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Standard: Human intestinal cancer organoids
Cell Regeneration volume 12, Article number: 24 (2023)
Abstract
Intestinal cancer is one of the most frequent and lethal types of cancer. Modeling intestinal cancer using organoids has emerged in the last decade. Human intestinal cancer organoids are physiologically relevant in vitro models, which provides an unprecedented opportunity for fundamental and applied research in colorectal cancer. “Human intestinal cancer organoids” is the first set of guidelines 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 for human intestinal cancer organoids, which apply to the production and quality control during the process of manufacturing and testing of human intestinal cancer organoids. It was 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 practocal protocols, and accelerate the international standardization of human intestinal cancer organoids for clinical development and therapeutic applications.
Scope
This document specifies the ethical requirements, technical requirements, and testing methods for human intestinal cancer organoids.
This standard applies to the production and testing of human intestinal cancer 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.
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WS 213 Diagnosis for Hepatitis C
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WS 293 Diagnostic Criteria for HIV/AIDS
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WS 299 Diagnostic Criteria for Viral Hepatitis B
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Pharmacopoeia of the People’s Republic of China (2020 Edition)
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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 original tissue (Clevers 2016; Fujii and Sato 2021; Kim et al. 2020; Sato et al. 2009).
Tumor organoids
Organoids that derived from tumor cells from tumor tissues or other specimens containing tumor cells from patients and cultured in vitro, and can expand and recapitulate tumor pathologic features, genetic characteristics, and treatment response (Fujii et al. 2016; Kolahi et al. 2020; Tuveson and Clevers 2019).
Human intestinal cancer organoids
Organoids that develop from intestinal tumor cells of patients with a pathological diagnosis of intestinal cancer, and can simulate the characteristics of intestinal cancer (Mo et al. 2022; Sato et al. 2011; van de Wetering et al. 2015).
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.
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 cancer organoids shall have a clear edge and cytoplasm when observed under the optical microscope. They shall be formed by cell clusters, exhibiting compact, loose, cystic, or mixed morphology (Betge et al. 2022; van de Wetering et al. 2015; Wang et al. 2022; Yao et al. 2020).
Culture and growth
The first generation of human intestinal cancer organoids developed from tissue or cells from patients, shall be kept alive in vitro for at least two months and shall be passaged for at least three generations (Lv et al. 2023; Sato et al. 2011).
Post-passage organoids shall be reconstructed in vitro into new analogous organoids, and their morphology and characteristics shall be consistent with those of the pre-passage organoids (Sato et al. 2011).
Viability
The organoid viability shall be ≥ 50% after thawing, and these living organoids shall be subcultured in vitro (Lv et al. 2023).
Microorganisms
Organoids shall be negative for fungi, bacteria, HBV, HCV, HIV, and exogenous viral factors.
Identity
The identity of organoids shall match that of the donor tissue by STR analysis (Lee et al. 2015).
Pathological features
Pathological features of organoids shall be recognized by qualified pathologists. These features shall be consistent with the typical features of tumor cells based on H&E staining images, such as hyperchromasia, abnormal mitosis, imbalanced nucleocytoplasmic ratio, etc. (De Angelis et al. 2022).
The immunohistochemical marker proteins CDX2 and CK20 of differentiated intestinal adenocarcinoma organoids shall be positive and the distribution of expression shall be non-polar and disordered (De Angelis et al. 2022; Ganesh et al. 2019).
Genetic characteristics
Genetic variant testing shall be performed on the organoids, and the test results shall be consistent with the results of the original tumor tissue. The tested genes shall include, but not limited to, KRAS, NRAS, HRAS, BRAF, APC, TP53, SMAD4, etc (Fujii et al. 2016; Ganesh et al. 2019; van de Wetering et al. 2015; Wanigasooriya et al. 2022; Zhao et al. 2022).
Test methods
Morphology
Observe organoid morphology by the inverted phase contrast microscope.
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 A shall be followed.
Microorganisms
Bacteria and fungi
The “1101 Sterility Inspection Method” in Pharmacopoeia of the People's Republic of China (2020 edition) shall be followed.
Mycoplasma
The “3301 Mycoplasma 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 of China (2020 edition) shall be followed.
STR
The method in Appendix B shall be followed.
Pathological features
The method in Appendix C shall be followed.
Genetic characteristics
The method in Appendix D shall be followed.
Availability of data and materials
Not applicable.
Abbreviations
- DMSO:
-
Dimethyl Sulfoxide
- DNA:
-
Deoxyribonucleic Acid
- HBV:
-
Hepatitis B Virus
- HCV:
-
Hepatitis C Virus
- HIV:
-
Human Immunodeficiency Virus
- H&E:
-
Hematoxylin and Eosin
- PBS:
-
Phosphate Buffer Saline
- STR:
-
Short Tandem Repeat
References
Betge J, Rindtorff N, Sauer J, Rauscher B, Dingert C, Gaitantzi H, et al. The drug-induced phenotypic landscape of colorectal cancer organoids. Nat Commun. 2022;13(1):3135. https://doi.org/10.1038/s41467-022-30722-9.
Clevers H. Modeling Development and Disease with Organoids. Cell. 2016;165(7):1586–97. https://doi.org/10.1016/j.cell.2016.05.082.
De Angelis ML, Francescangeli F, Nicolazzo C, Signore M, Giuliani A, Colace L, et al. An organoid model of colorectal circulating tumor cells with stem cell features, hybrid EMT state and distinctive therapy response profile. J Exp Clin Cancer Res. 2022;41(1):86. https://doi.org/10.1186/s13046-022-02263-y.
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.
Fujii M, Shimokawa M, Date S, Takano A, Matano M, Nanki K, et al. A colorectal tumor organoid library demonstrates progressive loss of Niche factor requirements during tumorigenesis. Cell Stem Cell. 2016;18(6):827–38. https://doi.org/10.1016/j.stem.2016.04.003.
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.
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.
Kolahi KS, Nakano M, Kuo CJ. Organoids as oracles for precision medicine in rectal cancer. Cell Stem Cell. 2020;26(1):4–6. https://doi.org/10.1016/j.stem.2019.12.003.
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.
Lv T, Shen L, Xu X, Yao Y, Mu P, Zhang H, et al. Patient-derived tumor organoids predict responses to irinotecan-based neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer. Int J Cancer. 2023;152(3):524-35. https://doi.org/10.1002/ijc.34302.
Mo S, Tang P, Luo W, Zhang L, Li Y, Hu X, et al. Patient-derived organoids from colorectal cancer with paired liver metastasis reveal tumor heterogeneity and predict response to Cchemotherapy. Adv Sci (Weinh). 2022;9(31):e2204097. https://doi.org/10.1002/advs.202204097.
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.
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. 2011;141(5):1762–72. https://doi.org/10.1053/j.gastro.2011.07.050.
Tuveson D, Clevers H. Cancer modeling meets human organoid technology. Science. 2019;364(6444):952–5. https://doi.org/10.1126/science.aaw6985.
van de Wetering M, Francies HE, Francis JM, Bounova G, Iorio F, Pronk A, et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell. 2015;161(4):933–45. https://doi.org/10.1016/j.cell.2015.03.053.
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. 2022;23(1):106. https://doi.org/10.1186/s13059-022-02673-3.
Wanigasooriya K, Barros-Silva JD, Tee L, El-Asrag ME, Stodolna A, Pickles OJ, et al. Patient derived organoids confirm that PI3K/AKT signalling is an escape pathway for radioresistance and a target for therapy in rectal cancer. Front Oncol. 2022;12:920444. https://doi.org/10.3389/fonc.2022.920444.
Yao Y, Xu X, Yang L, Zhu J, Wan J, Shen L, et al. Patient-derived organoids predict chemoradiation responses of locally advanced rectal cancer. Cell Stem Cell. 2020;26(1):17–26 e6. https://doi.org/10.1016/j.stem.2019.10.010.
Zhao Y, Zhang B, Ma Y, Zhao F, Chen J, Wang B, et al. Colorectal cancer patient-derived 2D and 3D models efficiently recapitulate inter- and intratumoral heterogeneity. Adv Sci (Weinh). 2022;9(22):e2201539. https://doi.org/10.1002/advs.202201539.
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
GQH, YGC, TBZ and AJM contributed to conception and design. HQL, YLW, CYC and YXQ 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.
Appendices
Appendix A
Normative Appendix: Organoid Viability Test (Calcein-AM Staining Method)
Instruments
Inverted microscope
Fluorescence microscope
Reagents
Unless otherwise specified, the reagents used shall be analytically pure, and the water used for testing shall be deionized water.
Dimethyl sulfoxide (DMSO) for cell culture
Phosphate-buffered saline (PBS): pH 7.4
Storage of Calcein-AM solution: 2 mmol/L in DMSO
Testing protocol
Organoid counting
Place the organoids under the microscope to observe their morphology and status. Determine whether the organoid morphology meets the requirements of 6.1 by visual observation, and count the organoids with a diameter ≥ 20 μm.
Living organoid counting
Add the Calcein-AM storage solution to the medium to a final concentration of 0.2 μmol/L, and incubate the mixture at 37℃ for 60 min. Then remove the medium with Calcein-AM gently with PBS and add fresh medium. The organoids are observed and photographed by fluorescence microscope at 490 nm excitation wavelength and 515 nm emission wavelength. Living organoids are in green with clear edges. Then count the living organoids ≥ 20 μm in diameter.
Organoid counting
Tomographically scan the organoids using a microscope and image acquisition software, with the interlayer height set to the range of 10 μm to 200 μm. Superimpose the scanned images to be a single planar map, and then count the organoids in the final map.
Organoid viability
Organoid viability is calculated according to Eq. (A.1):
In this equation:
X —Organoid viability,
Nalive—Number of living organoids,
Ntotal—Total number of organoids.
Calculation and analysis
Repeat the procedure twice more according to A.3, calculate the average of the three living organoid ratio results, and record it as the organoid viability.
Accuracy
The absolute difference between the results of three independent determinations obtained under reproducible conditions shall not exceed 10% of the arithmetic mean.
Appendix B
Normative Appendix: Organoid Authentication by STR Profiling
Instruments
Centrifuge
PCR-Cycler
Electrophoresis apparatus
Reagents
Cell DNA extraction kit
STR DNA profiling kit
Sample storage
The samples are prepared and stored below -80℃.
Testing protocol
Sample preparation
The organoids are cultured in the Matrigel to a stable growth state, and then they are mechanically pipetted out of the Matrigel. The mixture is collected in a centrifugal tube, the organoids are collected by centrifugation, and the supernatant is discarded.
Extraction of DNA
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A)
Perform genomic DNA extraction from organoids and primary tumor tissues according to the instructions of the cellular DNA extraction kit.
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B)
Measure the absorbance of extracted DNA by UV spectrophotometer to ensure that the ratio of A260/A280 is between 1.8 and 2.0.
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C)
DNA volume ≥ 20 μL, DNA concentration ≥ 50 ng/μL.
PCR amplification
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A)
Perform STR DNA amplification according to standard PCR amplification methods or the commercially approved kit instructions.
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B)
Set up a negative control group, a sample detection group, and a positive control group. Use sterile water as the template for PCR amplification in the negative control group; use the DNA extracted from organoid and primary tumor tissue samples as a template for PCR amplification in the sample detection group; use the DNA template for amplification in the positive control group.
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C)
Detect the PCR products of three groups by agarose gel electrophoresis. Clear target band shall be observed in the positive control but not in the negative control.
STR genotyping
Detect PCR products by capillary electrophoresis gene analyzer and STR genetic map data are obtained. The PCR banding pattern of organoids and primary tumor tissue shall be consistent.
Results analysis
When STR alleles contain the same number of repeats, only one allele peak shall appear in the profile, when they contain different numbers of repeats, two allele peaks appear in the profile. The test is considered valid when no allele peaks appeared in the negative control group and the positive control group is consistent with its standard genotyping data.
If more than two allelic peaks are present at the STR locus of the tested sample, the sample shall be determined to be cross-contaminated after repeated experiments to exclude interfering factors such as mutations in the primer binding region, provided that the test is valid.
Appendix C
Normative Appendix: Organoid histopathology testing (Paraffin Embedding Method)
Instruments
Paraffin-embedding machine
Paraffin-slicer
Reagents
Unless otherwise specified, the reagents used shall be analytically pure, and the water used for testing shall be deionized water.
Paraffin section preparation reagents: prepare reagents required for paraffin embedding according to the corresponding requirements, including fixing solution, dehydration solution, paraffin, dewaxing solution, rehydration solution, ethanol, xylene, and neutral resin.
H&E staining reagent: hematoxylin, eosin.
Immunohistochemical staining reagents: prepare antigen repair solution, primary antibody, secondary antibody, closure solution, PBS, DBA according to the corresponding requirements.
Testing protocol
Sample preparation and fixation
The organoids are mechanically pipetted out of Matrigel gently and transferred to a 15 mL centrifuge tube. Collect the organoids by centrifugation and discard the supernatant. The organoids are fixed with 4% paraformaldehyde for 15–30 min.
Paraffin section preparation of organoids
The organoid samples are fixed, dehydrated, hyalinized, immersed in paraffin, and embedded with a paraffin-embedding machine according to the method of paraffin section. Cut into standard thickness with paraffin-slicer.
H&E staining
Paraffin sections of organoids are dewaxed, rehydrated, stained with hematoxylin and eosin, then dehydrated with ethanol, hyalinized by xylene, and sealed by neutral resin.
Immunohistochemistry staining
Paraffin sections of organoids are dewaxed, rehydrated, antigen repaired and sealed with blocking solution, sections are incubated with primary antibody and then cleaned with PBS, sections are followed by incubated with second antibody and cleaned with PBS. Perform the color reaction with DAB and add water to stop the color reaction. The sections are lining dyed by hematoxylin, hyalinized with hydrochloric acid and flushing. Then dehydrated with ethanol, hyalinized by xylene, and sealed by neutral resin.
Results analysis
The test results obtained are analyzed and judged by personnel qualified in the pathological diagnosis, and these results shall be consistent with the results of the original tumor tissue.
Appendix D
Normative Appendix: Organoid Gene Mutation Testing
Instruments
Centrifuge
Reagents
Prepare the Cell DNA extraction kit according to the corresponding requirements.
Sample storage
The samples are prepared and stored below -80℃.
Testing Protocol
Sample preparation
The organoids are cultured in the Matrigel to a stable growth state, and then they are mechanically pipetted out of the Matrigel. The mixture is collected in a centrifugal tube, the organoids are collected by centrifugation, and the supernatant is discarded.
Extraction of DNA
Extract the genomic DNA of organoids according to the instructions of the cell DNA extraction kit.
DNA sequencing
Send the organoid DNA samples and original tumor tissues to institutions qualified for clinical genetic testing for 1st generation sequencing or 2nd generation sequencing testing, or the genetic loci will be tested by ARMS method or ddPCR method.
Results analysis
Analyze the mutant loci and compare the concordance between organoid sequencing and original tumor tissue sequencing results.
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Lin, H., Wang, Y., Cheng, C. et al. Standard: Human intestinal cancer organoids. Cell Regen 12, 24 (2023). https://doi.org/10.1186/s13619-023-00167-6
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DOI: https://doi.org/10.1186/s13619-023-00167-6