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LIVER Publication-derived

Acute Liver Failure Organoids for Transplantation

Full SOP with Academic or a plan

Source Nie et al., 2018 · Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine · 10.1186/s13287-017-0749-1

Authors: Yun-Zhong Nie, Yun-Wen Zheng, Miyuki Ogawa, Etsuko Miyagi, Hideki Taniguchi Duration: 30 days 6 phases Cell source: Patient-Derived iPSC (Umbilical Cord-derived Endothelial Cells and Mesenchymal Cells)

Abstract

This protocol generates functional single donor cell-derived liver organoids (SDC-LOs) from human induced pluripotent stem cells (hiPSCs) reprogrammed from umbilical cord endothelial cells, together with umbilical cord-derived endothelial and mesenchymal cells, for therapeutic transplantation in acute liver failure models. The resulting organoids rapidly assume hepatic functions and improve survival in acutely failed liver mice.

Cell source
Patient-Derived iPSC (Umbilical Cord-derived Endothelial Cells and Mesenchymal Cells)
Application
Disease modeling; Acute liver failure therapy

Protocol overview

50 steps across 6 phases

Phase 1 · EC and MC Isolation and Culture Day 0-7
  1. a Preparation of umbilical cord vein for EC isolation
  2. b Enzyme digestion of umbilical cord vein for EC extraction
  3. c Collection and centrifugation of EC-containing wash medium
  4. d Resuspension and plating of isolated ECs
  5. e Preparation of umbilical cord tissue for MC isolation
  6. f Enzyme cocktail digestion for MC extraction
  7. g Centrifugation and resuspension of isolated MCs
  8. h Seeding of isolated MCs
  9. i Maintenance of EC and MC cultures
Phase 2 · Generation of Nonviral Feeder-Free hiPSCs from UC-Derived ECs Day 0-30
  1. a Preparation of Matrigel-coated culture plates
  2. b Transfection of episomal iPSC reprogramming vectors into UC-ECs
  3. c Culture of transfected cells in mTeSR medium
  4. d Monitoring of hiPSC colony formation
  5. e Picking and expansion of individual hiPSC colonies
  6. f Maintenance of established hiPSC lines
Phase 3 · Hepatic Lineage Differentiation from hiPSCs Day 0-21
  1. a Preparation of serum-free differentiation (SFD) medium
  2. b Endodermal differentiation phase
  3. c Transition to hepatic lineage differentiation medium
  4. d Maturation to hepatic-like cells (HLCs)
Phase 4 · Liver Organoid (LO) Differentiation in 3D Microwell System Day 0-15
  1. a Preparation of SFD medium with growth factors for LO differentiation
  2. b Cell counting and preparation for 3D microwell plating
  3. c Preparation of 3D microwell plates
  4. d Simultaneous plating of the three cell types
  5. e Initial culture and organoid self-organization
  6. f Medium change and continued differentiation
  7. g Monitoring of organoid morphology and maturation
  8. h Collection of mature organoids at day 15
Phase 5 · Functional Characterization of Differentiated LOs Day 15-16
  1. a Sample preparation for ALB ELISA
  2. b Human albumin (ALB) production assay by ELISA
  3. c Urea production assay
  4. d Cytochrome P450 3A4 (CYP3A4) activity assay
  5. e RNA isolation from differentiated LOs
  6. f cDNA synthesis from isolated RNA
  7. g Quantitative PCR (qPCR) of hepatic genes
  8. h Immunocytochemical staining for hepatic markers
  9. i Glycogen storage assessment by PAS staining
  10. j Indocyanine green (ICG) uptake and release assay
Phase 6 · Transplantation of SDC-LOs into ALF Mice Day 0-14
  1. a Breeding and maintenance of Alb-TRECK/SCID mice
  2. b Induction of acute liver failure (ALF) in mice
  3. c Monitoring of ALF induction (48 hours post-DT)
  4. d Preparation of functional SDC-LOs for transplantation
  5. e Surgical transplantation of SDC-LOs into renal subcapsular space
  6. f Sham control surgery
  7. g Post-operative care and monitoring
  8. h Serum transaminase monitoring (day 2 post-transplantation)
  9. i Human albumin (hALB) detection in mouse serum
  10. j Survival monitoring and Kaplan–Meier analysis
  11. k Tissue collection and histological analysis (day 14)
  12. l Hematoxylin and eosin (H&E) staining of transplant and liver tissues
  13. m Immunofluorescence staining for human hepatic markers

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Attribution

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

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