Cell Culture / Organoids / Thyroid cancer PDO

Thyroid cancer PDO banking protocol

Patient-derived papillary thyroid and related organoids: embed in a thyroid epithelial niche (EGF / FGF / R-spondin / Noggin, A83-01…), score thyroid markers and iodine-uptake–related molecules, and run drug / RAI-related contrasts.[1][2]

Use cases

Thyroid PDO—not TPC-1 spheroids and not a full normal follicular expansion course.

UseFitReadoutNotes
PTC resection PDO vs RAI-refractory phenotypeRecommendedThyroid markers, iodine-uptake molecules, drugs[1]Sondorp et al. show PTC vs RAI-refractory organoids diverge at protein/transcript levels.
Long-term thyroid cancer organoid maintenanceContrastGenetic / phenotypic fidelity[2]Titrate per STAR Protocols; do not mix with normal follicular banks.
TPC-1 / BCPAP spheroids as PDODo not mixSpheroid diameterCell-line spheroids are not PDOs.

In one line

Thyroid-tumor clusters → dome → EGF/Rspo niche → brief Y-27632 → mechanical split → thyroid markers → drugs.

Protocol overview

  1. Ethics + cold chain; log histology (PTC / FTC / MTC / ATC) and BRAF / RAS status
  2. Trim necrosis; collagenase ± Dispase to clusters; brief RBC lysis (MC427) if bloody
  3. Matrix domes; thyroid epithelial niche (EGF, FGF, R-spondin, Noggin, A83-01…)[[1]][[2]]
  4. Brief Y-27632; score cystic growth
  5. Feed every 2–3 days; mechanical split; bank differentiated vs undifferentiated / medullary separately
  6. Thyroid markers (Tg / TTF-1 / PAX8 by study), EpCAM; mycoplasma
  7. Drug or RAI-related readouts with controls; in vivo uses transplant SOPs—not this page

Step 1 · Tissue and digest

  1. Log histology and molecular class; separate instruments for normal vs tumor.
  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 a literature thyroid recipe[1][2].
  2. Bank differentiated vs undifferentiated / medullary separately.
  3. Mechanical splits; log passage and BME lot.

Steps 4–5 · QC and drugs

  1. Thyroid markers + EpCAM; mycoplasma; match the primary.
  2. RAI / targeted drug arms with vehicle and controls[1].
  3. In vivo confirmation uses transplant models—not this page.

Niche reagents (titrate)

Literature starting points—not on-site prescribed doses.

ComponentRoleOn-site
EGF + Noggin + R-spondin ± FGFThyroid epithelial core[1][2]Open →
A83-01 / 烟酰胺Human epithelial stabilityOpen →
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
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 NogginMGF30002BMP antagonist; ENR / PDO niche; dual-SMAD neural inductionProteinOn request
Recombinant Human R-spondin-1MGF30003Wnt amplifier; long-term expansionProteinOn request
Recombinant Human FGF10MGF30007Prostate / pancreas / breast / lung / gastric / ovarian / liver / endometrial / kidney nicheProteinOn 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
PAX8PAX8 AntibodyMCAB5911Ovarian / Müllerian / endometrial / renal epithelial QC
CK7Cytokeratin 7 AntibodyMCAB5547Cholangiocyte / urothelial / some tubular / esophageal / thyroid / mesothelial epithelium 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
Inquire for this listCulture supplements

References

  1. [1] Patient-Derived Papillary Thyroid Cancer Organoids for Radioactive Iodine Refractory Screening. Cancers. 2020. PMID 33142750
  2. [2] Establishment and maintenance of thyroid organoids from human cancer cells. STAR Protoc. 2022. PMID 35600922

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.