Cell Culture / Organoids / Melanoma PDO

Melanoma PDO banking protocol

Patient-derived melanoma organoids / tumoroids: embed in a melanocytic–tumor niche (EGF / FGF2 core, ± Noggin / R-spondin / Wnt; brief Y-27632), score lineage markers, and run targeted / immuno-oncology contrasts; brain-met specimens can serve as matched contrasts.[1][2]

Use cases

Melanoma PDO—not A375 spheroids and not a full TIL expansion course.

UseFitReadoutNotes
Cutaneous / metastatic melanoma PDO biobank + targeted drugsRecommendedMolecular fidelity and drugs vs clinical response[2]Matched brain-met organoids can mirror divergent targeted responses.
PDO–immune co-culture / anti-tumor immune readoutsContrastImmune killing / cytokines[1]Co-culture is a separate project—do not mix with default expansion lots.
A375 / SK-MEL spheroids as PDODo not mixSpheroid diameterCell-line spheroids are not PDOs.

In one line

Melanoma clusters → dome → EGF/FGF2 niche → brief Y-27632 → mechanical split → lineage markers → targeted/IO drugs.

Protocol overview

  1. Ethics + cold chain; log primary / metastatic site and BRAF / NRAS / NF1 status
  2. Trim necrosis; collagenase ± Dispase to clusters; brief RBC lysis (MC427) if bloody
  3. Matrix domes; EGF / FGF2 core niche (± Noggin / R-spondin / Wnt by literature)[[1]][[2]]
  4. Brief Y-27632; score solid vs semi-cystic growth
  5. Feed every 2–3 days; mechanical split; bank primary vs brain-met separately
  6. Lineage markers (SOX10 / MITF / Melan-A by study), E-Cadherin; mycoplasma
  7. Targeted / IO arms with vehicle; in vivo uses transplant SOPs—not this page

Step 1 · Tumor processing

  1. Log primary / met and driver mutations; separate instruments.
  2. Collagenase (MC412) ± Dispase (MC413) to clusters; brief RBC lysis (MC427) if bloody.
  3. Do not grind to pure single cells.

Steps 2–3 · Niche and expansion

  1. Start with EGF / FGF2; add Wnt/Noggin/Rspo only if literature-backed[1][2].
  2. Bank primary vs brain-met separately; log passage and matrix lot.
  3. Prefer mechanical splits.

Steps 4–5 · QC and drugs

  1. Lineage markers + E-Cadherin; mycoplasma; match the primary.
  2. Include vehicle on targeted/IO arms; interpret vs clinical history[1][2].
  3. In vivo confirmation uses transplant SOPs—not this page.

Niche reagents (titrate)

Literature starting points—not on-site prescribed doses.

ComponentRoleOn-site
EGF + FGF2Melanoma core[1][2]Open →
Noggin / R-spondin ± Wnt（可选）Titrate by literature—not default gut ENROpen →
Y-27632 短期Seeding windowOpen →
红细胞裂解液Brief RBC lysis if bloodyOpen →
超低吸附 / 圆底读板Drug / immune contrast readoutsOpen →
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 FGF2MGF30006Prostate niches; also common in brain / PSC inductionProteinOn request
Recombinant Human VEGF-AMGF30015Vascular / endothelial co-culture extension; usually off in default epithelial PDO mediaProteinOn 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
E-CadherinE Cadherin AntibodyMCAB1155Epithelial integrity / EMT contrast
p53p53 Mouse mAbB10301Common in HGSOC / esophageal / HNC etc.; match to the primary
SOX10SOX10 AntibodyMCAB5826Melanoma / neural-crest lineage QC
MiTFMiTF AntibodyMCAB6661Melanocytic differentiation TF QC
Melan-AMelanA AntibodyMCAB5604Melanocytic differentiation marker; cutaneous / uveal melanoma 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

References

  1. [1] Modeling anti-tumor immune responses using patient-derived melanoma organoids. Cancer Immunol Immunother. 2025. PMID 41432764
  2. [2] Matched three-dimensional organoids and two-dimensional cell lines of melanoma brain metastases mirror response to targeted therapy. Sci Rep. 2024. PMID 39438602

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.