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LIVER Publication-derived

Optimized 3D Culture of Hepatic Cells for Liver Organoid Metabolic Assays

Source Gamboa et al., 2021 · Rutgers Cancer Institute of New Jersey, Rutgers University · 10.3390/cells10123280

👤 Christian Moya Gamboa, Yujue Wang, Huiting Xu, Katarzyna Kalemba, Fredric E. Wondisford, Hatem E. Sabaawy ⏱ 28 days 📋 7 phases 🧫 Human primary hepatocytes (adult donor-derived) and HepG2 cells

Abstract

This protocol establishes optimized conditions for 3D culture of hepatic organoids from adult human hepatocytes and HepG2 cells in serum-free, defined media supplemented with Forskolin (FSK) and Oncostatin M (OSM) to promote expansion while maintaining gluconeogenic gene expression. The resulting hepatic organoids (HepAOs and HepGOs) display hepatocyte-like function and metabolic competence for glucose production assays.

Cell source
Human primary hepatocytes (adult donor-derived) and HepG2 cells
Application
Disease modeling and metabolic assay development for gluconeogenesis

Protocol overview

49 steps across 7 phases

Primary Hepatocyte Isolation and Preparation Day 0–1
  1. 1 Receive and process fresh human hepatocyte suspension
  2. 2 Centrifuge hepatocyte suspension
  3. 3 Discard supernatant and wash cell pellet
  4. 4 Repeat centrifugation and washing
  5. 5 Resuspend pellet and count cells
  6. 6 Freeze or use hepatocytes immediately
Single Cell-Derived Hepatic Organoid Formation (Isolation Phase) Day 1–7
  1. 1 Prepare isolation medium
  2. 2 Prepare Matrigel-coated culture plates
  3. 3 Seed single cells in Matrigel
  4. 4 Incubate Matrigel dome
  5. 5 Add isolation medium overlay
  6. 6 Mark and monitor single cells
  7. 7 Monitor organoid formation
  8. 8 Replace media every 2 days
  9. 9 Count qualifying organoids at day 7
Organoid Expansion Phase Day 8–28
  1. 1 Replace isolation medium with expansion medium containing ROCK inhibitor
  2. 2 Replenish expansion medium
  3. 3 Expand organoids in EM for 14 days
  4. 4 Document expansion rates and morphology
  5. 5 Add BMP7 prior to differentiation
Hepatocyte Differentiation Phase Day 19–26
  1. 1 Replace media with hepatocyte differentiation medium
  2. 2 Replenish differentiation medium
  3. 3 Culture organoids in DM for 7 days
  4. 4 Monitor hepatocyte differentiation markers
Organoid Recovery from Matrigel and Dissociation As needed for analysis
  1. 1 Remove culture media from wells
  2. 2 Chill plate and add cold PBS
  3. 3 Incubate PBS to dissolve Matrigel
  4. 4 Centrifuge organoid suspension
  5. 5 Remove supernatant and add cell recovery solution
  6. 6 Dissolve pellet in recovery solution
  7. 7 Transfer to Eppendorf tubes and incubate on ice
  8. 8 Centrifuge and wash recovered organoids
HepG2 Hepatoma Cell Organoid (HepGO) Culture (Alternative to Primary Hepatocytes) Parallel to HepAO protocol
  1. 1 Prepare HepG2 cells in monolayer culture
  2. 2 Harvest HepG2 cells at 80–90% confluence
  3. 3 Prepare 48-well tissue culture plates
  4. 4 Seed HepG2 cells in Matrigel at optimized density
  5. 5 Overlay with expansion medium
  6. 6 Incubate plates under standard culture conditions
  7. 7 Change media every other day
  8. 8 Count HepGOs at day 7
  9. 9 Replace media with differentiation medium
  10. 10 Culture HepGOs in DM for 7 additional days
Glucose Production and Metabolic Competence Assay Upon completion of differentiation phase
  1. 1 Prepare organoid cultures for metabolic assay
  2. 2 Perform serum starvation
  3. 3 Prepare glucose production basal medium
  4. 4 Prepare test substrates
  5. 5 Perform glucose production assay
  6. 6 Measure glucose concentration enzymatically
  7. 7 Calculate glucose production rates

Full SOP

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Attribution

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

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