Cell Culture / Organoids / Cholangiocarcinoma PDO

Cholangiocarcinoma / biliary PDO banking protocol

Patient-derived intra-/extrahepatic cholangiocarcinoma and biliary tumor organoids: embed in a ductal niche (R-spondin / EGF / FGF10 / HGF, A83-01, forskolin…), bank separately from normal liver ductal expansion, QC with CK19 / CK7, and run drugs.[1][2]

Use cases

CCA PDO—not a full normal liver-ductal expansion course and not HepG2 / HuCCT1 spheroids.

UseFitReadoutNotes
CCA / biliary PDO biobank + drugsRecommendedGenomic fidelity and patient-specific drugs[1][2]Broutier / Saito platforms cover primary liver and biliary cancer drug screens.
Matched normal ductal expansion contrastContrastToxicity / selectivityNormal contrast can follow the liver ductal SOP; keep dishes separate.
HuCCT1 / HepG2 spheroids as CCA PDODo not mixSpheroid diameterCell-line spheroids are not PDOs.

In one line

Biliary-tumor clusters → dome → Rspo/EGF/FGF10/HGF niche → brief Y-27632 → mechanical split → CK19 → drugs.

Protocol overview

  1. Ethics + cold chain; log iCCA / eCCA / gallbladder site and molecular class
  2. Trim necrosis; collagenase ± Dispase to clusters; brief RBC lysis (MC427) if bloody
  3. Matrix domes; ductal niche (Rspo, EGF, FGF10, HGF, nicotinamide, A83-01, forskolin…)[[1]][[2]]
  4. Brief Y-27632; score cystic ductal growth
  5. Feed every 2–3 days; mechanical split; bank tumor vs matched normal duct separately
  6. CK19, CK7, EpCAM; mycoplasma; match the primary
  7. Drugs with vehicle; in vivo uses transplant SOPs—not this page

Step 1 · Tissue and digest

  1. Log anatomic site (intra-/extrahepatic / gallbladder) and molecular class.
  2. Collagenase (MC412) ± Dispase (MC413) to clusters; brief RBC lysis (MC427) if bloody.
  3. Separate instruments for tumor vs normal duct.

Steps 2–3 · Niche and expansion

  1. Start with a full ductal recipe[1][2]; bank separately from normal liver ductal lines.
  2. If stroma overtakes, raise A83-01 or shorten digest.
  3. Mechanical splits; log passage and BME lot.

Steps 4–5 · QC and drugs

  1. CK19 / CK7 / EpCAM; mycoplasma; match the primary.
  2. Include vehicle; interpret vs clinical regimens[1][2].
  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 + HGFDuctal / CCA core[1][2]Open →
烟酰胺 / A83-01 / ForskolinCommon in human ductal expansionOpen →
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
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] Human primary liver cancer–derived organoid cultures for disease modeling and drug screening. Nat Med. 2017. PMID 29131160
  2. [2] Establishment of Patient-Derived Organoids and Drug Screening for Biliary Tract Carcinoma. Cell Rep. 2019. PMID 31018139

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.