Cell Culture / Organoids / Gastric cancer PDO

Gastric cancer PDO banking protocol

Patient-derived gastric cancer organoids: embed in a gastric epithelial niche (Wnt / R-spondin / Noggin / EGF / FGF10 / gastrin), then QC with CK20/EpCAM and run drugs by subtype.[1][2][3]

Use cases

Gastric PDO—not a full normal gastric-gland course and not AGS spheroids.

UseFitReadoutNotes
Gastric resection / met PDO biobankRecommendedSubtype fidelity and drug response[1][3]Yan living biobank showed organoids can retain heterogeneity.
Normal gastric gland organoid contrastContrastToxicity contrast[2]Similar medium—keep separate from tumor drug plates.
AGS / MKN spheroidsDo not mixSpheroid diameterCell-line spheroids are not PDOs.

In one line

Gastric-tumor clusters → dome → Wnt/FGF10/gastrin niche → brief Y-27632 → mechanical split → CK/EpCAM → drugs.

Protocol overview

  1. Ethics + cold chain; log Lauren/WHO subtype and H. pylori history if known
  2. Trim necrosis; collagenase to clusters; keep matched normal mucosa separate
  3. Matrix domes; gastric niche (Wnt3a, R-spondin, Noggin, EGF, FGF10, gastrin)[[1]][[2]]
  4. Brief Y-27632 + A83-01; score cystic budding
  5. Feed every 2–3 days; mechanical split by growth
  6. CK19/20, EpCAM, E-Cadherin; mycoplasma; match the primary
  7. Drug assays with vehicle and standard-of-care arms[[3]]
  8. In vivo confirmation uses transplant SOPs

Step 1 · Tumor processing

  1. Separate instruments from normal margin; extra washes if mucus-rich.
  2. Collagenase to clusters; filter and spin; brief RBC lysis (MC427) if bloody.
  3. Diffuse-type tissue may mince finer—still avoid a pure single-cell grind.

Steps 2–3 · Gastric niche

  1. Bank in full Wnt/FGF10/gastrin medium[1][2].
  2. Only consider Wnt reduction after the line is stable—do not drop early to save reagent.
  3. Map Lauren subtype to each drug plate.

Steps 4–5 · QC and drugs

  1. Mechanical splits; CK / EpCAM / E-Cadherin before banking.
  2. Vehicle plus clinically relevant arms[3].
  3. In vivo take uses transplant protocols.

Niche reagents (titrate)

Literature starting points—not on-site prescribed doses.

ComponentRoleOn-site
Wnt3a + R-spondin + EGF + FGF10 + NogginGastric epithelial core[1][2]Open →
胃泌素 ICommon in human gastric recipesOpen →
A83-01TGF-β / stromaOpen →
Y-27632 短期Seeding windowOpen →
红细胞裂解液Brief RBC lysis after bloody digestsOpen →
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
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
Human Gastrin-IMGF30009Pancreatic ductal / gastric PDO peptidePeptideOn 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
CK20Cytokeratin 20 AntibodyMCAB5332CRC / some gastric / urothelial / esophageal QC
EpCAMEPCAM AntibodyMCAB5377Pan-epithelial; multi-tissue PDO
E-CadherinE Cadherin AntibodyMCAB1155Epithelial integrity / EMT contrast
Ki-67Ki67 Rabbit mAbB10315Proliferation index; pre/post-drug contrast
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

References

  1. [1] A Living Biobank of Patient-Derived Gastric Cancer Organoids. Cell Stem Cell. 2018. PMID 30343896
  2. [2] In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection. Gastroenterology. 2015. PMID 25768904
  3. [3] Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science. 2018. PMID 29420295

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.