Cell Culture / Organoids / Gallbladder cancer PDO

Gallbladder cancer / epithelium PDO banking protocol

Patient-derived gallbladder epithelial / cancer organoids: embed in a biliary niche (R-spondin / EGF / FGF10 ± HGF, A83-01, forskolin; optional Wnt stim/inhib arms), bank separately from liver ductal expansion and CCA PDOs, QC with CK7 / CK19, and run drugs.[1][2]

Use cases

Gallbladder PDO—not a full normal liver-ductal expansion course and not a renamed CCA PDO.

UseFitReadoutNotes
Gallbladder cancer PDO biobank + drugsRecommendedBiliary-marker fidelity and patient-specific drugs[1]Saito biliary platform covers gallbladder and related subtypes for drug screens.
Normal gallbladder epithelium Wnt phenotype contrastsContrastMorphology / lineage shifts with Wnt stim or inhibition[2]Separate from tumor dishes; do not treat inhibition arms as default expansion.
Copying CCA PDO steps as gallbladder cancerDo not mixMixed banksAnatomic site and Wnt windows differ—bank and log separately.

In one line

Gallbladder clusters → dome → Rspo/EGF/FGF niche → brief Y-27632 → mechanical split → CK7/19 → drugs.

Protocol overview

  1. Ethics + cold chain; log cancer vs stone-related inflammation and molecular class
  2. Trim necrosis; collagenase ± Dispase to clusters; brief RBC lysis (MC427) if bloody
  3. Matrix domes; biliary niche (Rspo, EGF, FGF10 ± HGF, A83-01, forskolin…)[[1]][[2]]
  4. Brief Y-27632; score cystic growth
  5. Feed every 2–3 days; mechanical split; bank tumor vs matched normal mucosa separately
  6. CK7, CK19, EpCAM; mycoplasma; Wnt stim/inhib on separate arms[[2]]
  7. Drugs with vehicle; in vivo uses transplant SOPs—not this page

Step 1 · Tissue and digest

  1. Log tumor vs inflamed mucosa; separate instruments.
  2. Collagenase (MC412) ± Dispase (MC413) to clusters; brief RBC lysis (MC427) if bloody.
  3. Do not grind to pure single cells.

Steps 2–3 · Niche and Wnt arms

  1. Start with a full biliary recipe[1][2]; bank separately from CCA / liver ductal lines.
  2. Wnt stim or inhibition on separate dishes—not mixed into default expansion lots.
  3. Mechanical splits; log passage and matrix lot.

Steps 4–5 · QC and drugs

  1. CK7 / CK19 / EpCAM; mycoplasma; match the primary.
  2. Include vehicle; interpret vs clinical regimens[1].
  3. In vivo confirmation uses transplant models—not this page.

Niche reagents (titrate)

Literature starting points—not on-site prescribed doses.

ComponentRoleOn-site
R-spondin + EGF + FGF10 ± HGFBiliary / gallbladder core[1][2]Open →
烟酰胺 / A83-01 / ForskolinCommon in human biliary expansionOpen →
Wnt3a / XAV939（对照臂）Stim or inhib on separate dishes[2]Open →
Y-27632 短期Seeding windowOpen →
红细胞裂解液Brief RBC lysis if bloodyOpen →
胶原酶 II 型Digest tissue/tumor to epithelial clustersOpen →
Dispase IIGentle dissociation with collagenaseOpen →
类器官冻存液Banking cryopreservationOpen →

Small molecules for this SOP

Deep-links to catalog SKUs; all prices on request. Growth factors (EGF/Noggin/R-spondin, etc.) are proteins—see that category.

CompoundSKURolePrice
Y-27632 2HClMCK1042ROCK block; brief survival boost after seed/splitOn request
SB 202190MCK1279p38 block; human epithelial organoid stabilityOn request
A83-01MCK1825ALK4/5/7; limit stromal overgrowthOn request
XAV939MCK1831Tankyrase; suppress Wnt; CRC / gut contrast armsOn request
ForskolinMCK1832Adenylate cyclase; cAMP; common in ovarian recipesOn request
NicotinamideMCK1836Common in human epithelial organoid mediaOn request
Inquire for this listAll small molecules

Proteins / peptides for this SOP

Growth factors and gastrin deep-linked by SKU; all prices on request. Activity/reconstitution per lot COA.

ProductSKURoleTypePrice
Recombinant Human EGFMGF30001Core epithelial growth factorProteinOn request
Recombinant Human NogginMGF30002BMP antagonist; ENR / PDO niche; dual-SMAD neural inductionProteinOn request
Recombinant Human R-spondin-1MGF30003Wnt amplifier; long-term expansionProteinOn request
Recombinant Human Wnt3aMGF30004Human colon / CRC / ductal / gastric / ovarian / liver WntProteinOn request
Recombinant Human FGF10MGF30007Prostate / pancreas / breast / lung / gastric / ovarian / liver / endometrial / kidney nicheProteinOn request
Recombinant Human HGFMGF30010Human liver ductal organoid keystoneProteinOn request
Inquire for this listProteins / enzymes / peptides

QC antibodies for this SOP

Common IHC/IF markers before banking or drugs; deep-linked catalog antibodies. Dilution/species per datasheet.

MarkerProductSKURole
CK19Cytokeratin 19 AntibodyMCAB5312Ductal-like epithelium; pancreas, some lung / ovarian / bladder / HNC
EpCAMEPCAM AntibodyMCAB5377Pan-epithelial; multi-tissue PDO
E-CadherinE Cadherin AntibodyMCAB1155Epithelial integrity / EMT contrast
CK7Cytokeratin 7 AntibodyMCAB5547Cholangiocyte / urothelial / some tubular / esophageal / thyroid / mesothelial epithelium QC
Inquire for this listAntibodies

Culture supplements for this SOP

Existing cell-culture SKUs (B-27 / N-2 / glutamine / antibiotics); titrate per insert and literature.

ProductSKURole
Advanced DMEM/F12 MediumMC410Serum-free organoid basal medium
B-27 Supplement (50×)MC317Serum-free basal supplement for epithelial / neural organoids
Stable glutamine (Ala-Gln, 200 mM)MC301Glutamine source for Advanced DMEM/F12 systems
Penicillin-Streptomycin (100×)MC306Early banking sterility; consider withdrawal after lines stabilize
Organoid Basement Membrane Extract (BME)MC4113D dome / sandwich embedding
Collagenase Type IIMC412Digest tissue/tumor to epithelial clusters
Dispase IIMC413Gentle dissociation with collagenase
Accutase Cell Detachment SolutionMC414Optional gentle enzymatic passaging
Organoid Cryopreservation MediumMC415Banking cryopreservation
Mycoplasma Detection KitMC416Pre-banking sterility QC
HEPES Buffer (1 M)MC305Organoid medium buffering; add to final concentration per recipe
Collagenase Type IVMC421Dissociation alternative; contrast with Type II
Organoid Primary Antimicrobial (Primocin-class)MC423Early primary / PDO / PSC banking contamination control
Ultra-Low Attachment 96-Well PlateMC425EB formation / spheroids / drug plates
U-Bottom 96-Well PlateMC430Spheroid settling / co-culture plates; complements ULA
RBC Lysis BufferMC427RBC removal after bloody digests
Inquire for this listCulture supplements

Drugs ↔ spheroid / modeling / transplant roles

PDO drug assays stay in-matrix on this SOP. Cell-line spheroid / 2D viability lives on the spheroid hub and disease models; in vivo take and PDX use transplant models. Do not mix the three workflows.

Spheroid cultureDisease modelsTransplant models

References

  1. [1] Establishment of Patient-Derived Organoids and Drug Screening for Biliary Tract Carcinoma. Cell Rep. 2019. PMID 31018139
  2. [2] Wnt Stimulation and Inhibition in the Development and Phenotype of Patient-Derived Gallbladder Organoids. Biol Cell. 2026. PMID 42388006

Related

Other SOPs

Disclaimer: Research use only (RUO). Not clinical guidance or an institutional SOP. Human tissue requires ethics approval. Inline [n] maps to the reference list. Titrate concentrations and digest times locally.