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LIVER Publication-derived

Human Multi-Lineage Liver Organoid Formation, Culture, and Exposure to Graphene Oxide

Source Romaldini et al., 2024 · Istituto Italiano di Tecnologia (IIT), Genoa, Italy · 10.3390/cells13181542

👤 Alessio Romaldini, Raffaele Spanò, Marina Veronesi, Benedetto Grimaldi, Tiziano Bandiera, Stefania Sabella ⏱ 29 days 📋 5 phases 🧫 Human iPSC-derived hepatocytes (upcyte UHHs), human primary endothelial cells (upcyte LSECs), human bone marrow-derived mesenchymal stromal cells (hbmMSCs)

Abstract

This protocol describes the formation and 29-day culture of human liver organoids (LOs) using upcyte hepatocytes, liver sinusoidal endothelial cells, and mesenchymal stromal cells in a 1:1:0.2 ratio on Matrigel-coated plates. LOs develop stable 3D architecture with functional hepatic structures (bile canalicular networks and sinusoid-like structures) and maintain hepatocellular function (CYP3A4 activity, albumin production) over extended periods, permitting acute and repeated-dose toxicity studies of nanomaterials including graphene oxide.

Cell source
Human iPSC-derived hepatocytes (upcyte UHHs), human primary endothelial cells (upcyte LSECs), human bone marrow-derived mesenchymal stromal cells (hbmMSCs)
Application
Disease modeling; Hepatotoxicity assessment; Nanomaterial toxicity screening

Protocol overview

25 steps across 5 phases

Cell Expansion and Preparation Pre-culture (variable duration)
  1. 1 Thaw and expand cryopreserved UHHs
  2. 2 Expand cryopreserved LSECs
  3. 3 Expand cryopreserved hbmMSCs
Liver Organoid Formation Day 0–10 (maturation phase)
  1. 1 Mix cell populations and prepare LO cell suspension
  2. 2 Resuspend cells in liver-organoid medium (LOM)
  3. 3 Coat 24-well plates with Matrigel diluted in LSEC medium
  4. 4 Seed cells into Matrigel-coated 24-well plates
  5. 5 Incubate cells to permit self-assembly into 3D structures
  6. 6 Daily media changes during maturation (Days 1–10)
  7. 7 Confirm organoid maturation by size stability
Organoid Culture Extension and Functional Characterization (Days 10–29) Day 10–29
  1. 1 Continue daily media changes from day 10 to day 29
  2. 2 Perform size monitoring and morphological assessment
  3. 3 Monitor hepatocellular function (optional: days 10, 29)
Treatment with Graphene Oxide (Acute and Repeated Exposure) Day 10–29 (after maturation)
  1. 1 Prepare graphene oxide (GO) suspension in LOM
  2. 2 Apply acute (single) dose treatment
  3. 3 Apply repeated-dose treatment
  4. 4 Prepare control (untreated) organoids
  5. 5 Optional: Apply rifampicin positive control (CYP3A4 induction)
Sample Collection and Analysis Days 1–29 (throughout culture)
  1. 1 Collect and process conditioned media for exo-metabolomics
  2. 2 Harvest organoids for gene expression analysis (qPCR)
  3. 3 Harvest organoids for protein analysis (Western blot)
  4. 4 Assess cell viability (resazurin reduction assay)
  5. 5 Measure CYP3A4 metabolic activity (BFC assay)
  6. 6 Measure albumin secretion (ELISA)
  7. 7 Assess apoptosis and senescence markers

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Romaldini et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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