Cell Culture / Organoids / Brain / neural organoids

Brain / neural organoid protocol (PSC → cerebral outline)

Human PSC-derived cerebral organoid outline: ULA-plate EBs → neural induction (optional dual-SMAD: Noggin/LDN + SB431542) → matrix embed → spinning/static expansion; titrate to Lancaster / Chambers.[1][2][3]

Use cases

PSC-derived brain organoids—not 2D primary neurons and not glioma PDO.

UseFitReadoutNotes
Human PSC cerebral organoids for development / diseaseRecommendedNeuroepithelium, laminar features, disease phenotypes[1][2]Lancaster workflow fits a standard TC room; windows need pilots.
2D neural-progenitor contrast after dual-SMADContrastPAX6 / SOX2 positivity[3]2D contrast helps tell whether 3D failure is induction itself.
Primary 2D neurons or glioma spheroids as “brain organoids”Do not mixAdherent networks / spheroid diameterNot self-organized PSC cerebral organoids.

In one line

PSC → ULA EB → neural induction ± dual-SMAD → BME embed → Neurobasal/B-27/N-2 expand → SOX2/PAX6/MAP2 QC.

Protocol overview

  1. Confirm PSC/iPSC pluripotency and mycoplasma clearance; ethics and line licenses in place
  2. Seed ULA 96-well plates for EBs; brief Y-27632
  3. Neural induction: dual-SMAD (Noggin or LDN193189 + SB431542) or Lancaster default; optional CHIR / FGF2 windows[[1]][[3]]
  4. Embed EBs in BME droplets; move to spinning or static expansion
  5. Feed Neurobasal ± DMEM/F12 + B-27 + N-2; log lots
  6. From weeks 2–4 score ventricle-like neuroepithelium; SOX2 / PAX6 / Nestin
  7. Longer culture for MAP2 / βIII-Tubulin; add patterning factors only when regionalization is required
  8. Disease modeling and drugs after morphology stabilizes; in vivo take is a transplant SOP

Step 1 · EB formation

  1. Gently singularize PSCs with TrypLE (MC426) or similar, count, and seed ULA 96-well plates (titrate density to the line)[1].
  2. Include brief Y-27632; score EB roundness the next day.
  3. Do not form EBs on adherent dishes—attachment breaks the 3D self-organization start.

Steps 2–3 · Neural induction and embedding

  1. Run neural induction per chosen recipe: Lancaster default or dual-SMAD (Noggin/LDN + SB431542)[1][3].
  2. After induction, embed EBs in BME droplets; polymerize at 37 °C then move to expansion medium.
  3. Add patterning factors (SHH / WNT / FGF…) only when regionalization is required—do not pile them on at day 0.

Steps 4–5 · Expansion, QC, and use

  1. Feed Neurobasal ± DMEM/F12 + B-27 + N-2 on schedule; spinning helps oxygenation[1].
  2. QC: SOX2 / PAX6 / Nestin (progenitors) and MAP2 / βIII-Tubulin (neurons); mycoplasma.
  3. Drugs or electrophysiology after morphology stabilizes; in vivo take uses transplant SOPs.

Niche reagents (titrate)

Literature starting points—not on-site prescribed doses.

ComponentRoleOn-site
超低吸附 96 孔板EB formation[1]Open →
Neurobasal + B-27 + N-2Neural expansion basal[1]Open →
Noggin / LDN193189 + SB431542Dual-SMAD induction (optional)[3]Open →
Activin A / BMP4Lineage contrast or alternate windowsOpen →
CHIR-99021 / FGF2Wnt / FGF windows by studyOpen →
基底膜基质（BME）EB embeddingOpen →
Y-27632 短期Split / EB windowOpen →
原代抗菌（Primocin 类）Optional early bankingOpen →
TrypLE（PSC 温和解离）Singularization / passaging alternativeOpen →
VEGF-A（血管共培养扩展）Off in default induction; endothelial co-culture separateOpen →
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
SB 431542MCK1038ALK4/5/7; dual-SMAD / TGF-β blockOn request
DAPTMCK1021γ-secretase / Notch; differentiation windowsOn request
PD0325901MCK1028MEK; PSC dual inhibition / lineage switchesOn request
ThiazovivinMCK1827ROCK; single-cell survival alternative (vs Y-27632)On request
(-)-BlebbistatinMCK1828Myosin II; reduce hyper-contractionOn request
LDN193189MCK1829BMP; dual-SMAD / lineage inductionOn request
All-trans Retinoic Acid (ATRA)MCK1837Lung airway–alveolar bias windows; usually off in default expansionOn 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 NogginMGF30002BMP antagonist; ENR / PDO niche; dual-SMAD neural inductionProteinOn request
Recombinant Human FGF2MGF30006Prostate niches; also common in brain / PSC inductionProteinOn request
Recombinant Human Activin AMGF30011PSC / brain induction windows (dual-SMAD lineage contrast)ProteinOn request
Recombinant Human BMP4MGF30012BMP axis; lineage induction / Noggin·LDN contrastProteinOn 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
SOX2SOX2 AntibodyMCAB5268Neural progenitor / pluripotency marker
PAX6PAX6 AntibodyMCAB5886Neuroectoderm / forebrain lineage
NestinNestin AntibodyMCAB5655Neural progenitor intermediate filament; brain / GBM
MAP2MAP2 (MXB20476) Rabbit mAbMDAB12803Mature neuronal dendritic marker; optional brain / GBM
βIII-Tubulinbeta III Tubulin (MXB14148) Rabbit mAbMDAB12328Early neuronal cytoskeleton
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
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
Organoid Primary Antimicrobial (Primocin-class)MC423Early primary / PDO / PSC banking contamination control
Neurobasal MediumMC424Brain / neural / GBM organoid basal
Ultra-Low Attachment 96-Well PlateMC425EB formation / spheroids / drug plates
U-Bottom 96-Well PlateMC430Spheroid settling / co-culture plates; complements ULA
TrypLE Express EnzymeMC426Gentle PSC / organoid passaging alternative
Inquire for this listCulture supplements

References

  1. [1] Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc. 2014. PMID 25188634
  2. [2] Cerebral organoids model human brain development and microcephaly. Nature. 2013. PMID 23995685
  3. [3] Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009. PMID 19252478

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.