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LIVER Publication-derived

UTOpiA: Extracorporeal whole blood circulation system combining HLA-depleted liver organoids with granulocyte-monocyte apheresis for reversal of ACLF and ALF

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

๐Ÿ‘ค Hitomi Yamaguchi, Yosuke Yoneyama, Kentaro Ichimura, Kanae Ohtsu, Mika Soen, Chiharu Moriya, Maki Kumagai, Robert P. Myers, G. Mani Subramanian, Takanori Takebe โฑ 72 days ๐Ÿ“‹ 10 phases ๐Ÿงซ 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

iPSC Culture and Foregut Differentiation Days 1โ€“6
  1. 1 Culture human iPSCs
  2. 2 Detach and seed iPSCs for foregut differentiation
  3. 3 Day 2 medium change
  4. 4 Day 3 medium change
  5. 5 Days 4โ€“6 foregut induction
  6. 6 Freeze foregut cells
Hepatocyte-like Cell (iHLC) Organoid Formation Days 1โ€“14
  1. 1 Thaw and resuspend foregut cells
  2. 2 Initial organoid culture with growth factors
  3. 3 Y-27632 treatment and spheroid formation
  4. 4 Retinoic acid and A83โ€“01 treatment
  5. 5 Retinoic acid-only treatment
  6. 6 Hepatocyte maturation in HCM medium
HLA Depletion Verification and Flow Cytometry Day 14 (prior to animal treatment)
  1. 1 Dislodge and treat iHLCs with IFN-ฮณ
  2. 2 Dissociate organoids into single cells
  3. 3 Prepare cells for flow cytometry
  4. 4 Stain with HLA antibodies
  5. 5 Analyze by flow cytometry
Alginate Encapsulation of iHLCs Prior to extracorporeal circulation
  1. 1 Prepare iHLCs for encapsulation
  2. 2 Encapsulate iHLCs in alginate
  3. 3 Prepare BAL columns
Animal Model Induction (BDL and LPS) Day 1 (surgery); Day 21 (treatment day)
  1. 1 Perform common bile duct ligation (BDL)
  2. 2 Allow 21-day recovery and cirrhosis development
  3. 3 Induce acute endotoxemia with LPS
Extracorporeal Circulation with UTOpiA System Treatment day (1 h post-LPS injection)
  1. 1 Anesthetize rat and establish vascular access
  2. 2 Prime extracorporeal circuit
  3. 3 Initiate whole blood circulation
  4. 4 Collect blood samples
  5. 5 Discontinue extracorporeal circulation and awaken rat
Post-Treatment Monitoring and Assessment Up to 72 h post-treatment
  1. 1 Assess coma severity
  2. 2 Monitor rats and record survival
  3. 3 Harvest tissues for histological analysis
Biochemical and Molecular Analysis Concurrent with post-treatment monitoring
  1. 1 Measure plasma biochemistry
  2. 2 Measure plasma cytokines
  3. 3 Measure hepatic growth factor, albumin, and AFP
Histological and Immunological Analysis Following tissue harvest
  1. 1 Perform H&E staining
  2. 2 Perform immunohistochemical staining
  3. 3 Perform immunofluorescence staining
  4. 4 Perform TUNEL staining
  5. 5 Calculate histological activity index
RNA-seq and Transcriptomic Analysis Following tissue harvest
  1. 1 Extract total RNA from snap-frozen liver tissue
  2. 2 Prepare RNA-seq libraries
  3. 3 Sequence on DNBSEQ-G400
  4. 4 Quality control and read processing
  5. 5 Quantify gene expression
  6. 6 Perform gene ontology and pathway analysis
  7. 7 Infer transcription factor activity

Full SOP

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Attribution

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

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