01 February 2021 Article history PDF Split View Cite Permissions Share Abstract Context Papillary thyroid cancer (PTC) has been one of the most frequent endocrine malignancies around the world. How to grow patient-derived organoids (PDOs) | Molecular Devices The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. 6C). Scale bar, 200 m. An official website of the United States government. Copy number landscape of patient, Figure 1. 8600 Rockville Pike Bartfeld Results: We demonstrate that patient-derived cancer organoids represent the cancers from which they were derived, including key histologic and molecular features. Preclinical Models of Follicular Cell-Derived Thyroid Cancer: An Overview from Cancer Cell Lines to Mouse Models. Tg, a well-established differentiated biomarker for the thyroid, was highly expressed in all PTC- and NTG-derived organoids and their parental tumors excepting PTC-3_T and PTC-3_O (see Fig. 12634-010] containing 1% HEPES [N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid; Gibco, No. From this experiment, we found that the addition of EGF and SB202190 are sufficient for the formation and growth of human PTC organoids (Supplementary Fig. Somatic single-nucleotide variants (SNVs) were detected by GATK (version 3.6). Arrow, continuous expansion; Dot, passage; PTC, papillary thyroid cancer; NTG, nodular thyroid goiter; O, organoid. de Biase HY-103456), G-1 (G proteincoupled estrogen receptor [GPER]-selective agonist, No. Published by Oxford University Press on behalf of the Endocrine Society. Application of human liver organoids as a patient-derived - eLife Drug sensitivity assays of PTC organoids demonstrate patient-specific drug responses, and large correlations with the respective mutational profiles. All patients gave informed consent under the auspices of the medical ethics committee of Shenzhen Second Peoples Hospital as per the Declaration of Helsinki. Notably, PTC organoids preserve the histopathological profiles and genomic heterogeneity of the originating tumors. Herein, we describe our successful creation of 43 tumor organoid cultures and nine adjacent normal tissue organoid cultures derived from patients with endometrial or ovarian cancer. The assay was performed with 3 technical replicates. Furthermore, the differences in drug response between organoid cultures (PTC-2-I_O/PTC-2-II_O and PTC-4-I_O/PTC-4-II_O) derived from different areas of the same tumor imply intratumor heterogeneity (see Fig. Immune organoids: from tumor modeling to precision oncology We describe a 3-dimensional culture system for the long-term expansion of patient-derived PTC organoid lines. Vlachogiannis Summary of organoid establishment, sources and applications. 2022;2:94. doi: 10.1038/s43586-022-00174-y. WES was performed to median depths of 140, 240, and 241 in the blood, primary tumor tissue, and matched organoid, respectively. Patient-derived organoids as a platform for drug screening in We detected interesting sensitivities to several drugs that demonstrated large correlations with their mutational profiles. 5 and Supplementary Fig. The lack of in vitro models to reflect the primary disease has become a bottleneck for further study on thyroid cancer. Bethesda, MD 20894, Web Policies Organoid Cultures Derived From Patients With Papillary Thyroid Cancer ab75635), ER (1:300, Abcam, No. It was a pleasant surprise to find that the intratumor heterogeneity in human PTCs was also reflected in their derived organoids. ObjectivesPartial or total resistance to preoperative chemoradiotherapy occurs in more than half of locally advanced rectal cancer patients. D, Expansion potential of PTC- and nodular thyroid goiter (NTG)-derived organoid lines. D, Effects of 100 nM E2, in the presence or absence of 1 M fulvestrant, tamoxifen citrate, MPP (1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole), PHTPP (4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl)phenol), or GPER-selective agonist G15, on cell proliferation of PTC organoid lines. Endocrinol Metab (Seoul). Colorectal carcinoma is a highly prevalent and heterogeneous gastrointestinal malignancy. Surgically resected PTC primary tissues were dissociated and processed for organoid derivation. Organoids were then resuspended in 2% Matrigel/organoid complete medium (15000 organoids/mL) and dispensed into round bottom, ultra-low attachment 96-well plates in triplicates. Tissues were resuspended in 5 mL of TrypLE Express (Gibco, No. Tissues and organoids were fixed in 10% neutral-buffered formalin for 24 hours, embedded in paraffin, and serial-sectioned at 4-m in thickness. MAB-0572), thyroglobulin (Tg, 1:200, Abcam, No. 120-38), A83-01 (500 nM, Sigma-Aldrich, No. Drugs were selected based on their clinical relevance to thyroid cancer treatment, and investigational agents being tested in clinical trials, or their ability to target key signaling pathways. The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Establishment of patient-derived cancer organoids for drug-screening Time-lapse phase-contrast imaging of the cohesive PTC organoids showed that many organoids seem to be clonally derived (Fig. For example, PTC-1_O, PTC-2-I_O, and PTC-4-I_O contain mutations in the BRAF gene (see Fig. See also Supplementary Table 5 (20). Patient-derived xenografts and organoids model therapy response in prostate cancer. Zhao Z, Chen X, Dowbaj AM, Sljukic A, Bratlie K, Lin L, Fong ELS, Balachander GM, Chen Z, Soragni A, Huch M, Zeng YA, Wang Q, Yu H. Nat Rev Methods Primers. MY Although most PTC patients have excellent overall survival, a subgroup of patients die, particularly when disease recurrence occurs. Servant R, Garioni M, Vlajnic T, Blind M, Pueschel H, Mller DC, Zellweger T, Templeton AJ, Garofoli A, Maletti S, Piscuoglio S, Rubin MA, Seifert H, Rentsch CA, Bubendorf L, Le Magnen C. J Pathol. Results: The Author(s) 2021. drug screen; estradiol; estrogen receptor ; genetic heterogeneity; organoid; papillary thyroid cancer. , Gardner L, Hughes J, et al. These data suggest that these newly developed PTC-derived organoids may be an excellent preclinical model for studying clinical response to anticancer drugs in a personalized way, as well as provide a potential strategy to develop prevention and treatment options for thyroid cancer with ER-specific antagonists. Loss of p53 and RB tumor suppressor pathway function are the most common feature shared across the organoid lines. For instance, we observed that PTC-1_O and PTC-2_O appeared as dense spheres consisting of multiple layers of cells, whereas PTC-3_O exhibited a hollow morphology with cells that appear to be wreathed (Fig. , Bayram T, van de Wetering M, et al. 2021 Feb 18;12(1):1117. doi: 10.1038/s41467-021-21300-6. The mean sequencing depth for blood samples were approximately 100 (~10 Gb per sample), and for tumor and organoid samples were approximately 200 (~20 Gb per sample). It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide, This PDF is available to Subscribers Only. Patient-Derived Cancer Organoids as Predictors of Treatment Response When required, the organoids were thawed using standard thawing procedures and cultured as described earlier. 4A and Supplementary Table 1 [20]). Development of glioblastoma organoids and their applications in personalized therapy. This is probably due to the intratumor heterogeneity. Organoid models derived from patients with malignant phyllodes tumor of the breast. Histological characterization of papillary thyroid cancer (PTC) organoids. 6D). Patient-Derived Organoids for Precision Cancer Immunotherapy 3D organ-on-a-chip: The convergence of microphysiological systems and organoids. Passage numbers of the organoid lines were PTC-1_O, P3; PTC-2-I_O, P3; PTC-2-II_O, P3; PTC-3_O, P4; PTC-4-I_O, P3; PTC-4-II_O, P3; PTC-5_O, P4; PTC-6-A_O, P4 (culturing for 56 days); PTC-6-B_O, P9 (culturing for 136 days); PTC-7_O, P4; PTC-8_O, P4; NTG-1_O, P6; NTG-2_O, P3; NTG-3_O, P5. 3505-0061], and 1% antibiotic-antimycotic [Gibco, No. In contrast to any PTC cell lines grown in 2D, tumor-derived organoids established in our study can recapitulate the histological features and molecular hallmarks of the matched primary tumors, even after long-term expansion in the same culture system. Cancer Res . 3 and Supplementary Fig. Drug sensitivity assays of PTC organoids demonstrated patient-specific drug responses (Fig. A, Flow diagram of establishment and characterization of PTC organoids, including the generation of PTC organoid lines from patients undergoing surgery using optimized medium components, as well as the histological characterization, DNA-sequencing, drug screen, and cell proliferation assay on PTC organoids. FOIA Before 3 [20]). Y However, study research on colorectal cancer organoids found that 6 of 14 lines with the KRAS mutation remained dependent on EGF (27). B, Heat map of gene mutation variations in driver genes of parental tumors and the corresponding organoids. Drug sensitivity testing and organoid cell . The PDOs can be used to build a biobank and perform single-cell sequencing, drug screening and genetic analyses. A brief history of organoids | American Journal of Physiology-Cell To understand thyroid cancer and translate this knowledge to clinical applications, patient-derived PTC organoids as a promising new preclinical model were established. Karkampouna S, La Manna F, Benjak A, Kiener M, De Menna M, Zoni E, Grosjean J, Klima I, Garofoli A, Bolis M, Vallerga A, Theurillat JP, De Filippo MR, Genitsch V, Keller D, Booij TH, Stirnimann CU, Eng K, Sboner A, Ng CKY, Piscuoglio S, Gray PC, Spahn M, Rubin MA, Thalmann GN, Kruithof-de Julio M. Nat Commun. 10099-141), followed by centrifugation at 300g. 2023 Jun 29. doi: 10.1007/s00109-023-02340-5. Published by Oxford University Press on behalf of the Endocrine Society. E, Representative gene copy number variations (CNVs) of PTC tumors and the paired organoids (red, gain; blue, loss; green, diploid). All stock solutions were diluted to the desired concentrations with prewarmed culture medium before use. Significance was set at P less than .05. M7068) was purchased from Sigma-Aldrich, and fulvestrant (ICI 182 780, a potent ER antagonist, No. Please enable it to take advantage of the complete set of features! Patient-Derived Organoids of Colorectal Cancer: A Useful Tool for 2017 Dec;23(12):1424-1435. doi: 10.1038/nm.4438. , Sinha R, Levine DA, Sander C, Schultz N. Sato 17101-015) in the presence of the rho-associated protein kinase inhibitor Y-27632 dihydrochloride (10 M, AbMole Bioscience, No. This is probably the biggest advantage of organoids compared with cancer cell lines. 13 Citations 7 Altmetric Metrics Abstract Multiple three-dimensional (3D) tumour organoid models assisted by multi-omics and Artificial Intelligence (AI) have contributed greatly to preclinical drug development and precision medicine. B and F, Representative confocal images of cell proliferation in PTC organoids induced by 10 nM and 100 nM E2, 100 nM propylpyrazole triol (PPT), 100 nM DPN (2,3-bis(4-hydroxyphenyl)-propionitrile), and 100 nM GPER-selective agonist (G-1). 2). Would you like email updates of new search results? G9683) according to the manufacturers instructions, and luminescence was detected in the VICTOR Nivo Multimode Plate Reader (PerkinElmer Inc). Loss of tumor suppressors PTEN,, Figure 5. sharing sensitive information, make sure youre on a federal The sequencing data were filtered by adaptor and low-quality reads by fastP (version 0.12.6). The methodology described here should enable the generation of a large repertoire of patient-derived prostate cancer lines amenable to genetic and pharmacologic studies. , Sasaki N, Lee-Six H, et al. As a novel-type model, organoid has significant advantages compared with conventional tumor research models, characterized with the high success rate of construction and the high matching with the original . Although most PTC patients have a favorable prognosis, a subgroup of patients die, especially when disease recurrence occurs. During the past decades, numerous anticancer drugs developed from screening 2D cancer cell lines failed in clinical trials. Human gastric cancer organoid culture and drug tests were completed with the cooperation of Chongqing Kingbiotech [10]. Front Endocrinol (Lausanne). The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Dissociated tissues were spun down at 300g for 5 minutes, washed once with AdDMEM/F12+++ medium (Advanced DMEM [Dulbeccos modified Eagles medium]/F12 medium [Gibco, No. Federal government websites often end in .gov or .mil. Scale bar, 100 m. An organoid is a self-organized 3D tissue that is typically derived from stem cells (pluripotent, fetal or adult), and which mimics the key functional, structural and biological complexity of. Our in vitro studies with ER-subtypeselective agonists and antagonists demonstrated that E2 and PPT stimulated cell proliferation of PTC organoids, which can be attenuated by ER or ER antagonists. , Dong W, Li J, Zhang H, Shan Z, Teng W. Vivacqua We then verified whether the somatic mutations of PTC organoid could be maintained in long-term culture. Institute of Shenzhen Translational Medicine, Shenzhen Second Peoples Hospital, the First Affiliated Hospital of Shenzhen University, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. Organoids are not organs: Sources of variation and misinformation inorganoid biology. 2019YFA0906000), the National Natural Science Foundation of China (grant No. PTC, papillary thyroid cancer; NTG, nodular thyroid goiter; Normal, nontumor normal tissue; T, tumor; O, organoid. Patient-derived organoids as a predictive biomarker for - Nature Using a 3D organoid system, we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. Praharaj PP, Bhutia SK, Nagrath S, Bitting RL, Deep G. Biochim Biophys Acta Rev Cancer. Esophageal organoids: applications and future prospects. Organoid Cultures Derived from Patients with Advanced - ScienceDirect Then, organoids provided the breakthrough required to generate models from multiple human cancer types, including pancreatic cancer (7), gastric cancer (8), liver cancer (9), bladder cancer (10), endometrial cancer (11), breast cancer (12), ovarian cancer (13), and prostate cancer (14). The cell pellets were resuspended in ice-cold Matrigel (Corning, No. Loss of tumor suppressors PTEN, TP53, RB1 , and CDKN2A in prostate organoid, Figure 6. The https:// ensures that you are connecting to the Nat Methods. The whole fluorescence intensity (FI) of EdU and Hoechst 33342 in each well was measured by the VICTOR Nivo Multimode Plate Reader (PerkinElmer Inc), and the proliferation levels of PTC organoids were presented by percentage of EdU-FI/Hoechst-FI. 2020 May;80(6):518-526. doi: 10.1002/pros.23966. SOP40104: Cryopreservation of Patient-Derived Organoid (PDOrg) Cultures; SOP30108: Implantation of Patient-Derived in vitro Material (2D and 3D cell cultures) for Generation of Cell Line Xenografts (CLXs) PDMR Genomics. Several studies have been conducted using two-dimensional cultured cell lines; however, these cells do not represent a study model of patient tumors very well. Previous research indicated that p38 is also involved in the regulation of apoptosis as a tumor suppressor (29). Circulating tumor cell-derived organoids: Current challenges and promises in medical research and precision medicine. Frontiers | Organoid: Next-Generation Modeling of Cancer Research and O Matano This improved efficiency of generation, together with the predictive value of organoid assays in terms of patient outcome, suggests two ways by which tumor organoids may serve clinical need: (i) organoid biobanks can be utilized for preclinical screening of new drugs [21] and (ii) individual patient-derived organoids can predict the response of . By contrast, no ER proteins were detected in PTC-8 tumors and the derived organoids (Fig. Organoid cultures conserve the genetic and phenotypic characteristics of the original tumor and maintain its heterogeneity, allowing their application in many research fields. Host-mouse Reference Sequence *.vcf file (NOD. Epub 2022 Dec 26. In total, we established 10 PTC-derived organoid lines from 8 patients, and 3 nodular thyroid goiter (NTG)-derived organoid lines from 3 patients. 4A and 4B, Supplementary Table 1 and 2 [20]). Organoid cultures derived from patients with advanced prostate cancer Disclosures: The authors have nothing to disclose. PTC, papillary thyroid cancer; NTG, nodular thyroid goiter. 2018 Apr;1869(2):117-127. doi: 10.1016/j.bbcan.2017.12.005. Cell viability was detected after 6 days of incubation by using the CellTiter-Glo 3D Reagent (Promega, No. 4 [20]). 2 [20]). Prostate cancer organoids exhibit diverse, Figure 4. The process, from receiving the sample in the research lab, to having a culture in the incubator, in preparation for the organoids to form, takes around 4 hours. eCollection 2022. 354253) and digested for organoid number counting. 7076S) for 1 hour at room temperature and finally developed with 3,3-diaminobenzidine (DAB) for 30 to 60 seconds, counterstained with hematoxylin, mounted, and digitally photographed with the Olympus IX73 microscope. Epub 2021 Jun 2. Clinical translation of patient-derived tumour organoids- bottlenecks After being rinsed with phosphate-buffered saline (PBS), the slides were then incubated with the secondary antibody, horseradish peroxidaselinked antirabbit immunoglobulin G (1:1000, Cell Signaling Technology, No. Drug screens were performed in technical (same screening run) and biological (2 different passage) duplicates. Dong Chen and others, Organoid Cultures Derived From Patients With Papillary Thyroid Cancer, The Journal of Clinical Endocrinology & Metabolism, Volume 106, Issue 5, May 2021, Pages 14101426, https://doi.org/10.1210/clinem/dgab020. Organoids | Nature Reviews Methods Primers A patient-derived tumor organoid created in the Skardal Biofabrication Lab. 4E and Supplementary Table 5 [20]). 12373), ER (1:200, Abcam, No. 6 and Supplementary Fig. HHS Vulnerability Disclosure, Help They can recapitulate the in vivo architecture, functionality, and genetic feature of parental tissues. 2019 Jul;79(10):1156-1165. doi: 10.1002/pros.23830. Growth of the organoids takes around 2 weeks from tissue preparations. Left behind was a cancer organoid - a 3D mass . As expected, after 4 months of culture, we found that the mutational landscape and CNVs of PTC-6_O (PTC-6-A_O: early passage, PTC-6-B_O: late passage) were stably maintained (see Fig. 6A, Supplementary Fig. In our study, we described a 3D culture system for successful and efficient generation of patient-derived PTC organoid lines in vitro. Patient-derived tumor organoids have been deployed in a wide variety of cancer types for chemotherapy screening and even prediction of clinical outcomes in some patients. The 3 ER proteins were shown to be expressed in tumor-organoid pairs of PTC-2, PTC-3, and PTC-6 (see Fig. Organoids can be derived from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs) through a process similar to the acquisition of their distinctive organization ( 1 ). Magr L, Verstegen MMA, Buschow S, van der Laan LJW, Peppelenbosch M, Desai J. J Immunother Cancer. Results from all these studies suggest that ER is the predominant mediator for the E2-induced cell proliferation in organoid lines. Yang H, Liang Q, Zhang J, Liu J, Wei H, Chen H, Wei W, Chen D, Zhao Y. Groups sharing the same alphabets (a, b) above the bars are not significantly different from one another (P>.05), whereas those not sharing the same alphabets represent significant difference at P less than .05 by ANOVA plus Tukey multiple comparison test. No significant changes in cell proliferation were seen in the DPN- and G-1treated groups (Fig. , Hedayat S, Vatsiou A, et al. In PTC-6_O, fulvestrant, tamoxifen, and MPP showed no significant effects on basal cell proliferation, but blocked E2-induced cell proliferation in organoid lines (Fig. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes, including TMPRSS2-ERG fusion, SPOP mutation, SPINK1 overexpression, and CHD1 loss. 2023 May;11(5):e006290. Thus, the normal thyroid organoids derived from nontumor tissues were actually not successfully established in our present study. A, Heat map of gene mutation variations in the most significantly mutated genes of PTC tumors and the matched organoids. a patient-derived gastric organoid coculture grown in Matrigel with DCs and CTLs purified from the patient's blood, . C, Histogram showing the different contributions of point mutation types in PTC organoids and the corresponding tumors. Epub 2014 Sep 23. ab39742) were applied to sections and incubated at 4 C overnight. 1: Establishment of an organoid biobank and its applications. A synopsis of prostate organoid methodologies, applications, and limitations. Bars represent meansSEM of 3 independent experiments. , Mastrogiovanni G, Verstegen MM, et al. Federal government websites often end in .gov or .mil. In a limited number of cases that were retrospectively studied, the response of patient-derived organoids to therapies resembled prior findings in the clinic (35). Patientderived organoids (PDOs) represent promising preclinical models in various tumor types. Organoid Cultures Derived from Patients with Advanced Prostate Cancer Highlights Generation of prostate cancer organoids from metastasis and circulating tumor cells Organoids retain the histological and molecular features of the patient specimen Organoids recapitulate the diversity of castration-resistant prostate cancer However, PTC-6_O (both 2 passages A and B) were resistant to selumetinib and trametinib despite containing the BRAF mutations. Top: Significant genomic aberrations in the prostate oncogenome from the MSKCC CGH dataset adopted from (Taylor et al., 2010). Trypsinization was stopped by the addition of 5 mL AdDMEM/F12+++ medium containing 20% fetal bovine serum (FBS; Gibco, No. Epub 2018 Jan 31. eCollection 2023. Furthermore, we screened different culture media to find the minimum requirement for the establishment of PTC organoid lines. 5A and 5B). Frontiers | Patient Derived Organoids Confirm That PI3K/AKT Signalling Dong Chen and Yawen Tan contributed equally to this work. To determine whether cell proliferation of organoids will respond to estrogen stimulation and to study estrogen receptors that are involved in this process, we treated PTC organoid lines derived from female patients with E2, either in the presence or absence of the selective ER antagonists, or with the selective ER agonists. For patients 2 and 4, in addition to the organoid lines (PTC-2-I_O and PTC-4-I_O) derived from tumor sites close to the parental tumors (PTC-2_T and PTC-4_T), we also established organoid lines (PTC-2-II_O and PTC-4-II_O) from tumor sites distant from the parental tumors. Establishing Pure Cancer Organoid Cultures: Identification, Selection 12648-010) and frozen following standard procedures. The ongoing study of generating xenografts in NOD-scid gamma mice with PTC organoids will be more helpful for in vivo drug testing. In addition to histological conservation, subsequent marker expression analysis revealed that organoids and their matched tumors exhibited similar staining patterns for CK19, galectin-3, Tg, Ki-67, CD117, and TTF-1 (see Fig. 5 and Supplementary Fig. In accordance with previous reports regarding a variety of tumor types (33), PTC organoids also retain a remarkable intratumor mutational heterogeneity of the originating tumors. a Applications for next-generation organoid biobanks in translational research. Zeng Organoids are in vitroself-assembling, organ-like, three-dimensional cellular structures that stably retain key characteristics of the respective organs. The whole fluorescence intensity (FI) of EdU and Hoechst 33342 in each well were measured using the VICTOR Nivo Multimode Plate Reader (PerkinElmer Inc), and the proliferation levels were presented as a percentage of EdU-FI/Hoechst-FI. PTC is commonly treated by single or a combination of treatments, including thyroidectomy, thyroid hormone therapy, and radioactive iodine treatments. Domcke 2 [20]). M1817) for 45 minutes in a water bath at 37 C with gentle shaking. 1B and 1C), suggesting that the efficient culture of PTC organoids requires the 2 factors. Primary antibodies against cytokeratin 19 (CK19, 1:200, Maixin Biotech, No. All rights reserved. , Konopka T, Gacquer D, et al. Hernndez-Llodr S, Segals L, Safont A, Juanpere N, Lorenzo M, Fumad L, Rodrguez-Vida A, Cecchini L, Bellmunt J, Lloreta-Trull J. Prostate. Before Gastric cancer samples were removed from the preservation solution and repeatedly rinsed with D-PBS (Solarbio, D1040) containing 100 U/mL penicillin-streptomycin-amphotericin B until the D-PBS was clear . SF Thyroid cancer has been the most frequent endocrine malignancy with an increasing prevalence in the last decade all over the world (1). 4C and 4D, Supplementary Table 3 and 4 [20]). This site needs JavaScript to work properly. Organoid culture medium was refreshed every 3 days. Digest patient-derived cell lines, cancer specimens and stem cells into separated cells. The minced tissues were digested with collagenase type II (5 mg/mL, Gibco, No. The site is secure. The emergence of organoid technology has provided a new direction for colorectal cancer research. See also Supplementary Table 2 (20). Organoids were passaged at a 1:2 to 1:4 dilution every 2 weeks. Accessibility Organoids are in vitro self-assembling, organ-like, three-dimensional cellular structures that stably retain key characteristics of the respective organs.

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