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LIVER Publication-derived

Biliary Organoids for Disease Modelling with Fluorescent Reporters

Full SOP with Academic or a plan

Source Koike et al., 2019 · Cincinnati Children's Hospital Medical Center · 10.1038/s41586-019-1598-0

Authors: Hiroyuki Koike, Kentaro Iwasawa, rie Ouchi, Mari Maezawa, Kirsten Giesbrecht, Norikazu Saiki, Autumn Ferguson, Masaki Kimura, Wendy L. thompson, James M. Wells, Aaron M. Zorn, takanori takebe Duration: 90 days 7 phases Cell source: Human iPSC, Human ESC

Abstract

This protocol establishes a three-dimensional in vitro model of hepato-biliary-pancreatic (HBP) organogenesis by co-culturing anterior and posterior gut spheroids derived from human pluripotent stem cells. The anteroposterior boundary interaction between foregut and midgut tissues enables spontaneous, retinoic acid-dependent specification and morphogenesis of interconnected hepatic, biliary, and pancreatic structures, without exogenous inductive factors.

Cell source
Human iPSC, Human ESC
Application
Disease modeling, Developmental study

Protocol overview

25 steps across 7 phases

Phase 1 · PSC Maintenance and Definitive Endoderm Induction D0-D3
  1. a Maintain undifferentiated PSCs in feeder-free conditions
  2. b Isolate PSC colonies and plate for endoderm induction
  3. c Apply day 1 endoderm induction medium
  4. d Apply day 2 endoderm induction medium
  5. e Apply day 3 endoderm induction medium
Phase 2 · Anterior and Posterior Gut Spheroid Specification D4-D7
  1. a Culture cells in anterior gut growth medium
  2. b Culture cells in posterior gut growth medium
Phase 3 · Anterior–Posterior Boundary Spheroid Formation D7-D9
  1. a Dissociate anterior and posterior gut cells to single cells
  2. b Plate anterior and posterior cells separately to form spheroids
  3. c Co-culture anterior and posterior spheroids to form fused boundary spheroids
  4. d Embed fused spheroids in Matrigel
Phase 4 · HBP Progenitor Emergence and Early Organoid Development D10-D13
  1. a Culture embedded spheroids and monitor HBP marker expression
  2. b Assess PROX1 reporter activity at boundary (if using PROX1::tdTomato iPSCs)
  3. c Prepare organoids for microdissection at day 13
Phase 5 · HBPO Air–Liquid Interface Culture and Maturation D13-D90
  1. a Microdissect PROX1-positive domain from day 13 organoids
  2. b Embed dissected PROX1-positive domain in Matrigel and transfer to Transwell
  3. c Culture in air-liquid interface system with daily medium replenishment
  4. d Continue long-term culture to day 90 for full maturation
Phase 6 · Quality Control: Immunofluorescence and Gene Expression Analysis D10-D90
  1. a Perform whole-mount immunofluorescence at day 11-13
  2. b Assess HBP marker expression by RT-qPCR at days 8, 11, and 12
  3. c Perform RNA-seq on dissected regions
  4. d Confirm boundary-specific retinoic acid signaling
  5. e Immunofluorescence staining of mature day 30-90 organoids
Phase 7 · Functional Assays (Optional) D30-D90
  1. a Assess pancreatic exocrine secretory function via CCK stimulation
  2. b Assess biliary function via fluorescein-labelled bile acid incorporation

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Attribution

This SOP was written by Organthis based on the published method in Koike et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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