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American Journal of Physiology. Cell... Jul 2020In vitro cell cultures are crucial research tools for modeling human development and diseases. Although the conventional monolayer cell cultures have been widely used in... (Review)
Review
In vitro cell cultures are crucial research tools for modeling human development and diseases. Although the conventional monolayer cell cultures have been widely used in the past, the lack of tissue architecture and complexity of such model fails to inform the true biological processes in vivo. Recent advances in the organoid technology have revolutionized the in vitro culture tools for biomedical research by creating powerful three-dimensional (3D) models to recapitulate the cellular heterogeneity, structure, and functions of the primary tissues. Such organoid technology enables researchers to recreate human organs and diseases in a dish and thus holds great promises for many translational applications such as regenerative medicine, drug discovery, and precision medicine. In this review, we provide an overview of the organoid history and development. We discuss the strengths and limitations of organoids as well as their potential applications in the laboratory and the clinic.
Topics: Animals; Biomedical Research; Cell Culture Techniques; Humans; Models, Biological; Organ Culture Techniques; Organoids
PubMed: 32459504
DOI: 10.1152/ajpcell.00120.2020 -
Current Protocols in Immunology Sep 2020Human intestinal organoids derived from adult stem cells are miniature ex vivo versions of the human intestinal epithelium. Intestinal organoids are useful tools for the...
Human intestinal organoids derived from adult stem cells are miniature ex vivo versions of the human intestinal epithelium. Intestinal organoids are useful tools for the study of intestinal physiology as well as many disease conditions. These organoids present numerous advantages compared to immortalized cell lines, but working with them requires dedicated techniques. The protocols described in this article provide a basic guide to establishment and maintenance of human intestinal organoids derived from small intestine and colon biopsies. Additionally, this article provides an overview of several downstream applications of human intestinal organoids. © 2020 The Authors. Basic Protocol 1: Establishment of human small intestine and colon organoid cultures from fresh biopsies Basic Protocol 2: Mechanical splitting, passage, and expansion of human intestinal organoids Alternate Protocol: Differentiation of human intestinal organoids Basic Protocol 3: Cryopreservation and thawing of human intestinal organoids Basic Protocol 4: Immunofluorescence staining of human intestinal organoids Basic Protocol 5: Generation of single-cell clonal intestinal organoid cultures Support Protocol 1: Production of Wnt3A conditioned medium Support Protocol 2: Production of Rspo1 conditioned medium Support Protocol 3: Extraction of RNA from intestinal organoid cultures.
Topics: Adult Stem Cells; Biomarkers; Biopsy; Cell Culture Techniques; Cell Differentiation; Cell Separation; Cells, Cultured; Colon; Cryopreservation; Culture Media, Conditioned; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Immunophenotyping; Intestinal Mucosa; Intestine, Small; Organoids; Tissue Culture Techniques
PubMed: 32940424
DOI: 10.1002/cpim.106 -
Journal of Animal Science Aug 2020Cultured meat grown in vitro from animal cells has the potential to address many of the ethical, environmental, and public health issues associated with conventional... (Review)
Review
Cultured meat grown in vitro from animal cells has the potential to address many of the ethical, environmental, and public health issues associated with conventional meat production. However, as well as overcoming technical challenges to producing cultured meat, producers and advocates of the technology must consider a range of social issues, including consumer appeal and acceptance, media coverage, religious status, regulation, and potential economic impacts. Whilst much has been written on the prospects for consumer appeal and acceptance of cultured meat, less consideration has been given to the other aspects of the social world that will interact with this new technology. Here, each of these issues is considered in turn, forming a view of cultured meat as a technology with a diverse set of societal considerations and far-reaching social implications. It is argued that the potential gains from a transition to cultured meat are vast, but that cultural phenomena and institutions must be navigated carefully for this nascent industry to meet its potential.
Topics: Animals; Consumer Behavior; Culture; Food Preferences; Food Technology; Humans; Meat; Tissue Culture Techniques
PubMed: 32745186
DOI: 10.1093/jas/skaa172 -
Nature Protocols Feb 2016This protocol describes a strategy for the generation of 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FACS-sorted single...
This protocol describes a strategy for the generation of 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FACS-sorted single luminal and basal cells), metastatic prostate cancer lesions and circulating tumor cells. Organoids derived from healthy material contain the differentiated luminal and basal cell types, whereas organoids derived from prostate cancer tissue mimic the histology of the tumor. We explain how to establish these cultures in the fully defined serum-free conditioned medium that is required to sustain organoid growth. Starting with the plating of digested tissue material, full-grown organoids can usually be obtained in ∼2 weeks. The culture protocol we describe here is currently the only one that allows the growth of both the luminal and basal prostatic epithelial lineages, as well as the growth of advanced prostate cancers. Organoids established using this protocol can be used to study many different aspects of prostate biology, including homeostasis, tumorigenesis and drug discovery.
Topics: Animals; Cells, Cultured; Culture Media, Serum-Free; Epithelial Cells; Humans; Male; Mice; Organ Culture Techniques; Organoids; Prostate; Time
PubMed: 26797458
DOI: 10.1038/nprot.2016.006 -
Nature Protocols Apr 2021Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are...
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.
Topics: Animals; Biological Specimen Banks; Breast; Breast Neoplasms; Cell Culture Techniques; Clone Cells; Female; Genetic Techniques; Humans; Mice; Organoids; Time Factors; Transplantation, Heterologous
PubMed: 33692550
DOI: 10.1038/s41596-020-00474-1 -
Cell Research Mar 2021The capacity of 3D organoids to mimic physiological tissue organization and functionality has provided an invaluable tool to model development and disease in vitro....
The capacity of 3D organoids to mimic physiological tissue organization and functionality has provided an invaluable tool to model development and disease in vitro. However, conventional organoid cultures primarily represent the homeostasis of self-organizing stem cells and their derivatives. Here, we established a novel intestinal organoid culture system composed of 8 components, mainly including VPA, EPZ6438, LDN193189, and R-Spondin 1 conditioned medium, which mimics the gut epithelium regeneration that produces hyperplastic crypts following injury; therefore, these organoids were designated hyperplastic intestinal organoids (Hyper-organoids). Single-cell RNA sequencing identified different regenerative stem cell populations in our Hyper-organoids that shared molecular features with in vivo injury-responsive Lgr5 stem cells or Clu revival stem cells. Further analysis revealed that VPA and EPZ6438 were indispensable for epigenome reprogramming and regeneration in Hyper-organoids, which functioned through epigenetically regulating YAP signaling. Furthermore, VPA and EPZ6438 synergistically promoted regenerative response in gut upon damage in vivo. In summary, our results demonstrated a new in vitro organoid model to study epithelial regeneration, highlighting the importance of epigenetic reprogramming that pioneers tissue repair.
Topics: Animals; Benzamides; Biphenyl Compounds; Cells, Cultured; Colitis; Culture Media, Conditioned; Dextran Sulfate; Disease Models, Animal; Female; Intestinal Mucosa; Intestines; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Morpholines; Organoids; Pyridones; Radiation Injuries; Regeneration; Signal Transduction; Stem Cells; Tissue Culture Techniques; Treatment Outcome; Valproic Acid
PubMed: 33420425
DOI: 10.1038/s41422-020-00453-x -
Theranostics 2022The advent of human pluripotent stem cells (hPSCs) presented a new paradigm to employ hPSC-derived cardiomyocytes (hPSC-CMs) in drug screening and disease modeling.... (Review)
Review
The advent of human pluripotent stem cells (hPSCs) presented a new paradigm to employ hPSC-derived cardiomyocytes (hPSC-CMs) in drug screening and disease modeling. However, hPSC-CMs differentiated in conventional two-dimensional systems are structurally and functionally immature. Moreover, these differentiation systems generate predominantly one type of cell. Since the heart includes not only CMs but other cell types, such monolayer cultures have limitations in simulating the native heart. Accordingly, three-dimensional (3D) cardiac tissues have been developed as a better platform by including various cardiac cell types and extracellular matrices. Two advances were made for 3D cardiac tissue generation. One type is engineered heart tissues (EHTs), which are constructed by 3D cell culture of cardiac cells using an engineering technology. This system provides a convenient real-time analysis of cardiac function, as well as a precise control of the input/output flow and mechanical/electrical stimulation. The other type is cardiac organoids, which are formed through self-organization of differentiating cardiac lineage cells from hPSCs. While mature cardiac organoids are more desirable, at present only primitive forms of organoids are available. In this review, we discuss various models of hEHTs and cardiac organoids emulating the human heart, focusing on their unique features, utility, and limitations.
Topics: Cell Differentiation; Humans; Myocytes, Cardiac; Organoids; Pluripotent Stem Cells; Tissue Engineering
PubMed: 35401829
DOI: 10.7150/thno.67661 -
Nature Communications Mar 2022Matrigel, a mouse tumor extracellular matrix protein mixture, is an indispensable component of most organoid tissue culture. However, it has limited the utility of...
Matrigel, a mouse tumor extracellular matrix protein mixture, is an indispensable component of most organoid tissue culture. However, it has limited the utility of organoids for drug development and regenerative medicine due to its tumor-derived origin, batch-to-batch variation, high cost, and safety issues. Here, we demonstrate that gastrointestinal tissue-derived extracellular matrix hydrogels are suitable substitutes for Matrigel in gastrointestinal organoid culture. We found that the development and function of gastric or intestinal organoids grown in tissue extracellular matrix hydrogels are comparable or often superior to those in Matrigel. In addition, gastrointestinal extracellular matrix hydrogels enabled long-term subculture and transplantation of organoids by providing gastrointestinal tissue-mimetic microenvironments. Tissue-specific and age-related extracellular matrix profiles that affect organoid development were also elucidated through proteomic analysis. Together, our results suggest that extracellular matrix hydrogels derived from decellularized gastrointestinal tissues are effective alternatives to the current gold standard, Matrigel, and produce organoids suitable for gastrointestinal disease modeling, drug development, and tissue regeneration.
Topics: Animals; Collagen; Drug Combinations; Extracellular Matrix; Hydrogels; Laminin; Mice; Organoids; Proteoglycans; Proteomics
PubMed: 35354790
DOI: 10.1038/s41467-022-29279-4 -
Biomedicine & Pharmacotherapy =... Feb 2022Gastric cancer (GC) is the fifth most common type of cancer and the third leading cause of death due to cancer worldwide. The gastric mucosa often undergoes many years... (Review)
Review
Gastric cancer (GC) is the fifth most common type of cancer and the third leading cause of death due to cancer worldwide. The gastric mucosa often undergoes many years of precancerous lesions of gastric cancer (PLGC) stages before progressing to gastric malignancy. Unfortunately, there are no effective Western drugs for patients with PLGC. In recent years, traditional Chinese medicine (TCM) has been proven effective in treating PLGC. Classical TCM formulas and chemical components isolated from some Chinese herbal medicines have been administered to treat PLGC, and the main advantage is their comprehensive intervention with multiple approaches and multiple targets. In this review, we focus on recent studies using TCM treatment for PLGC, including clinical observations and experimental research, with a focus on targets and mechanisms of drugs. This review provides some ideas and a theoretical basis for applying TCM to treat PLGC and prevent GC.
Topics: Drugs, Chinese Herbal; Gastric Mucosa; Humans; Medicine, Chinese Traditional; Precancerous Conditions; Stomach Neoplasms
PubMed: 34929576
DOI: 10.1016/j.biopha.2021.112542 -
Biochimica Et Biophysica Acta. Reviews... Apr 2021An improved understanding of stem cell niches, organogenesis, and disease models has paved the way for developing a three-dimensional (3D) organoid culture system.... (Review)
Review
An improved understanding of stem cell niches, organogenesis, and disease models has paved the way for developing a three-dimensional (3D) organoid culture system. Organoid cultures can be derived from primary tissues (single cells or tissue subunits), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs), or embryonic stem cells (ESCs). As a significant technological breakthrough, 3D organoid models offer a promising approach for understanding the complexities of human diseases ranging from the mechanistic investigation of disease pathogenesis to therapy. Here, we discuss the recent applications, advantages, and limitations of organoids as in vitro models for studying metabolomics, drug development, infectious diseases, and the gut microbiome. We further discuss the use of organoids in cancer modeling using high throughput sequencing approaches.
Topics: Biomedical Research; Humans; Models, Biological; Organ Culture Techniques; Organoids
PubMed: 33640383
DOI: 10.1016/j.bbcan.2021.188527