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Article ; Online: Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids.

van Ineveld, Ravian L / Ariese, Hendrikus C R / Wehrens, Ellen J / Dekkers, Johanna F / Rios, Anne C

Journal of visualized experiments : JoVE

2020 , Issue 160

Abstract: Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing ... ...

Abstract	Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.
MeSH term(s)	Animals ; Humans ; Imaging, Three-Dimensional/methods ; Mice ; Organoids/diagnostic imaging ; Organoids/growth & development
Language	English
Publishing date	2020-06-05
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/60709
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Single-cell resolution three-dimensional imaging of intact organoids

van Ineveld, Ravian L / Ariese, Hendrikus C.R / Wehrens, Ellen J / Dekkers, Johanna F / Rios, Anne C

Journal of visualized experiments. 2020 June 05, , no. 160

2020

Abstract	Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.
Keywords	breast neoplasms ; colon ; confocal microscopy ; fluorescence microscopy ; humans ; image analysis ; kidneys ; liver ; mammary glands ; organoids
Language	English
Dates of publication	2020-0605
Size	p. e60709.
Publishing place	Journal of Visualized Experiments
Document type	Article
Note	NAL-AP-2-clean
ZDB-ID	2259946-0
ISSN	1940-087X
ISSN	1940-087X
DOI	10.3791/60709
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: BEHAV3D: a 3D live imaging platform for comprehensive analysis of engineered T cell behavior and tumor response.

Alieva, Maria / Barrera Román, Mario / de Blank, Sam / Petcu, Diana / Zeeman, Amber L / Dautzenberg, Noël M M / Cornel, Annelisa M / van de Ven, Cesca / Pieters, Rob / den Boer, Monique L / Nierkens, Stefan / Calkoen, Friso G J / Clevers, Hans / Kuball, Jürgen / Sebestyén, Zsolt / Wehrens, Ellen J / Dekkers, Johanna F / Rios, Anne C

Nature protocols

2024

Abstract: Modeling immuno-oncology by using patient-derived material and immune cell co-cultures can advance our understanding of immune cell tumor targeting in a patient-specific manner, offering leads to improve cellular immunotherapy. However, fully exploiting ... ...

Abstract	Modeling immuno-oncology by using patient-derived material and immune cell co-cultures can advance our understanding of immune cell tumor targeting in a patient-specific manner, offering leads to improve cellular immunotherapy. However, fully exploiting these living cultures requires analysis of the dynamic cellular features modeled, for which protocols are currently limited. Here, we describe the application of BEHAV3D, a platform that implements multi-color live 3D imaging and computational tools for: (i) analyzing tumor death dynamics at both single-organoid or cell and population levels, (ii) classifying T cell behavior and (iii) producing data-informed 3D images and videos for visual inspection and further insight into obtained results. Together, this enables a refined assessment of how solid and liquid tumors respond to cellular immunotherapy, critically capturing both inter- and intratumoral heterogeneity in treatment response. In addition, BEHAV3D uncovers T cell behavior involved in tumor targeting, offering insight into their mode of action. Our pipeline thereby has strong implications for comparing, prioritizing and improving immunotherapy products by highlighting the behavioral differences between individual tumor donors, distinct T cell therapy concepts or subpopulations. The protocol describes critical wet lab steps, including co-culture preparations and fast 3D imaging with live cell dyes, a segmentation-based image processing tool to track individual organoids, tumor and immune cells and an analytical pipeline for behavioral profiling. This 1-week protocol, accessible to users with basic cell culture, imaging and programming expertise, can easily be adapted to any type of co-culture to visualize and exploit cell behavior, having far-reaching implications for the immuno-oncology field and beyond.
Language	English
Publishing date	2024-03-19
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	2244966-8
ISSN	1750-2799 ; 1754-2189
ISSN (online)	1750-2799
ISSN	1754-2189
DOI	10.1038/s41596-024-00972-6
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Article ; Online: In vivo genome-editing screen identifies tumor suppressor genes that cooperate with Trp53 loss during mammary tumorigenesis.

Heitink, Luuk / Whittle, James R / Vaillant, François / Capaldo, Bianca D / Dekkers, Johanna F / Dawson, Caleb A / Milevskiy, Michael J G / Surgenor, Elliot / Tsai, Minhsuang / Chen, Huei-Rong / Christie, Michael / Chen, Yunshun / Smyth, Gordon K / Herold, Marco J / Strasser, Andreas / Lindeman, Geoffrey J / Visvader, Jane E

Molecular oncology

2022 Volume 16, Issue 5, Page(s) 1119–1131

Abstract: Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. To gain insight into mutations that drive breast tumorigenesis, we describe a pipeline for the identification and validation of tumor suppressor genes. ... ...

Abstract	Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. To gain insight into mutations that drive breast tumorigenesis, we describe a pipeline for the identification and validation of tumor suppressor genes. Based on an in vivo genome-wide CRISPR/Cas9 screen in Trp53
MeSH term(s)	Animals ; CRISPR-Cas Systems/genetics ; Cell Transformation, Neoplastic/genetics ; Gene Editing ; Genes, Tumor Suppressor ; Humans ; Mice ; Tumor Suppressor Protein p53/genetics ; Tumor Suppressor Protein p53/metabolism
Chemical Substances	Tumor Suppressor Protein p53
Language	English
Publishing date	2022-01-26
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2415106-3
ISSN	1878-0261 ; 1574-7891
ISSN (online)	1878-0261
ISSN	1574-7891
DOI	10.1002/1878-0261.13179
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Article ; Online: Identification of Enteroendocrine Regulators by Real-Time Single-Cell Differentiation Mapping.

Gehart, Helmuth / van Es, Johan H / Hamer, Karien / Beumer, Joep / Kretzschmar, Kai / Dekkers, Johanna F / Rios, Anne / Clevers, Hans

Cell

2019 Volume 176, Issue 5, Page(s) 1158–1173.e16

Abstract: Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real time at single-cell level using a novel knockin allele of ... ...

Abstract	Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real time at single-cell level using a novel knockin allele of Neurog3, the master regulator gene briefly expressed at the onset of enteroendocrine specification. A bi-fluorescent reporter, Neurog3Chrono, measures time from the onset of enteroendocrine differentiation and enables precise positioning of single-cell transcriptomes along an absolute time axis. This approach yielded a definitive description of the enteroendocrine hierarchy and its sub-lineages, uncovered differential kinetics between sub-lineages, and revealed time-dependent hormonal plasticity in enterochromaffin and L cells. The time-resolved map of transcriptional changes predicted multiple novel molecular regulators. Nine of these were validated by conditional knockout in mice or CRISPR modification in intestinal organoids. Six novel candidate regulators (Sox4, Rfx6, Tox3, Myt1, Runx1t1, and Zcchc12) yielded specific enteroendocrine phenotypes. Our time-resolved single-cell transcriptional map presents a rich resource to unravel enteroendocrine differentiation.
MeSH term(s)	Animals ; Basic Helix-Loop-Helix Transcription Factors/genetics ; Cell Differentiation/genetics ; Cell Lineage/genetics ; Cell Lineage/physiology ; Enteroendocrine Cells/metabolism ; Enteroendocrine Cells/physiology ; Fluorescent Dyes ; Gene Expression Profiling/methods ; Homeodomain Proteins/genetics ; Intestinal Mucosa/cytology ; Mice ; Mice, Knockout ; Nerve Tissue Proteins/genetics ; Optical Imaging/methods ; Organoids ; Phenotype ; Single-Cell Analysis/methods ; Stem Cells ; Transcription Factors/genetics ; Transcriptome/genetics
Chemical Substances	Basic Helix-Loop-Helix Transcription Factors ; Fluorescent Dyes ; Homeodomain Proteins ; Nerve Tissue Proteins ; Transcription Factors
Language	English
Publishing date	2019-01-31
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	187009-9
ISSN	1097-4172 ; 0092-8674
ISSN (online)	1097-4172
ISSN	0092-8674
DOI	10.1016/j.cell.2018.12.029
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Zs.A 1167: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids.

Dekkers, Johanna F / van Vliet, Esmée J / Sachs, Norman / Rosenbluth, Jennifer M / Kopper, Oded / Rebel, Heggert G / Wehrens, Ellen J / Piani, Carol / Visvader, Jane E / Verissimo, Carla S / Boj, Sylvia F / Brugge, Joan S / Clevers, Hans / Rios, Anne C

Nature protocols

2021 Volume 16, Issue 4, Page(s) 1936–1965

Abstract: Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various ... ...

Abstract	Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive). Additionally, we provide methods for genetic manipulation by Lipofectamine 2000, electroporation or lentivirus and subsequent organoid selection and clonal culture. Finally, we introduce an optimized method for orthotopic organoid transplantation in mice, which includes injection of organoids and estrogen pellets without the need for surgery. Organoid derivation from tissue fragments until the first split takes 7-21 d; generation of genetically manipulated clonal organoid cultures takes 14-21 d; and organoid expansion for xenotransplantation takes >4 weeks.
MeSH term(s)	Animals ; Biological Specimen Banks ; Breast/pathology ; Breast Neoplasms/pathology ; Cell Culture Techniques/methods ; Clone Cells ; Female ; Genetic Techniques ; Humans ; Mice ; Organoids/pathology ; Time Factors ; Transplantation, Heterologous
Language	English
Publishing date	2021-03-10
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2244966-8
ISSN	1750-2799 ; 1754-2189
ISSN (online)	1750-2799
ISSN	1754-2189
DOI	10.1038/s41596-020-00474-1
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Article ; Online: Transplantable programmed death ligand 1 expressing gastroids from gastric cancer prone Nfkb1

Low, Jun T / Ho, Gwo-Yaw / Scott, Mark / Tan, Chin Wee / Whitehead, Lachlan / Barber, Kathy / Yip, Hon Y K / Dekkers, Johanna F / Hirokawa, Yumiko / Silke, John / Burgess, Antony W / Strasser, Andreas / Putoczki, Tracy L / O'Reilly, Lorraine A

Cell death & disease

2021 Volume 12, Issue 12, Page(s) 1091

MeSH term(s)	Animals ; Apoptosis Regulatory Proteins/metabolism ; Disease Models, Animal ; Humans ; Male ; Mice ; NF-kappa B p50 Subunit/metabolism ; Stomach Neoplasms/genetics
Chemical Substances	Apoptosis Regulatory Proteins ; NF-kappa B p50 Subunit ; Nfkb1 protein, mouse (147257-52-1)
Language	English
Publishing date	2021-11-17
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2541626-1
ISSN	2041-4889 ; 2041-4889
ISSN (online)	2041-4889
ISSN	2041-4889
DOI	10.1038/s41419-021-04376-2
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Article: Novel opportunities for CFTR-targeting drug development using organoids.

Dekkers, Johanna F / van der Ent, Cornelis K / Beekman, Jeffrey M

Rare diseases (Austin, Tex.)

2013 Volume 1, Page(s) e27112

Abstract: Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR mutations lead to production of non-functional CFTR, reduced amounts of normal functioning CFTR or misfolded CFTR with defects in ... ...

Abstract	Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR mutations lead to production of non-functional CFTR, reduced amounts of normal functioning CFTR or misfolded CFTR with defects in trafficking or function. For decades, CF treatment has been focused on the symptoms of CF, but pharmacotherapy using small molecules that target the basic defect of CF, the mutant CFTR protein, is now possible for a limited amount of subjects with CF. This raises the exciting possibility that the majority of people with CF may receive effective treatment targeting the different CFTR mutants in the future. We recently described a functional CFTR assay using rectal biopsies from subjects with CF that were cultured in vitro into self-organizing mini-guts or organoids. We here describe how this model may assist in the discovery of new CFTR-targeting drugs, the subjects that may benefit from these drugs, and the mechanisms underlying variability in CFTR genotype-phenotype relations.
Language	English
Publishing date	2013-11-11
Publishing country	United States
Document type	Journal Article
ZDB-ID	2817861-0
ISSN	2167-5511
ISSN	2167-5511
DOI	10.4161/rdis.27112
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Article ; Online: Taxanes trigger cancer cell killing in vivo by inducing non-canonical T cell cytotoxicity.

Vennin, Claire / Cattaneo, Chiara M / Bosch, Leontien / Vegna, Serena / Ma, Xuhui / Damstra, Hugo G J / Martinovic, Moreno / Tsouri, Efi / Ilic, Mila / Azarang, Leyla / van Weering, Jan R T / Pulver, Emilia / Zeeman, Amber L / Schelfhorst, Tim / Lohuis, Jeroen O / Rios, Anne C / Dekkers, Johanna F / Akkari, Leila / Menezes, Renee /

Medema, Rene / Baglio, Serena R / Akhmanova, Anna / Linn, Sabine C / Lemeer, Simone / Pegtel, Dirk M / Voest, Emile E / van Rheenen, Jacco

Cancer cell

2023 Volume 41, Issue 6, Page(s) 1170–1185.e12

Abstract: Although treatment with taxanes does not always lead to clinical benefit, all patients are at risk of their detrimental side effects such as peripheral neuropathy. Understanding the in vivo mode of action of taxanes can help design improved treatment ... ...

Abstract	Although treatment with taxanes does not always lead to clinical benefit, all patients are at risk of their detrimental side effects such as peripheral neuropathy. Understanding the in vivo mode of action of taxanes can help design improved treatment regimens. Here, we demonstrate that in vivo, taxanes directly trigger T cells to selectively kill cancer cells in a non-canonical, T cell receptor-independent manner. Mechanistically, taxanes induce T cells to release cytotoxic extracellular vesicles, which lead to apoptosis specifically in tumor cells while leaving healthy epithelial cells intact. We exploit these findings to develop an effective therapeutic approach, based on transfer of T cells pre-treated with taxanes ex vivo, thereby avoiding toxicity of systemic treatment. Our study reveals a different in vivo mode of action of one of the most commonly used chemotherapies, and opens avenues to harness T cell-dependent anti-tumor effects of taxanes while avoiding systemic toxicity.
MeSH term(s)	Humans ; T-Lymphocytes ; Taxoids/pharmacology ; Apoptosis ; Epithelial Cells ; Extracellular Vesicles ; Neoplasms/drug therapy
Chemical Substances	Taxoids
Language	English
Publishing date	2023-06-11
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2078448-X
ISSN	1878-3686 ; 1535-6108
ISSN (online)	1878-3686
ISSN	1535-6108
DOI	10.1016/j.ccell.2023.05.009
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Zs.A 5650: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (2.OG) ab Jg. 2022: Lesesaal (EG)
Zs.MG 104: Show issues

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Article ; Online: Derivation of a robust mouse mammary organoid system for studying tissue dynamics.

Jamieson, Paul R / Dekkers, Johanna F / Rios, Anne C / Fu, Nai Yang / Lindeman, Geoffrey J / Visvader, Jane E

Development (Cambridge, England)

2017 Volume 144, Issue 6, Page(s) 1065–1071

Abstract: Advances in stem cell research have enabled the generation of 'mini organs' or organoids that recapitulate phenotypic traits of the original biological specimen. Although organoids have been demonstrated for multiple organ systems, there are more limited ...

Abstract	Advances in stem cell research have enabled the generation of 'mini organs' or organoids that recapitulate phenotypic traits of the original biological specimen. Although organoids have been demonstrated for multiple organ systems, there are more limited options for studying mouse mammary gland formation
MeSH term(s)	Animals ; Cell Lineage ; Clone Cells ; Epithelial Cells/cytology ; Female ; Flow Cytometry ; Genes, Reporter ; Imaging, Three-Dimensional ; Mammary Glands, Animal/cytology ; Mammary Glands, Animal/growth & development ; Mice ; Microscopy, Confocal ; Organoids/cytology ; Tissue Culture Techniques/methods
Language	English
Publishing date	2017--15
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	90607-4
ISSN	1477-9129 ; 0950-1991
ISSN (online)	1477-9129
ISSN	0950-1991
DOI	10.1242/dev.145045
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