Cell Culture / Organoids / Soft-tissue sarcoma PDO

Soft-tissue sarcoma PDO banking protocol

Patient-derived soft-tissue sarcoma organoids / tumoroids (including small round-cell sarcoma, MPNST, rhabdomyosarcoma…): embed in a mesenchymal–tumor niche (EGF / FGF2 ± VEGF; brief Y-27632), QC by subtype (Desmin / Myogenin / S100…), run drugs, and keep separate from osteosarcoma.[1][2]

Use cases

Soft-tissue sarcoma PDO—not MG-63 / HT-1080 spheroids and not a renamed osteosarcoma SOP.

UseFitReadoutNotes
Small round-cell sarcoma / MPNST PDO biobank + targeted drugsRecommendedFusion fidelity and drugs (e.g. MCL-1 / SHH-related)[1][2]Tumoroid biobanks show actionable vulnerabilities in subtypes such as CIC::DUX4.
Fusion-positive rhabdomyosarcoma organoid contrastsContrastMyogenic markers / fusion-dependent drugsBank separately from osteosarcoma and epithelial PDOs; log matrix lots alone.
HT-1080 / RD spheroids as PDODo not mixSpheroid diameterCell-line spheroids are not PDOs.

In one line

Sarcoma clusters → dome → EGF/FGF2 niche → brief Y-27632 → passage → subtype markers → drugs.

Protocol overview

  1. Ethics + cold chain; log histologic subtype, fusions (e.g. CIC::DUX4), and site
  2. Trim necrosis; gentle collagenase to clusters; brief RBC lysis (MC427) if bloody
  3. BME / collagen embed (lock type and lot); EGF / FGF2 ± VEGF[[1]][[2]]
  4. Brief Y-27632; score solid growth
  5. Feed every 2–3 days; mechanical or gentle enzymatic splits; bank subtypes separately
  6. Subtype markers (Desmin / Myogenin / S100 by study); mycoplasma; match the primary
  7. Drugs with vehicle; in vivo uses transplant SOPs—not this page

Step 1 · Tissue and digest

  1. Log subtype and fusion status; separate instruments.
  2. Gentle collagenase (MC412) to clusters; brief RBC lysis (MC427) if bloody.
  3. Do not grind to pure single cells.

Steps 2–3 · Matrix and niche

  1. Lock BME or collagen I lot before banking.
  2. EGF / FGF2 ± VEGF; keep lots separate from epithelial PDOs.
  3. Bank subtypes separately; mechanical or gentle enzymatic splits.

Steps 4–5 · QC and drugs

  1. Subtype markers + morphology; mycoplasma; match the primary.
  2. Include vehicle; interpret vs fusion/pathway hypotheses[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 + FGF2 ± VEGFSoft-tissue sarcoma core[1][2]Open →
BME / 胶原 I 型（选定一种）Matrix type alters growth and drugsOpen →
Y-27632 短期Seeding windowOpen →
超低吸附 / 圆底读板Drug 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 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
p53p53 Mouse mAbB10301Common in HGSOC / esophageal / HNC etc.; match to the primary
DesminDesmin AntibodyMCAB5218Myogenic intermediate filament; RMS and related soft-tissue sarcoma QC
MyogeninMyogenin AntibodyMCAB8830RMS myogenic TF QC
S100S100 AntibodyMCAB6849MPNST / nerve-sheath support 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
Collagen Type I (rat tail / bovine)MC431Osteosarcoma / soft-tissue sarcoma matrix contrast; lock lots vs BME
Inquire for this listCulture supplements

References

  1. [1] Small round cell sarcoma tumoroid biobank reveals CIC::DUX4 sarcoma vulnerability to MCL-1 inhibition. Nat Commun. 2025. PMID 40841360
  2. [2] Establishment of a patient-derived malignant peripheral nerve sheath tumor organoid model reveals SHH pathway activation and therapeutic vulnerability. Hum Cell. 2026. PMID 42658353

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.