Cell Culture / Organoids / Mesothelioma PDO

Mesothelioma PDO banking protocol

Patient-derived pleural / peritoneal malignant mesothelioma organoids from resection or malignant effusion: embed in a mesothelial–tumor niche (EGF / FGF2 ± HGF, brief Y-27632), QC epithelioid vs sarcomatoid identity, and run platinum and related drug contrasts, including HIPEC-oriented pilots.[1][2]

Use cases

Mesothelioma PDO—not NCI-H28 spheroids and not a renamed lung PDO.

UseFitReadoutNotes
Pleural / peritoneal meso PDO vs platinum-tolerance phenotypesRecommendedMetabolism / drugs vs clinical exposure[1]High-fidelity platforms can mirror dynamic cisplatin tolerance.
Peritoneal meso HIPEC-oriented drug-combination pilotsContrastCombination sensitivity[2]In vivo HIPEC PK is a separate project—ex vivo is concentration contrast only.
NCI-H28 / MSTO spheroids as PDODo not mixSpheroid diameterCell-line spheroids are not PDOs.

In one line

Meso clusters / effusion cells → dome → EGF/FGF2 niche → brief Y-27632 → passage → EpCAM/CK → platinum drugs.

Protocol overview

  1. Ethics + cold chain; log pleural / peritoneal site, epithelioid / sarcomatoid / biphasic, and prior platinum exposure
  2. Mince/digest resections or enrich malignant effusion cells; brief RBC lysis (MC427) if bloody
  3. Matrix domes; EGF / FGF2 ± HGF mesothelial–tumor niche[[1]][[2]]
  4. Brief Y-27632; score cystic vs solid growth
  5. Feed every 2–3 days; mechanical split; bank histologic subtypes separately
  6. EpCAM, CK, E-Cadherin by subtype; mycoplasma; match the primary
  7. Platinum / HIPEC-related arms with vehicle; in vivo uses transplant SOPs—not this page

Step 1 · Tissue and digest

  1. Log site and histologic subtype; keep effusion vs solid-tumor workflows separate.
  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 ± HGF[1][2]; bank epithelioid vs sarcomatoid separately.
  2. Do not mix lots with lung PDO media.
  3. Mechanical splits; log passage and matrix lot.

Steps 4–5 · QC and drugs

  1. EpCAM / CK / E-Cadherin; mycoplasma; match the primary.
  2. Include vehicle on platinum / combination 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 ± HGFMesothelial–tumor core[1][2]Open →
Y-27632 短期Seeding windowOpen →
红细胞裂解液Brief RBC lysis for bloody / effusion samplesOpen →
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 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 FGF10MGF30007Prostate / pancreas / breast / lung / gastric / ovarian / liver / endometrial / kidney nicheProteinOn request
Recombinant Human HGFMGF30010Human liver ductal organoid keystoneProteinOn 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
EpCAMEPCAM AntibodyMCAB5377Pan-epithelial; multi-tissue PDO
E-CadherinE Cadherin AntibodyMCAB1155Epithelial integrity / EMT contrast
p53p53 Mouse mAbB10301Common in HGSOC / esophageal / HNC etc.; match to the primary
CK7Cytokeratin 7 AntibodyMCAB5547Cholangiocyte / urothelial / some tubular / esophageal / thyroid / mesothelial epithelium QC
CalretininCalretinin AntibodyMCAB7078Common QC for epithelioid mesothelioma
WT1WT1 (MXB12127) Rabbit mAbMDAB15817Mesothelioma support QC; match to the primary
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

Drugs ↔ spheroid / modeling / transplant roles

PDO drug assays stay in-matrix on this SOP. Cell-line spheroid / 2D viability lives on the spheroid hub and disease models; in vivo take and PDX use transplant models. Do not mix the three workflows.

Spheroid cultureDisease modelsTransplant models

References

  1. [1] A High-Fidelity Patient-Derived Organoid Platform Recapitulates the Dynamic Metabolic Landscape of Cisplatin Tolerance in Mesothelioma. Cancers. 2026. PMID 42192862
  2. [2] Patient-Derived Organoid Models and Precision HIPEC in Diffuse Malignant Peritoneal Mesothelioma: Modeling Heterogeneity to Address Recurrence. Int J Mol Sci. 2026. PMID 42737808

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.