Skip to content
← Back to browse
LIVER Publication-derived

miR-122 Inhibition in Human Liver Organoids: Construction, Characterization, and Disease Modeling

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

👤 Hossein Sendi, Ivy Mead, Meimei Wan, Marjan Mehrab-Mohseni, Kenneth Koch, Anthony Atala, Herbert L. Bonkovsky, Colin E. Bishop ⏱ 28 days 📋 8 phases 🧫 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

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

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Sendi et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence