Cell Culture / Organoids / Osteosarcoma PDO

Osteosarcoma PDO banking protocol

Patient-derived osteosarcoma organoids: digest biopsy/resection tissue, embed in a chosen matrix, expand in a mesenchymal–tumor niche (EGF / FGF2 ± VEGF; brief Y-27632), and score invasion plus genome-informed drug / immuno contrasts—matrix type alters invasion and response, so lock lots.[1][2]

Use cases

Osteosarcoma PDO—not MG-63 spheroids and not a generic epithelial PDO recipe.

UseFitReadoutNotes
Osteosarcoma PDO biobank, invasion, and genome-informed drugsRecommendedMatrix-dependent invasion and drug differences[1]Changing matrix type resets the experiment—re-titrate.
PDX–organoid interactive banks and immune-infiltration contrastsContrastT-cell infiltration / immunotherapy pilots[2]Co-culture is a separate project—separate from default expansion dishes.
MG-63 / U2OS spheroids as PDODo not mixSpheroid diameterCell-line spheroids are not PDOs.

In one line

Osteosarcoma clusters → chosen-matrix embed → EGF/FGF2 niche → brief Y-27632 → passage → invasion/drugs.

Protocol overview

  1. Ethics + cold chain; log anatomic site, grade, and drivers if known
  2. Trim necrosis and gross mineralized debris; gentle collagenase to clusters
  3. Embed in a chosen matrix (BME / collagen by study); log type and lot[[1]]
  4. EGF / FGF2 ± VEGF; brief Y-27632
  5. Feed every 2–3 days; mechanical or gentle enzymatic splits; avoid over-digestion
  6. Osteoblastic / mesenchymal lineage markers by study; mycoplasma; match the primary
  7. Invasion and drug / immuno arms with vehicle; in vivo uses transplant SOPs—not this page

Step 1 · Tissue and digest

  1. Log site and molecular data; minimize mineralized-debris interference.
  2. Gentle collagenase (MC412) to clusters; do not grind to pure single cells.
  3. Brief RBC lysis (MC427) if needed.

Steps 2–3 · Matrix and niche

  1. Lock matrix type and lot before banking[1].
  2. EGF / FGF2 ± VEGF; keep lots separate from epithelial PDO recipes.
  3. Mechanical or gentle enzymatic splits; log passage.

Steps 4–5 · QC and drugs

  1. Lineage markers by study + morphology; mycoplasma; match the primary.
  2. Include vehicle on invasion and drug / immuno arms[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 ± VEGFOsteosarcoma core[1][2]Open →
基底膜基质 / 胶原（选定一种）Matrix type alters invasion and drugs[1]Open →
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
SATB2SATB2 AntibodyMCAB5103Osteosarcoma / osteoblastic lineage support QC
RUNX2RUNX2 AntibodyMCAB5864Osteoblastic TF; osteosarcoma 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] Matrix type influences embedded patient-derived osteosarcoma organoid invasion and response to treatment. Front Pharmacol. 2026. PMID 42311411
  2. [2] Reconstruction of T cell infiltration in an osteosarcoma PDX-organoid interactive biobank for personalized immunotherapy. Cell Rep Med. 2026. PMID 41763214

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.