Cell Culture / Organoids / Glioblastoma PDO

Glioblastoma PDO banking protocol

Patient-derived glioblastoma organoids (GBOs): mince/digest fresh resections, embed in a neural-tumor niche (Neurobasal / B-27 / N-2, EGF / FGF2…), preserve heterogeneity and invasion phenotypes, and run drug / CAR-T contrasts—separate from PSC cerebral organoids.[1][2]

Use cases

GBM PDO—not U87 spheroids and not a full PSC cerebral-organoid induction course.

UseFitReadoutNotes
GBM resection PDO biobank + drug / immunotherapy pilotsRecommendedHeterogeneity retention, drugs, and CAR-T contrasts[1][2]Jacob / STAR Protocols support banking and mouse orthotopic follow-up.
Co-culture / invasion contrasts with PSC brain organoidsContrastInvasion distance, lineage markersPSC brain organoids are a separate page—do not import induction steps into GBM banking.
U87 / LN229 spheroids as GBM PDODo not mixSpheroid diameterCell-line spheroids are not PDOs.

In one line

GBM mince → dome/suspension → EGF/FGF2 neural-tumor niche → brief Y-27632 → passage → SOX2/Nestin → drugs.

Protocol overview

  1. Ethics + cold chain; log IDH / MGMT status and resection site
  2. Trim necrosis; mince ± gentle digest to clusters (not a pure single-cell grind)
  3. Matrix embed or literature suspension; Neurobasal / B-27 / N-2 + EGF / FGF2[[1]][[2]]
  4. Brief Y-27632; score solid / invasive-like growth
  5. Feed every 2–3 days; mechanical split; log passage and medium lots
  6. SOX2, Nestin, ±MAP2; mycoplasma; match the primary
  7. Drug / CAR-T arms with vehicle; orthotopic grafts use transplant SOPs—not this page

Step 1 · Tissue and dissociation

  1. Log molecular class; separate instruments for necrosis vs normal brain when possible.
  2. Prefer mincing; brief collagenase (MC412) ± Dispase (MC413) only if needed.
  3. Filter large debris; do not chase pure single cells.

Steps 2–3 · Niche and expansion

  1. Start with Neurobasal / B-27 / N-2 + EGF / FGF2[1][2].
  2. Keep separate from PSC brain-induction lots; log factor lots.
  3. Mechanical splits; avoid over-digestion that erases heterogeneity.

Steps 4–5 · QC and drugs

  1. SOX2 / Nestin ± MAP2; mycoplasma; match the primary.
  2. Include vehicle on drug / CAR-T arms[1].
  3. Orthotopic take uses transplant SOPs—not this page.

Niche reagents (titrate)

Literature starting points—not on-site prescribed doses.

ComponentRoleOn-site
Neurobasal + B-27 / N-2Neural-tumor basal[1][2]Open →
EGF + FGF2GBO core growth factorsOpen →
Y-27632 短期Seeding windowOpen →
超低吸附板Optional suspension / drug contrastsOpen →
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
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
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
SOX2SOX2 AntibodyMCAB5268Neural progenitor / pluripotency marker
NestinNestin AntibodyMCAB5655Neural progenitor intermediate filament; brain / GBM
MAP2MAP2 (MXB20476) Rabbit mAbMDAB12803Mature neuronal dendritic marker; optional brain / GBM
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
N-2 Supplement (100×)MC318Neural / some epithelial recipes; use with or vs B-27 by system
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
Neurobasal MediumMC424Brain / neural / GBM organoid basal
Ultra-Low Attachment 96-Well PlateMC425EB formation / spheroids / drug plates
U-Bottom 96-Well PlateMC430Spheroid settling / co-culture plates; complements ULA
Inquire for this listCulture supplements

References

  1. [1] Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR T cell testing. Nat Protoc. 2020. PMID 33169003
  2. [2] Generation of glioblastoma patient-derived organoids and mouse brain orthotopic xenografts for drug screening. STAR Protoc. 2021. PMID 33665625

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.