Cell Culture / Organoids / Breast cancer PDO

Breast cancer PDO banking protocol

Patient-derived breast tumor organoids: digest, embed, expand in Sachs-type medium with neuregulin-1, then add hormone or targeted arms by ER/HER2 subtype.[1][2]

Use cases

Breast PDO—not MCF-7 spheroids and not a full normal-mammary morphogenesis course.

UseFitReadoutNotes
Resection breast PDO biobankRecommendedSubtype fidelity and drug response[1]Sachs living biobank showed organoids can retain heterogeneity.
Normal / high-risk breast organoidsContrastArchitecture and colony formation[2][3]Similar medium, but keep dishes separate from tumor drug screens.
MCF-7 / MDA-MB-231 spheroidsDo not mixSpheroid viabilityCell-line 3D is not a PDO.

In one line

Breast-tumor clusters → dome → NRG1 + EGF/FGF/Noggin/Rspo → brief Y-27632 → ±E2 by subtype → CK/ER/HER2 QC then drugs.

Protocol overview

  1. Ethics + cold chain; record ER / PR / HER2 / Ki-67
  2. Trim fat; collagenase to epithelial clusters
  3. Matrix domes; Sachs-type medium with NRG1[[1]][[2]]
  4. Brief Y-27632; estradiol for ER+; do not force hormone on TNBC
  5. Feed every 2–3 days; split at 1–3 weeks by growth
  6. CK8/18 ± ER/HER2 vs clinical IHC; mycoplasma
  7. Drug arms by subtype: endocrine, anti-HER2, chemo[[1]]
  8. In vivo take is a transplant SOP—not this page

Step 1 · Tissue and digest

  1. Log clinical subtype; trim fat so oil does not foul the matrix.
  2. Collagenase to epithelial clusters; avoid a pure single-cell grind[2].

Steps 2–3 · NRG1 niche

  1. After domes set, add complete medium with neuregulin-1[1].
  2. E2 for ER+; add anti-HER2 on assay arms—not as a maintenance factor at therapeutic dose.
  3. Brief Y-27632; chronic ROCK block shifts clone composition.

Steps 4–5 · QC and drugs

  1. Prefer mechanical splits; IHC/IF for ER, HER2, CK before banking.
  2. Run TNBC, HER2+, and luminal drug plates separately—one chemo plate does not represent the bank[1].

Niche reagents (titrate)

Literature starting points—not on-site prescribed doses.

ComponentRoleOn-site
Advanced DMEM/F12 + B-27 + NRG1Sachs medium keystone[1][2]Open →
EGF / FGF7 / FGF10 / Noggin / R-spondinMammary epithelial nicheOpen →
雌二醇（ER+）Hormone-dependent lines; skip by default in TNBCOpen →
孕酮（PR+ 激素臂）With E2 on separate dishes; skip by default in TNBCOpen →
Y-27632、SB202190、烟酰胺Seeding and human epithelium stability[2]Open →
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
17β-EstradiolMCK1835Hormone arm for ER+ breast organoidsOn request
ProgesteroneMCK1838Breast / endometrial hormone arms; with E2 on separate dishesOn 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 NRG1MGF30005Sachs breast keystone; optional in some lung recipesProteinOn request
Recombinant Human FGF10MGF30007Prostate / pancreas / breast / lung / gastric / ovarian / liver / endometrial / kidney nicheProteinOn request
Recombinant Human FGF7MGF30008Common in breast and airway epithelial recipesProteinOn request
Recombinant Human IGF-1MGF30013Seen in some epithelial / breast / liver recipesProteinOn 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
ERαER alpha AntibodyMCAB5041Breast / endometrial subtype vs clinical pathology
PRProgesterone Receptor AntibodyMCAB5590Breast / endometrial hormone-receptor QC
HER2ErbB 2 AntibodyMCAB5210HER2+ breast PDO QC and drug arms
EpCAMEPCAM AntibodyMCAB5377Pan-epithelial; multi-tissue PDO
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
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
N-Acetyl-L-cysteine (NAC)MC417Antioxidant in prostate and related recipes
HydrocortisoneMC418Additive in breast / airway epithelial recipes
Charcoal-Stripped FBS (CSS)MC419Serum + hormone manipulation only; not for default serum-free banking
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
TrypLE Express EnzymeMC426Gentle PSC / organoid passaging alternative
Inquire for this listCulture supplements

References

  1. [1] A living biobank of breast cancer organoids captures disease heterogeneity. Cell. 2018. PMID 29224780
  2. [2] Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids. Nat Protoc. 2021. PMID 33692550
  3. [3] Organoid cultures from normal and cancer-prone human breast tissues yield comparable breast epithelial disease models. Nat Commun. 2020. PMID 32398863

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.