Cell Culture / Organoids / Cervical cancer PDO

Cervical cancer PDO banking protocol

Patient-derived cervical epithelial / cancer organoids from biopsy, resection, or cytobrush: embed in a cervical niche (EGF / FGF / Noggin / R-spondin ± Wnt, A83-01…), log HPV status, QC squamous vs glandular identity, and run chemo/RT contrasts.[1][2]

Use cases

Cervical PDO—not HeLa / SiHa spheroids and not a full viral life-cycle course.

UseFitReadoutNotes
Cervical cancer / HSIL PDO biobank + chemo/RTRecommendedTissue dynamics, HPV-related phenotypes, drugs[1][2]Lõhmussaar / STAR Protocols cover surgical and cytobrush workflows.
Matched normal cervical epithelium contrastContrastToxicity / selectivityKeep normal and tumor dishes separate; do not mix drug plates.
HeLa / SiHa spheroids as PDODo not mixSpheroid diameterCell-line spheroids are not PDOs.

In one line

Cervical clusters → dome → EGF/Rspo niche → brief Y-27632 → mechanical split → squam/gland + HPV log → drugs.

Protocol overview

  1. Ethics + cold chain; log histology (SCC / ADC / CIN) and HPV type
  2. Dissociate biopsy / resection / cytobrush to clusters; brief RBC lysis (MC427) if bloody
  3. Matrix domes; cervical niche (EGF, FGF, Noggin, R-spondin ± Wnt, A83-01…)[[1]][[2]]
  4. Brief Y-27632; score cystic vs solid growth
  5. Feed every 2–3 days; mechanical split; bank cancer vs pre-cancer / normal separately
  6. EpCAM, CK, E-Cadherin; mycoplasma; match the primary
  7. Drug / RT arms with vehicle; in vivo uses transplant SOPs—not this page

Step 1 · Tissue and digest

  1. Log HPV and histology; separate instruments for pre-cancer / cancer / normal.
  2. Collagenase (MC412) ± Dispase (MC413) to clusters; follow STAR Protocols for cytobrush[2].
  3. Brief RBC lysis (MC427) if bloody; do not grind to pure single cells.

Steps 2–3 · Niche and expansion

  1. Start with a full cervical recipe[1][2]; bank SCC vs ADC separately.
  2. If stroma overtakes, raise A83-01 or shorten digest.
  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 drug/RT arms; interpret vs clinical history[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 + Noggin + R-spondin ± Wnt3aCervical epithelial core[1][2]Open →
FGF2 / FGF10（按文献）Common in epithelial nichesOpen →
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 →
超低吸附 / 圆底读板Drug / RT contrast 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 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 FGF2MGF30006Prostate niches; also common in brain / PSC inductionProteinOn 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
CK19Cytokeratin 19 AntibodyMCAB5312Ductal-like epithelium; pancreas, some lung / ovarian / bladder / HNC
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
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 organoids model cervical tissue dynamics and viral oncogenesis in cervical cancer. Cell Stem Cell. 2021. PMID 33852917
  2. [2] Protocol for culturing patient-derived organoids of cervical cancer. STAR Protoc. 2024. PMID 39356641

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.