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GASTRIC Publication-derived

Signet-Ring Cell Carcinoma Gastric Tumouroids

Full SOP with Academic or a plan

Source Li et al., 2019 · Fudan University Shanghai Cancer Center · 10.2139/ssrn.3360093

Authors: Yaqi Li, Renjie Wang, Dan Huang, Xiaoji Ma, Shaobo Mo, Qiang Guo, Guoxiang Fu, Yuanchuang Li, Xiaoya Xu, Xiang Hu, Yi Zhou, Yun Deng, Long Zhang, Honghong Chen, Jianjun Gao, Zhen Zhang, Sanjun Cai, Guoqiang Hua, Junjie Peng Duration: 120 days 8 phases Cell source: Patient-Derived Carcinoma (Signet-ring cell carcinoma, primary colon tumor with ovarian metastases)

Abstract

This protocol describes the establishment and characterization of YQ-173, a patient-derived organoid line from a rare human colon signet-ring cell carcinoma (SRCC). The method involves tissue digestion, 3D culture in Matrigel, optimization of culture media, and subsequent 2D monolayer derivation. The organoid line is validated for morphological, histological, genetic, and functional characteristics, and applied for personalized drug screening to identify JAK2 inhibitors as potential therapeutic agents.

Cell source
Patient-Derived Carcinoma (Signet-ring cell carcinoma, primary colon tumor with ovarian metastases)
Application
Disease modeling and drug screening for rare signet-ring cell carcinoma

Protocol overview

41 steps across 8 phases

Phase 1 · Tissue Collection and Tumor Cell Isolation Day 0-1
  1. a Obtain surgical sample and consent
  2. b Prepare tissue for histology and DNA isolation
  3. c Wash tissue with PBS and digestion
  4. d Isolate crypts and cell aggregates
Phase 2 · Three-Dimensional Organoid Culture Optimization Day 1-10
  1. a Embed isolated aggregates in Matrigel
  2. b Overlay with culture medium
  3. c Test four culture media formulations
  4. d Test additional growth factors
  5. e Select optimal medium
Phase 3 · Routine 3D Organoid Culture and Maintenance Day 10-30+
  1. a Maintain and observe organoid growth
  2. b Refresh culture medium every 2 days
  3. c Passage organoids at split ratios 1:5 or 1:10
  4. d Bank organoid line and confirm stability
Phase 4 · Transfer to 2D Monolayer Culture Day 30-40
  1. a Prepare organoids for 2D transfer
  2. b Dissociate organoids to single cells or small aggregates
  3. c Seed cells in tissue culture flasks
  4. d Maintain 2D cultures
Phase 5 · Morphological and Molecular Characterization Throughout culture (sampling at Days 10, 20, 30)
  1. a Histology (Hematoxylin & Eosin staining)
  2. b Immunohistochemistry (IHC) staining
  3. c Immunofluorescence (IF) staining
  4. d RNA extraction and qRT-PCR
  5. e Electron microscopy
  6. f Chromosome analysis (Karyotyping)
Phase 6 · Genomic Characterization: RNA-seq and DNA Sequencing Days 30-60
  1. a RNA isolation and RNA-seq library preparation
  2. b RNA-seq data analysis
  3. c Genomic DNA extraction
  4. d Capture-based targeted DNA sequencing
  5. e Mutation and copy-number analysis
Phase 7 · In Vitro Drug Screening and Validation Days 40-60
  1. a Prepare organoids for initial drug screening
  2. b Screen compound library
  3. c Measure organoid viability after drug screening
  4. d Identify hit compounds and select for validation
  5. e Dose-response validation of selected drugs
  6. f Determine IC₅₀ values and select lead compound
Phase 8 · In Vivo Xenograft Model and Drug Efficacy Testing Days 60-120
  1. a Prepare cells and animals for xenotransplantation
  2. b Subcutaneous xenotransplantation
  3. c Monitor tumor growth (untreated phase)
  4. d Randomize and treat with AT9283
  5. e Monitor tumor growth and body weight during treatment
  6. f Euthanize mice and harvest tumors
  7. g Histological analysis of excised tumors

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Attribution

This SOP was written by Organthis based on the published method in Li et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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