Cell Culture / Organoids / Intestinal organoids

Adult intestinal organoid protocol

Adult small-intestine/colon crypt organoids: EDTA crypt release → matrix embed → EGF / Noggin / R-spondin (add Wnt3a for human colon) to expand Lgr5 cells. Titrate to Sato.[1][2][3]

Use cases

Adult crypt expansion—not PSC gut tubes and not CRC PDO (see that SOP).

UseFitReadoutNotes
Adult SI / colon crypt expansionRecommendedBudding, take rate, Lgr5-related readouts[1][3]Mouse SI is classic ENR; human colon is more Wnt-dependent[[2]].
CRC PDOContrastTumor morphology and drug responseAPC-mutant lines often drop Wnt; use the CRC PDO SOP.
PSC gut tubes / organ-on-chipDo not mixLayering and peristalsis-like structureInduction windows (Activin → FGF/Wnt) are a different book from adult crypts.

In one line

Release crypts → dome → ENR (+ Wnt for human colon) → brief Y-27632 → split ~5–7 d → bud morphology + mycoplasma, then bank.

Protocol overview

  1. Ethics (human) or IACUC (mouse); tissue on ice
  2. Strip mucosa; EDTA-release crypts (not a single-cell grind)
  3. Filter and count crypts; mix with matrix on ice; spot domes
  4. ENR medium; add Wnt3a / nicotinamide for human colon[[2]]
  5. Y-27632 for 48–72 h after seed; then withdraw
  6. Feed every 2–3 days; mouse cultures often split at 5–7 days
  7. Budding/cyst morphology; mycoplasma then cryo
  8. Differentiation means dropping Wnt/R-spondin—do not mix with expansion medium

Step 1 · Crypt isolation

  1. Mouse: flush SI/colon with cold PBS; open longitudinally and clear mucus.
  2. Human: separate mucosa and keep on ice; seed the same day[2].
  3. Release crypts in 2–5 mM EDTA at 4 °C for 20–40 min; harsh trituration to single cells drops take[3].
  4. Filter ~70 μm; low-speed spin to pellet crypts.

Step 2 · Embedding

  1. Mix on ice at target density (tens to hundreds of crypts per ~50 μL gel is common).
  2. Spot domes; after 37 °C set, add pre-warmed ENR without washing them away.

Step 3 · ENR / Wnt niche

  1. Mouse SI expands long-term in EGF + Noggin + R-spondin[1].
  2. Human colon typically needs Wnt3a and nicotinamide ± A83-01 / p38 block[2].
  3. Y-27632 only for 1–3 days after seed or split.
  4. For goblet/enteroendocrine differentiation, drop Wnt or switch dishes—do not mix with expansion.

Step 4 · Passaging and banking

  1. When buds crowd or cores darken, shear to microclusters and re-embed 1:3–1:6[3].
  2. Bank in organoid-compatible freezing medium; optional Y-27632 on thaw day 1.
  3. QC: budding morphology, mycoplasma; STR for human lines when possible.

Niche reagents (titrate)

Literature starting points—not on-site prescribed doses.

ComponentRoleOn-site
Advanced DMEM/F12 + B-27 ± N-2 + 双抗BasalOpen →
隐窝释放 EDTA 工作液Adult crypt release[3]Open →
基底膜基质（BME）Dome embeddingOpen →
EGF + Noggin + R-spondin-1（ENR）Core mouse SI triad[1]Open →
Wnt3a（人结肠 / 近端扩增）Often required for human colon[2]Open →
烟酰胺、A83-01、SB202190（人）Common in Sato human epithelium[2]Open →
Y-27632Briefly after crypt seedingOpen →
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
DAPTMCK1021γ-secretase / Notch; differentiation windowsOn request
A83-01MCK1825ALK4/5/7; limit stromal overgrowthOn request
IWP-2MCK1830Porcupine; block Wnt secretion; CRC Wnt-withdraw contrastOn request
XAV939MCK1831Tankyrase; suppress Wnt; CRC / gut contrast armsOn 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
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
CK20Cytokeratin 20 AntibodyMCAB5332CRC / some gastric / urothelial / esophageal QC
CDX2CDX2 AntibodyMCAB5117Intestinal lineage TF; CRC / gut
EpCAMEPCAM AntibodyMCAB5377Pan-epithelial; multi-tissue PDO
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
HEPES Buffer (1 M)MC305Organoid medium buffering; add to final concentration per recipe
Collagenase Type IVMC421Dissociation alternative; contrast with Type II
Crypt-Release EDTA Working SolutionMC422Adult intestinal crypt EDTA release
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
Inquire for this listCulture supplements

References

  1. [1] Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009. PMID 19329995
  2. [2] Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology. 2011. PMID 21841781
  3. [3] Growing self-organizing mini-guts from a single intestinal stem cell: methodology and applications. Nat Protoc. 2013. PMID 23787894

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.