Cell Culture / Organoids / Prostate cancer PDO

Prostate cancer PDO banking protocol

Foolproof in-vitro workflow for patient-derived prostate cancer organoids: digest → Matrigel dome → DHT niche medium → passaging/banking and AR QC. Titrate doses to Gao/Drost and local pilots; not a product-insert substitute.[1][2]

Use cases

This page is in-vitro PDO only. Cell-line spheroids and in-vivo CDX are contrast rows—do not mix steps.

UseFitReadoutNotes
Patient-derived PCa PDORecommendedGlandular morphology, AR/CK, ±DHT growth and drug response[1][2]This SOP: biopsy/resection embedding and banking. DHT by default for AR+ adenocarcinoma; do not copy for NEPC/double-negative.
LNCaP / 22Rv1 3D spheroidsContrastSpheroid diameter/viability; AR+ drug-response contrastCell-line 3D is not a PDO. Follow the line datasheet; do not use this tissue-digest SOP.
LNCaP s.c. / orthotopic CDXDo not mixTumor volume, serum PSAIn vivo CDX has its own transplant SOP; a Matrigel plug is not an organoid dome.

In one line

Fresh PCa clusters → matrix dome → DHT + Wnt/Noggin/EGF + A83-01 → brief Y-27632 → mechanical split → bank after AR/mycoplasma QC.

Protocol overview

  1. Ethics + cold-chain tissue (resection/biopsy; seed same day)
  2. Trim fat/necrosis; PBS wash; mince ~1 mm³
  3. Collagenase ± Dispase to small clusters (not a pure single-cell grind)
  4. Filter, spin; mix with matrix on ice; spot domes; polymerize at 37 °C
  5. Add DHT-containing prostate niche medium + brief Y-27632
  6. Feed every 2–3 days; passage at 7–14 days (mechanical microclusters first)
  7. Morphology + AR/CK ± PSA/PSMA; mycoplasma then bank
  8. Drug assays: ±DHT, AR antagonists; in vivo take uses transplant SOPs

Step 1 · Tissue and digest

  1. Ethics in place; keep tissue at 4 °C and seed the same day if possible.
  2. On ice, trim fat and frank necrosis; PBS-wash blood.
  3. Mince ~1 mm³; collagenase ± Dispase 30–90 min at 37 °C to small clusters, not a pure single-cell grind[2].
  4. Filter 70–100 μm, low-speed spin; brief RBC lysis (MC427) if bloody.

Step 2 · Matrix embedding (dome)

  1. Chill tips and plates; keep matrix liquid; avoid freeze–thaw.
  2. Mix clusters with matrix on ice; ~20–40 μL/dome in 24-well plates.
  3. After 37 °C set, add pre-warmed complete medium along the wall without washing the dome away[2].

Step 3 · DHT niche culture

  1. AR+ adenocarcinoma: basal + B-27 + DHT + EGF/Noggin/R-spondin + A83-01 (see reagent table; titrate to[1][2]). Default is serum-free—charcoal-stripped FBS is usually not required.
  2. Add Y-27632 after seeding; withdraw once survival stabilizes—do not leave ROCK inhibitor on chronically.
  3. Half-feeds early, then full feeds every 2–3 days; score glandular architecture.
  4. Do not default DHT for NEPC or AR− phenotypes. For serum-containing recipes or strict androgen depletion/add-back, see the charcoal-stripped FBS section below.

Step 4 · Passaging

  1. Passage when cores necrose, diameter is excessive, or medium acidifies fast (often 7–14 days).
  2. Release matrix on ice or with gentle enzyme; prefer mechanical microclusters[2].
  3. Brief TrypLE (MC426) or similar only if single cells are required. Re-embed 1:2–1:4; optional Y-27632 for 24 h.

Step 5 · Bank, QC, and assays

  1. Bank healthy, clean cultures; organoid-compatible freezing medium; controlled-rate then LN2.
  2. Before banking: morphology, passage, mycoplasma; AR+ expected CK8/18, AR, ±PSA/PSMA[1][3].
  3. Drug assays include ±DHT and AR-antagonist arms (in serum-free medium, simply withdraw/add DHT). In vivo take uses transplant SOPs, not this page.

Niche reagents (titrate)

Literature starting points—not on-site prescribed doses.

ComponentRoleOn-site
Advanced DMEM/F12 + B-27 + GlutaMAX / L-Gln + 双抗Basal + serum-free supplementOpen →
DHT 1–10 nMAndrogen for AR+ prostate[1][2]Open →
EGF / Noggin / R-spondinEpithelial growth and Wnt amplification[2][3]Open →
A83-01TGF-β block; limit stromal overgrowth[2]Open →
FGF2 / FGF10、烟酰胺、NAC（Drost 体系）Titrate per Drost; not a prescription[2]Open →
Y-27632 ~10 μMOnly ~2–7 days after seed/split[2]Open →
Charcoal-stripped FBS（CSS）Not for default banking; serum+hormone cases only—see dedicated sectionOpen →
KSFM（替代无血清基底）Only CSS+KSFM alternate recipes—see dedicated sectionOpen →
TrypLE（温和酶解备选）Brief single-cell needs; prefer mechanical triturationOpen →
胶原酶 II 型Digest tissue/tumor to epithelial clustersOpen →
Dispase IIGentle dissociation with collagenaseOpen →
类器官冻存液Banking cryopreservationOpen →

When charcoal-stripped FBS (CSS) is needed

Default banking here follows Drost/Gao serum-free medium (B-27 + defined DHT)—CSS is usually not purchased or added. Use charcoal-stripped FBS only for the serum-containing + hormone-manipulation cases below, then add DHT back at a defined concentration.

ScenarioNeed CSS?Plan
PDO banking / routine expansion (this SOP)NoAdvanced DMEM/F12 + B-27 + niche factors + DHT; neither regular FBS nor CSS[2].
±DHT / AR-antagonist assays in serum-free mediumNoSimply withdraw vs add DHT (1–10 nM); lock the B-27 lot. CSS does not replace a defined ±DHT arm.
Switching to an FBS-containing expansion/assay mediumYesSwitch to CSS (often ~5%, titrate to literature), starve 24–72 h, then add DHT back; never claim androgen depletion with untreated FBS.
KSFM-style “SFM base + low serum” alternative organoid recipesYesKSFM (MC428) or similar base + CSS (MC419) + DHT (often ~10 nM in literature); keep dishes separate from the B-27 SOP—do not mix lots.
LNCaP / 22Rv1 2D or spheroid androgen response (contrast, not PDO)YesRPMI-class medium + CSS, then DHT/antagonists after deprivation; follow the line datasheet—not this tissue-digest SOP.
Regular FBS for maintenance, mycoplasma screens, non-hormone readoutsNoRegular FBS is fine; hormone claims still require B-27±DHT or CSS±DHT systems.
  • ·CSS = charcoal-stripped FBS: charcoal removes steroids and other small molecules; lot-to-lot drift remains—log catalog and lot.
  • ·Reconstitute DHT in EtOH/DMSO; match vehicle in controls; titrate working levels still around 1–10 nM.
  • ·Do not “top up” B-27 complete medium with regular FBS—that reintroduces uncontrolled steroids and defeats defined DHT.
  • ·Inquire with SKU MC419 (charcoal-stripped FBS / CSS); distinct from regular FBS page products.

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 431542MCK1038ALK4/5/7; dual-SMAD / TGF-β blockOn request
SB 202190MCK1279p38 block; human epithelial organoid stabilityOn request
A83-01MCK1825ALK4/5/7; limit stromal overgrowthOn request
RepSoxMCK1826ALK5; TGF-β block alternative (vs A83-01)On request
ThiazovivinMCK1827ROCK; single-cell survival alternative (vs Y-27632)On request
Prostaglandin E2MCK1833Common in prostate organoid recipesOn request
5α-DihydrotestosteroneMCK1834Androgen for AR+ prostate organoidsOn 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 FGF2MGF30006Prostate niches; also common in brain / PSC inductionProteinOn request
Recombinant Human FGF10MGF30007Prostate / pancreas / breast / lung / gastric / ovarian / liver / endometrial / kidney nicheProteinOn 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
ARAndrogen Receptor AntibodyMCAB5030Prostate PDO epithelial / AR-pathway QC
PSAPSA/KLK3 AntibodyMCAB5775Prostate secretory-phenotype support QC
PSMAPSMA AntibodyMCAB5192Prostate epithelial marker (optional by study)
CK8Cytokeratin 8 AntibodyMCAB5383Luminal / epithelial lineage
CK18Cytokeratin 18 AntibodyMCAB5028Luminal epithelium; breast / prostate / ovarian / endometrial
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
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
RBC Lysis BufferMC427RBC removal after bloody digests
Keratinocyte Serum-Free Medium (KSFM)MC428Prostate CSS-style recipes; optional contrast for skin / epidermis
Inquire for this listCulture supplements

References

  1. [1] Organoid cultures derived from patients with advanced prostate cancer. Cell. 2014. PMID 25201530
  2. [2] Organoid culture systems for prostate epithelial and cancer tissue. Nat Protoc. 2016. PMID 27010729
  3. [3] Identification of multipotent luminal progenitor cells in human prostate organoid cultures. Cell. 2014. PMID 24905166

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.