-like structures after 3–4 days in Matrigel. After culture for 1–2 weeks, small-intestinal organoids developed buds and formed a mature structure. Cho-orgs from intrahepatic bile ducts grew more slowly but were longer lasting, expressed the cholangiocyte markers Krt19 and Krt7, and recapitulated the in vivo tissue organization.Conclusions: The protocol takes 2–4 weeks to establish a stable organoid growth system. Organoids could be stably passaged, cryopreserved, and recovered. The organoids retained tissue characteristics, including marker expression.

scholarly journals Culture and differentiation of rabbit intestinal organoids and organoid-derived cell monolayers

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Egi Kardia ◽  
Michael Frese ◽  
Elena Smertina ◽  
Tanja Strive ◽  
Xi-Lei Zeng ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
Cell Types ◽  
Productive Infection ◽  
Biological Processes ◽  
Cell Monolayers ◽  
Host Interactions ◽  
Monolayer Cultures ◽  
Intestinal Organoids ◽  
Small Intestinal ◽  
Conventional Cell

AbstractOrganoids emulate many aspects of their parental tissue and are therefore used to study pathogen-host interactions and other complex biological processes. Here, we report a robust protocol for the isolation, maintenance and differentiation of rabbit small intestinal organoids and organoid-derived cell monolayers. Our rabbit intestinal spheroid and monolayer cultures grew most efficiently in L-WRN-conditioned medium that contained Wnt, R-spondin and Noggin, and that had been supplemented with ROCK and TGF-β inhibitors. Organoid and monolayer differentiation was initiated by reducing the concentration of the L-WRN-conditioned medium and by adding ROCK and Notch signalling inhibitors. Immunofluorescence staining and RT-qPCR demonstrated that our organoids contained enterocytes, enteroendocrine cells, goblet cells and Paneth cells. Finally, we infected rabbit organoids with Rabbit calicivirus Australia-1, an enterotropic lagovirus that—like many other caliciviruses—does not grow in conventional cell culture. Despite testing various conditions for inoculation, we did not detect any evidence of virus replication, suggesting either that our organoids do not contain suitable host cell types or that additional co-factors are required for a productive infection of rabbit organoids with Rabbit calicivirus Australia-1.

Identity and cell fate of Ngn3-expressing population in small intestinal organoids

2018 ◽  
Author(s):  
Patricia Fonseca Pedro ◽  
Anastasia Tsakmaki ◽  
Gavin Bewick
Keyword(s):  
Cell Fate ◽  
Intestinal Organoids ◽  
Small Intestinal

scholarly journals Simultaneous paralogue knockout using a CRISPR-concatemer in mouse small intestinal organoids

2016 ◽  
Vol 420 (2) ◽  
pp. 271-277 ◽  
Author(s):  
Amanda Andersson-Rolf ◽  
Alessandra Merenda ◽  
Roxana C. Mustata ◽  
Taibo Li ◽  
Sabine Dietmann ◽  
...  
Keyword(s):  
Intestinal Organoids ◽  
Small Intestinal
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