Cell Culture / Organoids / Ovarian cancer PDO

Ovarian cancer PDO banking protocol

Patient-derived ovarian cancer organoids: embed in a Kopper-type epithelial niche (R-spondin / Noggin / EGF / FGF10 ± Wnt, nicotinamide, A83-01, forskolin…), then QC with PAX8 / CK / p53 and run platinum drugs by subtype.[1][2][3]

Use cases

Ovarian PDO—not SKOV3 spheroids and not a full normal fallopian-tube expansion course.

UseFitReadoutNotes
Ovarian resection / ascites PDO biobank + platinum drugsRecommendedSubtype fidelity and drug vs clinical response[1]Kopper platform covers major subtypes and supports drugs plus xenografting.
Normal fallopian / OSE contrastContrastToxicity contrastKeep normal and tumor dishes separate; similar medium, separate drug plates.
SKOV3 / OVCAR spheroidsDo not mixSpheroid diameterCell-line spheroids are not PDOs.

In one line

Ovarian tumor / ascites clusters → dome → Rspo/FGF10 niche → brief Y-27632 → mechanical split → PAX8/CK/p53 → platinum drugs.

Protocol overview

  1. Ethics + cold chain; log histology (HGSOC / LGSOC / clear-cell / mucinous…) and BRCA / HRD
  2. Trim necrosis from solid tumor; spin ascites for clusters; collagenase to small clumps
  3. Matrix domes; Kopper-type medium (Rspo, Noggin, EGF, FGF10 ± Wnt, nicotinamide, A83-01, forskolin…)[[1]]
  4. Brief Y-27632; score cystic vs solid growth
  5. Feed every 2–3 days; mechanical split by growth
  6. PAX8, CK8/18/19, EpCAM, p53; mycoplasma; match the primary
  7. Drugs: platinum ± PARP inhibitors as relevant; include vehicle[[1]][[2]]
  8. In vivo / PDX uses transplant SOPs—not this page

Step 1 · Specimen processing

  1. Log site (primary / omentum / ascites) and prior platinum exposure.
  2. Ascites: low-speed spin for clusters ± brief RBC lysis (MC427); solid tumor: collagenase to clumps.
  3. Wash mucus-rich specimens thoroughly before embedding.

Steps 2–3 · Niche and expansion

  1. Start with a full Kopper-type recipe so epithelium survives[1].
  2. Bank HGSOC vs LGSOC / clear-cell separately.
  3. If stroma overtakes, raise A83-01 or shorten digest.

Steps 4–5 · QC and drugs

  1. Mechanical microcluster splits; PAX8 / CK / p53 / EpCAM before banking.
  2. Dose platinum ± PARP after the line is stable; vehicle ± recurrent-lesion contrast[1][2].
  3. In vivo uses transplant models; cell-line viability lives on the disease-models hub—do not mix steps with this PDO SOP.

Niche reagents (titrate)

Literature starting points—not on-site prescribed doses.

ComponentRoleOn-site
R-spondin + Noggin + EGF + FGF10 ± Wnt3aKopper core[1]Open →
烟酰胺 / A83-01 / ForskolinCommon in human ovarian recipesOpen →
Y-27632 短期Seeding windowOpen →
B-27 补充剂Serum-free basalOpen →
红细胞裂解液Brief RBC lysis for ascites / bloody specimensOpen →
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
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
CK8Cytokeratin 8 AntibodyMCAB5383Luminal / epithelial lineage
CK18Cytokeratin 18 AntibodyMCAB5028Luminal epithelium; breast / prostate / ovarian / endometrial
CK19Cytokeratin 19 AntibodyMCAB5312Ductal-like epithelium; pancreas, some lung / ovarian / bladder / HNC
EpCAMEPCAM AntibodyMCAB5377Pan-epithelial; multi-tissue PDO
E-CadherinE Cadherin AntibodyMCAB1155Epithelial integrity / EMT contrast
PAX8PAX8 AntibodyMCAB5911Ovarian / Müllerian / endometrial / renal epithelial QC
p53p53 Mouse mAbB10301Common in HGSOC / esophageal / HNC etc.; match to the primary
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
N-2 Supplement (100×)MC318Neural / some epithelial recipes; use with or vs B-27 by system
Stable glutamine (Ala-Gln, 200 mM)MC301Glutamine source for Advanced DMEM/F12 systems
Penicillin-Streptomycin (100×)MC306Early banking sterility; consider withdrawal after lines stabilize
ITS Supplement (100×)MC311Insulin–transferrin–selenium; some epithelial recipes
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] An organoid platform for ovarian cancer captures intra- and interpatient heterogeneity. Nat Med. 2019. PMID 31011202
  2. [2] A living biobank of ovarian cancer ex vivo models reveals profound mitotic heterogeneity. Nat Commun. 2020. PMID 32054838
  3. [3] A living biobank of breast cancer organoids captures disease heterogeneity. Cell. 2018. PMID 29224780

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.