- Editorial
- Open access
- Published:
Standard: Human intestinal organoids
Cell Regeneration volume 12, Article number: 23 (2023)
Abstract
Organoids have attracted great interest for disease modelling, drug discovery and development, and tissue growth and homeostasis investigations. However, lack of standards for quality control has become a prominent obstacle to limit their translation into clinic and other applications. “Human intestinal organoids” is the first guideline on human intestinal organoids in China, jointly drafted and agreed by the experts from the Chinese Society for Cell Biology and its branch society: the Chinese Society for Stem Cell Research. This standard specifies terms and definitions, technical requirements, test methods, inspection rules for human intestinal organoids, which is applicable to quality control during the process of manufacturing and testing of human intestinal organoids. It was originally released by the Chinese Society for Cell Biology on 24 September 2022. We hope that the publication of this standard will guide institutional establishment, acceptance and execution of proper practical protocols and accelerate the international standardization of human intestinal organoids for applications.
Scope
This document specifies the ethical requirements, technical requirements, and testing methods for human intestinal organoids.
This standard applies to the production and testing of human intestinal organoids.
Normative references
The following referenced documents are indispensable for the application of these documents. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including all amendments) applies.
-
WS 213 Diagnosis for Hepatitis C
-
WS 293 Diagnostic Criteria for HIV/AIDS
-
WS 299 Diagnostic Criteria for Viral Hepatitis B
-
Pharmacopoeia of the People's Republic of China (2020 Edition)
-
National Guide to Clinical Laboratory Procedures
Terms and definitions
The following terms and definitions apply to this document.
Organoids
Three-dimensional (3D) structures that grow from stem cells or progenitor cells in vitro, consist of organ-specific cell types, and can mimic the in vivo architecture and specific function of the original tissue (Clevers 2016; Fujii and Sato 2021; Kim et al. 2020; Sato et al. 2009).
Human intestinal organoids
Organoids that develop from human intestinal stem cells of normal tissue, which are capable of self-formation, long-term growth and renewal, and possess a variety of mature intestinal epithelial cell types (Sato and Clevers 2013; Sato et al. 2011a).
Passage
Process of dissociating existing organoids into smaller fragments, or single cell via physical, chemical, or biological methods, and keeping them growing in vitro under the same culture conditions (Ganesh et al. 2019).
Cryopreservation
Freezing process by which organoids are maintained at low temperature in an inactive state for maintaining cellular composition, gene expression, and functional properties.
Thawing
Process of bringing frozen organoids from an inactive to an actively growing state.
Intestinal stem cells
Cells that can self-renew and possess the ability to differentiate into all types of intestinal epithelial cells (Barker 2014; Barker et al. 2012; Beumer and Clevers 2021; Gehart and Clevers 2019).
Intestinal stem cell differentiation
Process of intestinal stem cells dividing into their daughter cells, including enterocytes, goblet cells, Paneth cells, enteroendocrine cells, et al. (Beumer and Clevers 2021; Gehart and Clevers 2019).
Transit amplifying cells (TA cells)
Cells that are initially expanded from stem cells and have high proliferative capacity, and can initially differentiate into progenitor cells and further differentiate into various types of mature intestinal epithelial cells (Beumer and Clevers 2021; Gehart and Clevers 2019).
Enterocytes
Intestinal epithelial cells that are mainly responsible for the absorption of nutrients from the intestinal lumen, which are columnar with oval nuclei located at the base of the cells and with regularly arranged microvilli located at the luminal side (Beumer and Clevers 2021; Gehart and Clevers 2019).
Goblet cells
Intestinal epithelial cells that secret mucus periodically, which are enlarged and cup-shaped with the filled mucus particles at the top and the nucleus at the bottom (Beumer and Clevers 2021; Gehart and Clevers 2019; Gustafsson and Johansson 2022).
Paneth cells
Intestinal epithelial cells that usually gather at the bottom of the small intestinal crypts and intermingle with intestinal stem cells, which are conical in shape with thick eosinophilic secretory granules on the top and the round nucleus at the base (Beumer and Clevers 2021; Bevins and Salzman 2011; Clevers 2013; Gehart and Clevers 2019; Porter et al. 2002).
Enteroendocrine cells
Intestinal epithelial cells that secret intestinal hormones in response to luminal food stimulation or pH changes, which are irregularly conical with a large number of secretory particles at the bottom (Beumer and Clevers 2021; Gehart and Clevers 2019; Gribble and Reimann 2019).
Ethics requirements
A legal and valid informed consent shall be signed by the donor who provides the tissue to develop the organoid. The consent form includes, but not limited to, potential research and therapeutic applications under the appropriate conditions, potential commercial applications of research results, and other issues applicable.
The production and research project of human intestinal cancer organoids shall be approved by the ethics review committee.
The personal information of donors shall be protected.
Technical requirements
Morphology
Human intestinal organoids shall be cystic or bud-like, with a cavity in the middle and tightly contacting columnar epithelial cells on the outside under optical microscopy. The cavities and the edges of the junctions shall be clear, and the cells shall be transparent (Sato et al. 2011a; Wang et al. 2022a).
Chromosomal karyotype
The chromosomal karyotype of human intestinal organoids shall be 46, XY or 46, XX.
Marker genes
Marker gene expressions shall be detected in human intestinal organoids, including the stem cell marker LGR5 (Barker et al. 2007), the goblet cell marker MUC2 (Chang et al. 1994), the enterocyte marker ALPI (Tetteh et al. 2016), the enteroendocrine cell marker CHGA (Zeve et al. 2022), and the proliferative cell marker MKI67 (Beumer and Clevers 2021). In addition, the Paneth cell marker LYZ should be detected in organoids derived from small intestine (Sato et al. 2011b; Wang et al. 2022b).
Cell composition
Human small intestinal organoids shall contain LGR5+ intestinal stem cells, KI67+ and LGR5− transit amplifying cells, ALPI+ enterocytes, MUC2+ goblet cells, LYZ+ Paneth cells and CHGA+ enteroendocrine cells, the percentage of enterocytes shall be no less than 30% (Tetteh et al. 2016; Wang et al. 2020).
Human large intestinal organoids shall contain LGR5+ intestinal stem cells, KI67+ and LGR5− transit amplifying cells, ALPI+ enterocytes, MUC2+ goblet cells and CHGA+ enteroendocrine cells, the percentage of goblet cells shall be no less than 30% (Gustafsson and Johansson 2022; Wang et al. 2020).
Functional parameters
Alkaline phosphatase and lysozyme should be detected in human small intestinal organoids (Sensoy and Oznurlu 2019; Wang et al. 2020).
Mucins should be detected in human large intestinal organoids (Chang et al. 1994; Walaas et al. 2023).
Culture and growth
Human intestinal organoids derived from healthy donors shall be able to be passaged for at least 5 generations in vitro after the initial culture, during which the total cell number shall not decrease. Compared to the last generation, the passaged organoids shall have the same morphology, cell composition, karyotype and other characteristics (Sato et al. 2011a).
When organoids are passaged, the cells shall be able to reconstruct into new organoids in vitro, and maintain the capacities of self-renewal and differentiation (Sato et al. 2011a).
Viability
The organoid viability shall be ≥ 50% after thawing, and these living organoids shall be subcultured in vitro.
Microorganisms
Organoids shall be negative for fungi, bacteria, mycoplasma, and virus.
Identity
The identity of organoids shall match that of the donor tissue by STR analysis (Lee et al. 2015).
Test methods
Morphology
Observe organoid morphology by the inverted phase contrast microscope.
Chromosomal karyotype
The method in “Preparation and quality control of animal cells for the production of biological products” from the Pharmacopoeia of the People's Republic of China (2020 edition) shall be followed.
Marker genes
The method in Appendix A shall be followed.
Cell composition
The method in Appendix B shall be followed.
Functional parameters
For lysozyme detection, the method in Appendix B shall be followed.
For alkaline phosphatase detection, the method in Appendix C shall be followed.
For mucin detection, the method in Appendix D shall be followed.
Quantity
Count the organoid number, defined by a pre-defined diameter threshold, from the images taken by an inverted phase contrast microscope that is attached with a scale bar.
Viability
Organoid viability testing shall be performed on the primary organoids and passaged organoids, and the method in Appendix E shall be followed.
Microorganisms
Mycoplasma
The “3301 Mycoplasma Inspection Method” in Pharmacopoeia of the People's Republic of China (2020 edition) shall be followed.
Bacteria and Fungi
The “1101 Sterility Inspection Method” in Pharmacopoeia of the People's Republic of China (2020 edition) shall be followed.
HIV
The method in WS 293 shall be followed.
HBV
The method in WS 299 shall be followed.
HCV
The method in WS 213 shall be followed.
Exogenous viral factors
The “3302 Exogenous Viral Factors Inspection Method” in Pharmacopoeia of the People's Republic (2020 edition) of China shall be followed.
STR
The method in Appendix F shall be followed.
Availability of data and materials
Not applicable.
Abbreviations
- Ct:
-
Cycle-threshold
- DAPI:
-
4,6-Diamino-2-phenyl indole
- DMSO:
-
Dimethyl Sulfoxide
- HBV:
-
Hepatitis B Virus
- HCV:
-
Hepatitis C Virus
- HIV:
-
Human Immunodeficiency Virus
- PBS:
-
Phosphate Buffer Saline
- PCR:
-
Polymerase Chain Reaction
- RNA:
-
Ribonucleic Acid
- STR:
-
Short Tandem Repeat
References
Barker N. Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration. Nat Rev Mol Cell Biol. 2014;15(1):19–33. https://doi.org/10.1038/nrm3721.
Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 2007;449(7165):1003–7. https://doi.org/10.1038/nature06196.
Barker N, van Oudenaarden A, Clevers H. Identifying the stem cell of the intestinal crypt: strategies and pitfalls. Cell Stem Cell. 2012;11(4):452–60. https://doi.org/10.1016/j.stem.2012.09.009.
Beumer J, Clevers H. Cell fate specification and differentiation in the adult mammalian intestine. Nat Rev Mol Cell Biol. 2021;22(1):39–53. https://doi.org/10.1038/s41580-020-0278-0.
Bevins CL, Salzman NH. Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis. Nat Rev Microbiol. 2011;9(5):356–68. https://doi.org/10.1038/nrmicro2546.
Chang SK, Dohrman AF, Basbaum CB, Ho SB, Tsuda T, Toribara NW, et al. Localization of mucin (MUC2 and MUC3) messenger RNA and peptide expression in human normal intestine and colon cancer. Gastroenterology. 1994;107(1):28–36. https://doi.org/10.1016/0016-5085(94)90057-4.
Clevers H. The intestinal crypt, a prototype stem cell compartment. Cell. 2013;154(2):274–84. https://doi.org/10.1016/j.cell.2013.07.004.
Clevers H. Modeling Development and Disease with Organoids. Cell. 2016;165(7):1586–97. https://doi.org/10.1016/j.cell.2016.05.082.
Fujii M, Sato T. Somatic cell-derived organoids as prototypes of human epithelial tissues and diseases. Nat Mater. 2021;20(2):156–69. https://doi.org/10.1038/s41563-020-0754-0.
Ganesh K, Wu C, O’Rourke KP, Szeglin BC, Zheng Y, Sauve CG, et al. A rectal cancer organoid platform to study individual responses to chemoradiation. Nat Med. 2019;25(10):1607–14. https://doi.org/10.1038/s41591-019-0584-2.
Gehart H, Clevers H. Tales from the crypt: new insights into intestinal stem cells. Nat Rev Gastroenterol Hepatol. 2019;16(1):19–34. https://doi.org/10.1038/s41575-018-0081-y.
Gribble FM, Reimann F. Function and mechanisms of enteroendocrine cells and gut hormones in metabolism. Nat Rev Endocrinol. 2019;15(4):226–37. https://doi.org/10.1038/s41574-019-0168-8.
Gustafsson JK, Johansson MEV. The role of goblet cells and mucus in intestinal homeostasis. Nat Rev Gastroenterol Hepatol. 2022;19(12):785–803. https://doi.org/10.1038/s41575-022-00675-x.
Kim J, Koo BK, Knoblich JA. Human organoids: model systems for human biology and medicine. Nat Rev Mol Cell Biol. 2020;21(10):571–84. https://doi.org/10.1038/s41580-020-0259-3.
Lee SH, Hong JH, Park HK, Park JS, Kim BK, Lee JY, et al. Colorectal cancer-derived tumor spheroids retain the characteristics of original tumors. Cancer Lett. 2015;367(1):34–42. https://doi.org/10.1016/j.canlet.2015.06.024.
Porter EM, Bevins CL, Ghosh D, Ganz T. The multifaceted Paneth cell. Cell Mol Life Sci. 2002;59(1):156–70. https://doi.org/10.1007/s00018-002-8412-z.
Sato T, Clevers H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science. 2013;340(6137):1190–4. https://doi.org/10.1126/science.1234852.
Sato T, Stange DE, Ferrante M, Vries RG, Van Es JH, Van den Brink S, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology. 2011a;141(5):1762–72. https://doi.org/10.1053/j.gastro.2011.07.050.
Sato T, van Es JH, Snippert HJ, Stange DE, Vries RG, van den Born M, et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature. 2011b;469(7330):415–8. https://doi.org/10.1038/nature09637.
Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459(7244):262–5. https://doi.org/10.1038/nature07935.
Sensoy E, Oznurlu Y. Determination of the changes on the small intestine of pregnant mice by histological, enzyme histochemical, and immunohistochemical methods. Turk J Gastroenterol. 2019;30(10):917–24. https://doi.org/10.5152/tjg.2019.18681.
Tetteh PW, Basak O, Farin HF, Wiebrands K, Kretzschmar K, Begthel H, et al. Replacement of Lost Lgr5-Positive Stem Cells through Plasticity of Their Enterocyte-Lineage Daughters. Cell Stem Cell. 2016;18(2):203–13. https://doi.org/10.1016/j.stem.2016.01.001.
Walaas GA, Gopalakrishnan S, Bakke I, Skovdahl HK, Flatberg A, Ostvik AE, et al. Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids. Front Immunol. 2023;14:1095812. https://doi.org/10.3389/fimmu.2023.1095812.
Wang R, Mao Y, Wang W, Zhou X, Wang W, Gao S, et al. Systematic evaluation of colorectal cancer organoid system by single-cell RNA-Seq analysis. Genome Biol. 2022a;23(1):106. https://doi.org/10.1186/s13059-022-02673-3.
Wang Y, Song W, Wang J, Wang T, Xiong X, Qi Z, et al. Single-cell transcriptome analysis reveals differential nutrient absorption functions in human intestine. J Exp Med. 2020;217(2). https://doi.org/10.1084/jem.20191130.
Wang Y, Song W, Yu S, Liu Y, Chen YG. Intestinal cellular heterogeneity and disease development revealed by single-cell technology. Cell Regen. 2022b;11(1):26. https://doi.org/10.1186/s13619-022-00127-6.
Zeve D, Stas E, de Sousa CJ, Mannam P, Qi W, Yin X, et al. Robust differentiation of human enteroendocrine cells from intestinal stem cells. Nat Commun. 2022;13(1):261. https://doi.org/10.1038/s41467-021-27901-5.
Acknowledgements
We thank Dong Gao, Yi Arial Zeng, Xia Wang, Xiaolei Yin, Shan Bian, Yongchun Zhang, Yan Liu, Zhiwei Cai, Huili Hu, Lei Chen, Ming Jiang, Ying Xi and Guang Yang for stimulating suggestions.
Funding
This work was supported by grants from the National Natural Science Foundation of China (31988101 to Y.-G.C.; 82173461 To G.Q.H.), Guangdong Basic and Applied Basic Research Foundation (2021A1515111215) to Y.L.W. and China Postdoctoral Science Foundation (2021M703230 and 2022T150653) to Y.L.W., National Key R&D Program of China (2018YFA0108400) to T.B.Z., the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16040501) to A.J.M..
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author information
Authors and Affiliations
Contributions
YGC, GQH, TBZ and AJM contributed to conception and design. YLW, HQL, LZZ and FH drafted and revised the manuscript. JH, ZZ, WQS, LHS, CXD, BZ, JNC, LW (Lei Wang), LW (Liu Wang), LML, WLC, CPY, ZJS, YYY, CLW, YZ, QYL and KL critically read and revised the manuscript.
Corresponding authors
Ethics declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing financial interests. Y.-G.C. is the Editor‑in‑Chief of Cell Regeneration. He was not involved in the review or decision related to this manuscript. This work was not sponsored by any commercial organizations, and all the other authors declare that they have no competing interests.
Supplementary Information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
About this article
Cite this article
Wang, Y., Lin, H., Zhao, L. et al. Standard: Human intestinal organoids. Cell Regen 12, 23 (2023). https://doi.org/10.1186/s13619-023-00168-5
Published:
DOI: https://doi.org/10.1186/s13619-023-00168-5