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LIVER Publication-derived

Liver Organoids for Drug Screening (2024)

Full SOP with Academic or a plan

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

Authors: Alessio Romaldini, Raffaele Spanò, Marina Veronesi, Benedetto Grimaldi, Tiziano Bandiera, Stefania Sabella Duration: 29 days 5 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Romaldini et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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