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LIVER Publication-derived

Fatty Liver Disease Organoids for Infection Studies

Full SOP with Academic or a plan

Source Sendi et al., 2018 · Wake Forest Institute of Regenerative Medicine / Wake Forest Baptist Medical Center · 10.1371/journal.pone.0200847

Authors: Hossein Sendi, Ivy Mead, Meimei Wan, Marjan Mehrab-Mohseni, Kenneth Koch, Anthony Atala, Herbert L. Bonkovsky, Colin E. Bishop Duration: 28 days 8 phases Cell source: Primary Human Hepatocytes, Kupffer Cells, Hepatic Stellate Cells, Liver Sinusoidal Endothelial Cells

Abstract

This protocol describes the construction and characterization of multicellular 3D human liver organoids containing primary hepatocytes (80%), Kupffer cells (10%), stellate cells (5%), and endothelial cells (5%) that remain viable and functional for 4+ weeks. The protocol includes methods for lentiviral transduction to inhibit miR-122 expression and comprehensive analysis of resulting inflammatory, fibrotic, and metabolic dysregulation as a model of non-alcoholic fatty liver disease (NAFLD).

Cell source
Primary Human Hepatocytes, Kupffer Cells, Hepatic Stellate Cells, Liver Sinusoidal Endothelial Cells
Application
Disease modeling (NAFLD/NASH pathogenesis and steatofibrosis)

Protocol overview

25 steps across 8 phases

Phase 1 · Cell Procurement and Preparation Day 0
  1. a Obtain and Thaw Cryopreserved Hepatic Cells
  2. b Prepare Cell Mixture
Phase 2 · Liver Organoid Formation and Initial Culture Day 0-Day 4
  1. a Form Organoids via Gravitational Aggregation
  2. b Transfer Organoids to Culture Medium (Hanging Drop Method)
  3. c Initialize Feeding Schedule (ULA Method)
Phase 3 · Lentiviral Transduction for miR-122 Inhibition Day 0 (immediately after cell mixing)
  1. a Add Lentiviral Particles Immediately After Cell Mixing
Phase 4 · Medium Changes and Long-Term Culture Day 4-Day 28
  1. a Perform Medium Changes Every Other Day
Phase 5 · Organoid Sampling and Analysis Day 4, Day 11, Day 18
  1. a Harvest and Pool Organoids
  2. b Assess Viability via Live/Dead Staining
  3. c Measure ATP Content for Viability
  4. d Extract Total RNA for qPCR
Phase 6 · Histological Processing and Immunostaining Day 4, Day 11, Day 18 (or after sampling)
  1. a Fix Organoids
  2. b Embed in HistoGel and Paraffin
  3. c Cut and Prepare Histological Sections
  4. d Perform Standard Immunohistochemistry (IHC) on Sections
  5. e Perform Whole Mount Immunofluorescence
Phase 7 · Real-Time PCR (qPCR) Gene Expression Profiling Day 4, Day 11 (post-transduction)
  1. a Synthesize cDNA
  2. b Prepare qPCR Master Mix
  3. c Load qPCR Arrays
  4. d Perform Thermal Cycling
  5. e Analyze Data
Phase 8 · Supporting Functional Assays (Optional) Day 1-Day 28
  1. a Transmission Electron Microscopy (TEM)
  2. b Measure Secreted Liver Function Markers
  3. c Assess Drug Metabolism Capacity
  4. d Perform Toxicity Assays (Optional)

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Attribution

This SOP was written by Organthis based on the published method in Sendi et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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