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LIVER Publication-derived

Liver Organoids for Disease Modelling (No. 2)

Full SOP with Academic or a plan

Source Yamaguchi et al. · Institute of Science Tokyo (Science Tokyo); The University of Osaka

Authors: Hitomi Yamaguchi, Yosuke Yoneyama, Kentaro Ichimura, Kanae Ohtsu, Mika Soen, Chiharu Moriya, Maki Kumagai, Robert P. Myers, G. Mani Subramanian, Takanori Takebe Duration: 72 days 10 phases Cell source: Human iPSC (HLA-A, HLA-B, CIITA triple knockout)

Abstract

This protocol describes the generation of hypoimmunogenic hepatocyte-like cell (iHLC) organoids from HLA-depleted iPSCs and their integration into an extracorporeal UTOpiA system combined with granulocyte-monocyte apheresis for treatment of acute-on-chronic liver failure (ACLF) and acute liver failure (ALF). The system demonstrates therapeutic efficacy through anti-inflammatory and metabolic support mechanisms.

Cell source
Human iPSC (HLA-A, HLA-B, CIITA triple knockout)
Application
Disease modeling (ACLF and ALF)

Protocol overview

46 steps across 10 phases

Phase 1 · iPSC Culture and Foregut Differentiation Days 1-6
  1. a Culture human iPSCs
  2. b Detach and seed iPSCs for foregut differentiation
  3. c Day 2 medium change
  4. d Day 3 medium change
  5. e Days 4-6 foregut induction
  6. f Freeze foregut cells
Phase 2 · Hepatocyte-like Cell (iHLC) Organoid Formation Days 1-14
  1. a Thaw and resuspend foregut cells
  2. b Initial organoid culture with growth factors
  3. c Y-27632 treatment and spheroid formation
  4. d Retinoic acid and A83-01 treatment
  5. e Retinoic acid-only treatment
  6. f Hepatocyte maturation in HCM medium
Phase 3 · HLA Depletion Verification and Flow Cytometry Day 14 (prior to animal treatment)
  1. a Dislodge and treat iHLCs with IFN-γ
  2. b Dissociate organoids into single cells
  3. c Prepare cells for flow cytometry
  4. d Stain with HLA antibodies
  5. e Analyze by flow cytometry
Phase 4 · Alginate Encapsulation of iHLCs Prior to extracorporeal circulation
  1. a Prepare iHLCs for encapsulation
  2. b Encapsulate iHLCs in alginate
  3. c Prepare BAL columns
Phase 5 · Animal Model Induction (BDL and LPS) Day 1 (surgery); Day 21 (treatment day)
  1. a Perform common bile duct ligation (BDL)
  2. b Allow 21-day recovery and cirrhosis development
  3. c Induce acute endotoxemia with LPS
Phase 6 · Extracorporeal Circulation with UTOpiA System Treatment day (1 h post-LPS injection)
  1. a Anesthetize rat and establish vascular access
  2. b Prime extracorporeal circuit
  3. c Initiate whole blood circulation
  4. d Collect blood samples
  5. e Discontinue extracorporeal circulation and awaken rat
Phase 7 · Post-Treatment Monitoring and Assessment Up to 72 h post-treatment
  1. a Assess coma severity
  2. b Monitor rats and record survival
  3. c Harvest tissues for histological analysis
Phase 8 · Biochemical and Molecular Analysis Concurrent with post-treatment monitoring
  1. a Measure plasma biochemistry
  2. b Measure plasma cytokines
  3. c Measure hepatic growth factor, albumin, and AFP
Phase 9 · Histological and Immunological Analysis Following tissue harvest
  1. a Perform H&E staining
  2. b Perform immunohistochemical staining
  3. c Perform immunofluorescence staining
  4. d Perform TUNEL staining
  5. e Calculate histological activity index
Phase 10 · RNA-seq and Transcriptomic Analysis Following tissue harvest
  1. a Extract total RNA from snap-frozen liver tissue
  2. b Prepare RNA-seq libraries
  3. c Sequence on DNBSEQ-G400
  4. d Quality control and read processing
  5. e Quantify gene expression
  6. f Perform gene ontology and pathway analysis
  7. g Infer transcription factor activity

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Attribution

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

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