Cell Culture / Organoids / Pancreatic NET PDO

Pancreatic / GEP neuroendocrine tumor PDO banking protocol

Patient-derived pancreatic neuroendocrine tumor (pNET) and GEP-NET / NEC organoids: embed in a neuroendocrine–epithelial niche (EGF / FGF2 ± R-spondin / Wnt, brief Y-27632), bank separately from PDAC ductal recipes, QC with CgA / synaptophysin, and run targeted / GSK3 drug contrasts.[1][2]

Use cases

pNET / GEP-NET PDO—not PDAC ductal organoids and not an islet-function expansion course.

UseFitReadoutNotes
pNET / GEP-NET PDO biobank + targeted drugsRecommendedNeuroendocrine-marker fidelity and drugs[1][2]Combinatory GSK3 inhibition shows anti-tumor potential in pNET 3D models.
NEC vs well-differentiated NET banking contrastsContrastGrade-linked drug differences[2]Even if NEC leans epithelial, keep dishes and logs separate from PDAC.
Copying the PDAC SOP as pNETDo not mixMixed banks / wrong nicheDuctal adenocarcinoma and NET differ in niche and QC.

In one line

NET clusters → dome → EGF/FGF2 niche → brief Y-27632 → mechanical split → CgA/Syn → drugs.

Protocol overview

  1. Ethics + cold chain; log site (pancreas / small bowel / colorectal…), grade (G1–G3 / NEC), and functional status
  2. Trim necrosis; collagenase ± Dispase to clusters; brief RBC lysis (MC427) if bloody
  3. Matrix domes; EGF / FGF2 ± R-spondin / Wnt—do not default to PDAC high-Wnt/FGF10 recipes[[1]][[2]]
  4. Brief Y-27632; score solid vs semi-cystic growth
  5. Feed every 2–3 days; mechanical split; bank NET separately from PDAC / islet contrasts
  6. Chromogranin A, synaptophysin; mycoplasma; match the primary
  7. Targeted / GSK3 arms with vehicle; in vivo uses transplant SOPs—not this page

Step 1 · Tissue and digest

  1. Log grade and functional status; separate instruments from PDAC specimens.
  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; add Wnt/Rspo only if literature-backed[1][2].
  2. Do not default to the full PDAC Wnt/FGF10/gastrin stack.
  3. Mechanical splits; log passage and matrix lot.

Steps 4–5 · QC and drugs

  1. CgA / synaptophysin; mycoplasma; match the primary.
  2. Include vehicle on targeted / GSK3 arms[1].
  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 ± R-spondinpNET / GEP-NET core[1][2]Open →
CHIR-99021（GSK3 对照臂）Not default expansion; drug arms on separate dishes[1]Open →
Y-27632 短期Seeding windowOpen →
红细胞裂解液Brief RBC lysis if bloodyOpen →
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
CHIR-99021MCK1040GSK-3 block → Wnt on; stemness/lineage inductionOn 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
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
CgAChromogranin A AntibodyMCAB6701Neuroendocrine QC; pNET / GEP-NET
SynaptophysinSynaptophysin AntibodyMCAB5245Neuroendocrine synaptic-vesicle protein QC
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
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] Anti-tumor potential of combinatory GSK3 inhibition in human 3D models of pancreatic neuroendocrine tumors and patient-derived GEP-NET primary cultures. Endocr Relat Cancer. 2025. PMID 40862396
  2. [2] Patient-Derived Organoid Models of Human Neuroendocrine Carcinoma. Front Endocrinol. 2021. PMID 33776923

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.