Cell Culture / Organoids / Lung cancer PDO

Lung cancer PDO banking protocol

Patient-derived lung cancer organoids: embed digested tumor in an airway/alveolar-like niche (EGF / FGF7·10 / Noggin / R-spondin ± Wnt), then QC with CK/EpCAM and run drugs by histology.[1][2][3]

Use cases

Lung PDO—not a full airway developmental course and not A549 spheroids.

UseFitReadoutNotes
Lung ADC / SCC resection PDO drugsRecommendedViability, morphology, vs clinical response[1][3]Driver annotation drives drug-arm design.
Normal airway organoid contrastContrastToxicity / differentiation contrast[2]Keep normal airway dishes separate; similar medium, separate drug plates.
A549 / H1299 spheroidsDo not mixSpheroid diameterCell-line spheroids are not PDOs.

In one line

Lung-tumor clusters → dome → FGF/EGF niche → brief Y-27632 → mechanical split → CK/EpCAM + mycoplasma → subtype drugs.

Protocol overview

  1. Ethics + cold chain; log histology (ADC / SCC / SCLC etc.) and drivers
  2. Trim necrosis and obvious cartilage; collagenase to epithelial clusters
  3. Matrix domes; airway-like medium (EGF, FGF7/10, Noggin, R-spondin ± Wnt)[[1]][[2]]
  4. Brief Y-27632 + A83-01 to limit stroma; score cystic vs solid growth
  5. Feed every 2–3 days; mechanical split at 1–3 weeks
  6. CK8/19, EpCAM, E-Cadherin; mycoplasma; match primary IHC/NGS
  7. Drug arms by driver (EGFR-TKI, chemo…); include vehicle
  8. In vivo confirmation uses transplant SOPs—not this page

Step 1 · Tumor processing

  1. Log site (lobe / met) and prior therapy.
  2. Collagenase ± Dispase to clusters; filter and spin; brief RBC lysis (MC427) if needed.
  3. Keep SCLC / poorly differentiated digests shorter—avoid a pure single-cell grind.

Steps 2–3 · Niche and expansion

  1. Start with a full airway-like recipe so epithelium survives[2].
  2. If stroma overtakes, raise A83-01 or shorten digest—do not flood serum.
  3. ADC vs SCC morphologies differ; bank separately.

Steps 4–5 · QC and drugs

  1. Mechanical microcluster splits; CK / EpCAM / E-Cadherin before banking.
  2. Dose after the line is stable; separate EGFR-mutant and chemo plates[1].
  3. Ki-67 can contrast proliferation pre/post drug.

Niche reagents (titrate)

Literature starting points—not on-site prescribed doses.

ComponentRoleOn-site
EGF + FGF7 / FGF10 + Noggin + R-spondinAirway-like core[1][2]Open →
Wnt3a（按体系）Needed in some banking startsOpen →
A83-01TGF-β / stromaOpen →
Y-27632 短期Seeding windowOpen →
红细胞裂解液Brief RBC lysis after bloody digestsOpen →
VEGF-A（血管共培养扩展）Off in default epithelial PDO; endothelial co-culture separateOpen →
ATRA（肺泡 / 谱系窗口）Off in default airway expansion; lineage bias on separate dishesOpen →
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
All-trans Retinoic Acid (ATRA)MCK1837Lung airway–alveolar bias windows; usually off in default expansionOn 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 NRG1MGF30005Sachs breast keystone; optional in some lung recipesProteinOn request
Recombinant Human FGF10MGF30007Prostate / pancreas / breast / lung / gastric / ovarian / liver / endometrial / kidney nicheProteinOn request
Recombinant Human FGF7MGF30008Common in breast and airway epithelial recipesProteinOn 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
CK8Cytokeratin 8 AntibodyMCAB5383Luminal / epithelial lineage
CK19Cytokeratin 19 AntibodyMCAB5312Ductal-like epithelium; pancreas, some lung / ovarian / bladder / HNC
EpCAMEPCAM AntibodyMCAB5377Pan-epithelial; multi-tissue PDO
E-CadherinE Cadherin AntibodyMCAB1155Epithelial integrity / EMT contrast
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
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
N-Acetyl-L-cysteine (NAC)MC417Antioxidant in prostate and related recipes
HydrocortisoneMC418Additive in breast / airway epithelial recipes
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] Patient-derived lung cancer organoids as in vitro cancer models for therapeutic screening. Nat Commun. 2019. PMID 31467275
  2. [2] Long-term expanding human airway organoids for disease modeling. EMBO J. 2019. PMID 30643021
  3. [3] Human Lung Adenocarcinoma-Derived Organoid Models for Drug Screening. Cell Rep. 2020. PMID 32771979

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.