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Malignant Pleural Mesothelioma Organoids

Full SOP with Academic or a plan

Source Eltjona et al., 2017 · University Hospital of Novara, Department of Health Sciences · 10.1186/1750-1172-3-34

Authors: Rrapaj Eltjona, Renzo Boldorini, Marisa Gariglio Duration: 180 days 5 phases Cell source: Patient-Derived Mesothelioma Biopsies (Malignant Pleural Mesothelioma)

Abstract

This protocol describes the generation and characterization of three-dimensional patient-derived organoids and patient-derived xenograft (PDX) models from human malignant pleural mesothelioma (MPM) biopsies. Human mesothelioma tissue fragments obtained via thoracoscopic biopsy are used to develop in vitro 3D organoid cultures and in vivo PDX models in immunodeficient mice, providing platforms for drug sensitivity testing, molecular characterization, and investigation of HMGB1 protein as a prognostic biomarker.

Cell source
Patient-Derived Mesothelioma Biopsies (Malignant Pleural Mesothelioma)
Application
Disease modeling, Drug screening, Biomarker validation

Protocol overview

31 steps across 5 phases

Phase 1 · Sample Collection and Preparation Day 0
  1. a Obtain fresh mesothelioma biopsies or tumor fragments
  2. b Transport and initial tissue assessment
  3. c Divide tissue for parallel in vitro and in vivo models
Phase 2 · In Vitro Organoid Generation and Culture Days 0-7
  1. a Prepare mesothelioma tissue for dissociation
  2. b Enzymatic and mechanical dissociation of tumor tissue
  3. c Assess cell viability and count
  4. d Prepare organoid culture matrix and medium
  5. e Establish 3D organoid cultures
  6. f Culture organoids with media changes
  7. g Characterize organoid morphology
  8. h Immunofluorescence characterization of organoid markers
  9. i Passage organoids in vitro
Phase 3 · In Vivo Patient-Derived Xenograft (PDX) Model Development Days 0-180
  1. a Prepare immunodeficient mice for implantation
  2. b Prepare tumor tissue for implantation
  3. c Surgical implantation of tumor tissue
  4. d Monitor tumor growth and measure dimensions
  5. e Conduct serial passages of PDX tumors
  6. f Immunophenotyping of PDX tumors
  7. g Perform histopathological and immunohistochemical analysis of PDX tumors
  8. h Bank tumor tissue and establish cryopreservation
Phase 4 · Drug Sensitivity Testing and Biomarker Studies Days 14-90
  1. a Prepare organoid and PDX-derived cell suspensions for drug testing
  2. b Establish baseline growth kinetics and proliferation assay
  3. c Conduct dose-response drug sensitivity studies
  4. d Evaluate HMGB1 expression in organoids and PDX tumors
  5. e Test HMGB1-targeting therapeutic agents
Phase 5 · Molecular Characterization and Data Analysis Days 90-180
  1. a Extract RNA from organoids and PDX tumors
  2. b Perform reverse transcription (RT) for cDNA synthesis
  3. c Perform RT-PCR for HMGB1 gene expression analysis
  4. d Quantify HMGB1 mRNA expression by qRT-PCR
  5. e Perform statistical analysis of data
  6. f Compare organoid/PDX characteristics with original patient tumor

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Attribution

This SOP was written by Organthis based on the published method in Eltjona et al., 2017. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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