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LIVER Publication-derived

Liver Organoids for Regenerative Medicine with Stromal Cells

Full SOP with Academic or a plan

Source Nigro et al., 2021 · Mediterranean Institute for Transplantation and Advanced Specialized Therapies (IRCCS ISMETT), Palermo, Italy · 10.3389/fmed.2021.746298

Authors: Antonio Lo Nigro, Alessia Gallo, Matteo Bulati, Giampiero Vitale, Diego Sebastian Paini, Mariangela Pampalone, Daniele Galvagno, Pier Giulio Conaldi, Vitale Miceli Duration: 22 days 11 phases Cell source: Human EpCAM+ liver progenitor cells (primary) + Human amnion-derived mesenchymal stromal/stem cells (hAMSCs)

Abstract

This protocol establishes human liver organoids from EpCAM+ primary liver cells and demonstrates that conditioned medium from human amnion-derived mesenchymal stromal/stem cells (hAMSCs), particularly from 3D spheroid cultures, enhances hepatic progenitor cell differentiation into mature hepatocytes. The paracrine factors in hAMSC secretome increase expression of hepatocyte markers (ALB, NTCP, CYP3A4) and functional activity, offering translational implications for liver repair and regeneration.

Cell source
Human EpCAM+ liver progenitor cells (primary) + Human amnion-derived mesenchymal stromal/stem cells (hAMSCs)
Application
Hepatocyte differentiation and liver regeneration modeling

Protocol overview

56 steps across 11 phases

Phase 1 · HepG2 and HDFa Cell Culture Maintenance Ongoing
  1. a Culture HepG2 cells in RPMI medium
  2. b Culture HDFa cells in DMEM medium
Phase 2 · Human Liver Organoid Expansion from EpCAM+ Cells Days 0-14+ (with 7-10 day passage cycles)
  1. a Tissue preparation and collagenase digestion
  2. b Stop digestion and filter cell suspension
  3. c Prepare cells for FACS sorting
  4. d FACS separation of EpCAM+ cells
  5. e Pellet and resuspend EpCAM+ cells
  6. f Prepare organoid culture in BME 2
  7. g Add expansion medium (EM) to organoid cultures
  8. h Culture organoids for expansion phase
Phase 3 · Isolation, Culture, and Characterization of Human Amnion-Derived MSCs (hAMSCs) Days 0-14+
  1. a Prepare amnion tissue from human term placenta
  2. b Decontaminate amnion fragments
  3. c Enzymatic digestion with dispase
  4. d Collagenase and DNase digestion
  5. e Filter and collect hAMSC suspension
  6. f Culture hAMSCs in 2D monolayer
  7. g Culture hAMSCs in 3D spheroids
  8. h Flow cytometry characterization of hAMSCs
Phase 4 · Differentiation Potential of hAMSCs (Optional Characterization) Days 0-14
  1. a Prepare cells for differentiation assay
  2. b Osteogenic and adipogenic differentiation
  3. c Chondrogenic differentiation
  4. d Immunofluorescence detection of differentiation markers
Phase 5 · Conditioned Medium (CM) Preparation from 2D and 3D hAMSC Cultures Days 0-5
  1. a Prepare hAMSCs for 2D CM collection
  2. b Switch to serum-free differentiation medium for 2D culture
  3. c Collect 2D hAMSC conditioned medium
  4. d Prepare hAMSCs for 3D CM collection
  5. e Switch 3D hAMSC spheroids to serum-free differentiation medium
  6. f Collect 3D hAMSC conditioned medium
Phase 6 · Human Liver Organoid Differentiation (7-Day Priming + 13-15 Day Differentiation) Days 0-22
  1. a Prepare organoids for differentiation protocol
  2. b Switch organoids to differentiation medium (unconditioned control)
  3. c Switch organoids to 2D hAMSC-CM (alternative differentiation condition)
  4. d Switch organoids to 3D hAMSC-CM (alternative differentiation condition)
  5. e Maintain organoids in differentiation conditions
  6. f Observe morphological changes
Phase 7 · Gene Expression Analysis by Real-Time PCR After 13-15 days differentiation
  1. a Extract total RNA from organoids
  2. b Synthesize cDNA from RNA
  3. c Perform real-time PCR
  4. d Calculate relative expression
Phase 8 · Functional Analysis: Albumin Secretion and CYP3A4 Activity After 13-15 days differentiation
  1. a Collect culture supernatant for albumin analysis
  2. b Measure albumin concentration by ELISA
  3. c Prepare organoids for CYP3A4 activity assay
  4. d Measure CYP3A4 activity using luminescence assay
Phase 9 · Cell Viability Analysis of hAMSCs in Serum-Free Conditions During CM preparation (Days 1-3)
  1. a Prepare hAMSC samples for viability assessment
  2. b Perform annexin V/7-AAD double staining
  3. c Analyze stained cells by flow cytometry
Phase 10 · Cytokine/Chemokine and Growth Factor Analysis in Conditioned Media After 3 days CM conditioning
  1. a Prepare conditioned media samples
  2. b Perform multiplex cytokine analysis using Luminex technology
  3. c Calculate factor concentrations
Phase 11 · Immunofluorescence of Liver Organoids (Optional Characterization) Days 0-7 (early organoid formation) or after differentiation
  1. a Seed organoids for immunofluorescence
  2. b Fix organoids
  3. c Permeabilize organoid tissue
  4. d Block non-specific binding
  5. e Apply primary antibody
  6. f Wash and apply secondary antibody
  7. g Mount organoid slides
  8. h Microscopy imaging

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Attribution

This SOP was written by Organthis based on the published method in Nigro et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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