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LIVER Publication-derived

3D Organoid modelling of hepatoblast-like and mesenchymal-like hepatocellular carcinoma cell lines

Source Oz et al., 2021 · Izmir Biomedicine and Genome Center; Dokuz Eylul University · 10.20517/2394-5079.2021.43

👤 Ozden Oz, Evin Iscan, Tugce Batur, Mehmet Ozturk ⏱ 16 days 📋 7 phases 🧫 HCC cell lines (Hep3B, Huh7, HepG2, SNU398, SNU449, SNU475)

Abstract

This protocol establishes a method to generate 3D organoid-like structures from hepatocellular carcinoma (HCC) cell lines representing hepatoblast-like and mesenchymal-like subgroups. Cells are cultured in Matrigel scaffolds for up to 9 days, then processed for paraffin embedding, histological and immunohistochemical analysis to characterize biomarker expression patterns and cellular organization.

Cell source
HCC cell lines (Hep3B, Huh7, HepG2, SNU398, SNU449, SNU475)
Application
Disease modeling; characterization of HCC cell line phenotypes in 3D culture

Protocol overview

22 steps across 7 phases

Monolayer Cell Culture and Preparation Days 1-7 (minimum 3 passages)
  1. 1 Thaw and expand cell lines
  2. 2 Prepare single-cell suspension
Matrigel Scaffold-Based 3D Culture Setup Day 8 (culture initiation)
  1. 3 Prepare culture media
  2. 4 Mix cells with Matrigel
  3. 5 Create Matrigel domes in non-attachment plate
  4. 6 Invert plates to form hanging drops
  5. 7 Solidify Matrigel domes
  6. 8 Return plates to normal position and add culture medium
3D Culture Maintenance and Monitoring Days 8–9 (culture period)
  1. 9 Incubate organoids
  2. 10 Monitor organoid formation on Day 3
  3. 11 Monitor and feed on Day 5
  4. 12 Monitor and feed on Day 7
Harvest and Fixation Day 9
  1. 13 Harvest organoid-like spheroids
  2. 14 Fix organoids in formalin
Paraffin Embedding and Sectioning Days 10–11
  1. 15 Process organoids for paraffin embedding
  2. 16 Cut paraffin sections
Hematoxylin-Eosin (H&E) Staining Days 12–13
  1. 17 Deparaffinize and rehydrate sections
  2. 18 Perform H&E staining
  3. 19 Pathological evaluation of H&E-stained sections
Immunohistochemical Staining Days 14–16
  1. 20 Prepare sections for immunohistochemistry
  2. 21 Automated immunohistochemical staining
  3. 22 Microscopic analysis and imaging

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Oz et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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