Skip to content
Back to browse
LIVER Publication-derived

Cholestasis-Induced Liver Fibrosis Organoids

Full SOP with Academic or a plan

Source Li et al., 2024 · School of Life Science, Beijing University of Chinese Medicine · 10.7150/ijbs.90441

Authors: Hongmei Li, Jingyi Li, Ting Wang, Ke Sun, Guangrui Huang, Yulin Cao, Fenfang Wu, Anlong Xu Duration: 56 days 10 phases Cell source: Human iPSC

Abstract

This protocol establishes the differentiation of human induced pluripotent stem cells (hiPSCs) into functional hepatobiliary organoids (HBOs) over approximately 35 days in vitro, followed by transplantation into a cynomolgus monkey model of cholestasis-induced liver fibrosis via subcapsular and submesenteric routes. The study evaluates the safety and efficacy of HBO transplantation in alleviating liver fibrosis through antiapoptotic, antifibrotic, and CTSV-mediated mechanisms.

Cell source
Human iPSC
Application
Disease modeling - cholestasis-induced liver fibrosis

Protocol overview

30 steps across 10 phases

Phase 1 · hiPSC Culture and Preparation Days 1-5 pre-differentiation
  1. a Plate hiPSCs on Matrigel-coated wells
  2. b Verify pluripotency markers
Phase 2 · Hepatobiliary Organoid Differentiation - Endoderm Induction Days 1-4
  1. a Dissociate hiPSCs and passage onto fresh Matrigel plates
  2. b Initiate differentiation with activin A and BMP4
  3. c Add mTeSR supplement and continue culture
  4. d Transition to endoderm specification
Phase 3 · Hepatobiliary Organoid Differentiation - Hepatic Progenitor Formation Days 5-16
  1. a Induce liver bud formation with BMP2 and FGF4
  2. b Culture with HGF and KGF for hepatic progenitor maturation
Phase 4 · Hepatobiliary Organoid Differentiation - Hepatobiliary Maturation Days 17-36
  1. a Hepatocyte and cholangiocyte differentiation with oncostatin M and cholesterol MIX
  2. b Mature HBO culture with ascorbic acid and transferrin supplement
  3. c Select Day 35 HBOs for transplantation
Phase 5 · Nonhuman Primate Bile Duct Ligation and HBO Transplantation - Preparation Day -5 (pre-transplant)
  1. a Initiate immunosuppressive therapy
Phase 6 · Bile Duct Ligation Surgery and HBO Transplantation - Day 0 Day 0 (surgery day)
  1. a Anesthetize monkey and prepare surgical field
  2. b Collect pre-surgery liver biopsy and verify normal liver
  3. c Dissect hepatic vasculature and ligate common bile duct
Phase 7 · HBO Transplantation - Liver Subcapsular Route Day 0 (within 30 minutes post-BDL)
  1. a Prepare Day 35 HBOs for subcapsular transplantation
  2. b Inject HBOs under liver capsule at 5 sites
  3. c Close liver incision and abdominal wall
Phase 8 · HBO Transplantation - Submesenteric Route (Alternative to Subcapsular) Day 0 (within 30 minutes post-BDL, alternative route)
  1. a Prepare HBOs for submesenteric transplantation
  2. b Identify colon mesentery and select 6 transplant sites
  3. c Graft HBOs into mesenteric sites and suture
  4. d Close abdominal wall
Phase 9 · Post-Transplantation Monitoring and Sample Collection Days 1-56 (8 weeks post-transplantation)
  1. a Collect baseline blood sample (Pre-Tx)
  2. b Weekly blood sampling and biochemical analysis (Weeks 1-8)
  3. c Abdominal ultrasound imaging
  4. d CT scan imaging at baseline, 4 weeks, and 8 weeks
Phase 10 · Terminal Procedures and Sample Collection Day 56 (8 weeks post-transplantation)
  1. a Euthanasia and gross organ examination
  2. b Quantify liver and spleen volumes
  3. c Tissue sampling for histopathology
  4. d Collect final blood sample for coagulation and safety parameters

Full SOP

Full SOPs need an account and a plan

Create an account, then get free Academic access (for verified academics) or choose a plan. Plans include step-by-step procedures, reagent concentrations, QC checklists and batch record templates.

Create an account

Already registered? Log in

Attribution

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

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