hPSC-Derived Cortical Brain Organoid Differentiation Kit
hPSC-Derived Cortical Brain Organoid Differentiation Kit
Price:
Regular price
$4,833 USD
Regular price
Sale price
$4,833 USD
Unit price
per
For shipping services or bulk orders, you may request a quotation.
Secure checkout with
View full details
Product Details
Product Details
Product Specification
| Usage |
This kit can generate more than 200 cortical brain organoids. It is recommended to start differentiation with one 6-well plate to produce 60–100 homogeneous neuroepithelial spheroids with an ideal formation efficiency of 60%.
1. Experimental Instruments and Materials: Instruments: Biological safety cabinet, cell incubator, horizontal centrifuge, inverted microscope, low-temperature refrigerator Materials: Centrifuge tubes (15 mL, 50 mL), pipettes (10 μL, 100 μL, 1000 μL, multi-channel 100 μL), sterile pipette tips (10 μL, 200 μL, 1000 μL), serological pipettes (10 mL, 50 mL), wide-bore 200 μL / 1 mL pipette tips, TC-treated non-low-adhesion 6-well plates, ultra-low attachment 6-well plates, ultra-low attachment 96-well U-bottom plates 2. Experimental Protocols: Preparation of hESCs / hiPSCsCulture hESCs / hiPSCs in TC-treated 6-well plates using hESC/iPSC Culture Kit until cell confluency reaches 70–80%.Neural Epithelial Differentiation (Two Induction Methods)Method 1: 2D Induction of Neural Epithelial Stem Cells (NESC) 1. Warm culture vessels, medium and reagents to room temperature (15–25 °C) before use. 2. Perform neural epithelial induction 2 days after hPSC passaging. Initiate induction when cells form small island-like colonies with density lower than 30%, without stressed single cells or spherical stress clusters. Aspirate complete hPSC medium, rinse cells twice with DMEM/F12 to remove floating cells, add 2 mL Basal Medium 1 per well, and refresh medium every 2 days until colony density reaches 80%. Under 10× microscope, qualified colonies feature smooth rounded edges, condensed and abundant nuclei. The induction period lasts 3–4 days. Note: Discard abnormal PSC colonies with spontaneous differentiation or floating aggregates; do not use them for induction. Thaw and expand a new batch of cells instead. Method 2: Direct Neural Epithelial Induction from Embryoid Bodies (EB) (Recommended for Beginners) 1. Warm culture vessels, medium and reagents to room temperature (15–25 °C) before use. 2. Initiate EB formation when hPSC colonies reach 80% density with clear smooth borders and normal nucleus-cytoplasm ratio. Wash cells twice with DPBS, add 500 μL Accutase or enzyme-free hESC/iPSC Passage Solution per well for digestion (6–10 min). Terminate digestion once colonies shrink, wrinkle and brighten with slight cell detachment upon plate shaking. 3. Aspirate digestion reagent as appropriate: If extensive cell detachment occurs with Accutase, retain digestion solution and neutralize with equal volume DMEM/F12. If enzyme-free passage solution is used, aspirate reagent and resuspend cells with 500 μL Basal Medium 2 per well, then transfer cell suspension into 15 mL or 50 mL centrifuge tubes according to digestion volume. 4. For Accutase-digested cells: Centrifuge at 120 g for 5 min, discard supernatant, resuspend pellet with Basal Medium 2 at 500 μL per digested well, then supplement with 1× Supplement A. For enzyme-free digested cells: Directly add Basal Medium 2 (500 μL per digested well) to the cell suspension, then supplement with 1× Supplement A. 5. Transfer cell suspension mixed with 1× Supplement A into a reagent reservoir or 10 cm culture dish, dispense 100 μL per well into ultra-low attachment 96-well U-bottom plates via multi-channel pipette. Seal plates with parafilm, spin on horizontal plate spinner at 1500 rpm for 5 min, then incubate at 37 °C overnight. Add an additional 100 μL Basal Medium 2 (without Supplement A) per well on Day 2. Total EB formation time is approximately 48 hours. 6. On Day 3, fully aspirate medium, add 200 μL Basal Medium 1 per well for 3 days of culture. On the second day, remove 100 μL spent medium and replenish with 100 μL fresh medium to eliminate metabolic waste.Neurosphere Formation (Skip Steps 2–4 for EB-derived neural epithelial protocol, proceed directly to Step 5)1. Warm culture vessels, medium and reagents to room temperature (15–25 °C) before use. 2. Pre-warm 6 mL Accutase to room temperature inside biosafety cabinet. Wash induced neural epithelial stem cells twice with 1 mL DPBS per well, add 800–1000 μL Accutase per well, incubate for 5–8 min and observe cell status every 4 min. Terminate digestion when colony edges curl, intercellular gaps brighten and minor floating cells appear. 3. Aspirate digestion reagent from side wall, resuspend cells with 1 mL Basal Medium 3 per well, adjust total volume to 12 mL Basal Medium 3 plus 12 μL 1× Supplement A. If over-digestion leads to massive cell detachment, neutralize with 1 mL DMEM/F12 per well, centrifuge at 120 g for 3 min to collect cells, then resuspend in 12 mL Basal Medium 3 plus 12 μL 1× Supplement A. Note: Prior to volume adjustment, perform trypan blue staining to verify cell viability above 80% before proceeding. Qualified EBs with expanded neuroectoderm feature translucent edges, diameter >400 μm, dense interior without cavities, and morphology similar to primary mouse neurospheres. 4. After centrifugation, cell pellets gather on one side of the tube. Incubate for 24 h, then add 100 μL Basal Medium 3 (without Supplement A) per well. Neurosphere formation induction lasts 2 days. 5. For EB-derived neural epithelial cultures: After 3 days of induction, fully aspirate Basal Medium 1 and add 250 μL Basal Medium 3 per well for 2 days of induction.Neural Rosette Expansion (Identical Protocol for Both Induction Schemes)1. Warm culture vessels, medium and reagents to room temperature (15–25 °C) before use. 2. Harvest neurospheres (200–600 μm in diameter) using wide-bore 200 μL pipette tips and transfer into ultra-low attachment 6-well plates, maximum 10 neurospheres per well. Prepare at least two 6-well plates for neural rosette expansion. 3. After transferring neurospheres into 6-well plates, gently add 2 mL Basal Medium 4 per well. Place plates on orbital shaker at 80 rpm for 18 days, refresh 2 mL medium per well every 1–2 days.Cortical Brain Organoid Maturation1. Warm culture vessels, medium and reagents to room temperature (15–25 °C) before use. 2. When dark punctate structures appear at the core and periphery of neurospheres, neural rosette expansion reaches optimal stage. Fully aspirate medium, add 2 mL Basal Medium 5 per well, culture on orbital shaker at 80 rpm for 18–26 days with medium exchange every 2–3 days. Organoids are ready for characterization and downstream assays after maturation. Immunofluorescence staining of mature organoids reveals TUJ1+/SOX2+ populations forming VZ and SVZ-like structures with high expression of glutamatergic transcripts. Long-term shaking culture in Basal Medium 5 supports culture up to 120 days without central core apoptosis. 3. When cortical organoids grow larger than 1 mm in diameter, dissect them to reduce internal cell death. Cut organoids >1 mm into 2–4 fragments with sharp No.2 surgical blade, wash fragments once with DPBS, transfer into 15 mL centrifuge tubes with wide-bore 1000 μL tips and allow natural sedimentation for 5–8 min. Aspirate DPBS supernatant, resuspend fragments in Basal Medium 4 plus 1× Supplement A, transfer into ultra-low attachment 6-well plates and shake at 80 rpm for 4 days with daily medium refreshment. After 4 days, switch to Basal Medium 5 and shake at 120 rpm for extended culture, maintaining organoid viability up to 180 days. |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Description |
This product is the hPSC-derived cortical brain organoid induction and differentiation kit. Human pluripotent stem cells (hPSCs) can be differentiated into cortical brain organoids via this standardized culture system optimized for human cerebral cortex development and functional research, which adopts ventral forebrain signaling pathway regulation technology.
This system sequentially activates Wnt/β-catenin signaling and gradually inhibits BMP/Smad signaling to drive directional differentiation of human PSCs into high-purity glutamatergic neurons (VGLUT1/2 positive rate >85%), and simultaneously induces radial glial cells (Pax6+/BLBP+) to form biomimetic ventricular zone structures. Layered cortical organoids with 1.5–2.0 mm stable diameter can be generated within 60 days, characterized by deep-layer cortical marker TBR1 (Layers V-VI) and superficial SATB2+ neurons (Layers II-IV), accompanied by compact myelination mediated by MBP+/MOG+ oligodendrocytes (LFB staining positive rate >70%).
High-throughput culture (≥96 organoids per batch) is achievable in 3D rotating bioreactors or ultra-low attachment plates (3DRBC, oxygen gradient maintained at 8–12%). This model is applicable to neurodegenerative disease modeling (e.g. tau pathology in Alzheimer’s disease), NMDA receptor functional analysis in schizophrenia, and synaptic plasticity research for autism spectrum disorders, and is compatible with single-cell RNA sequencing and dynamic calcium imaging tracking technology.
Operators must possess hands-on hPSC culture experience and fundamental understanding of organoid technology.
Product Composition: 1. Kit Reagents
2. Supporting Reagents & Consumables
Notice: For research use only. Shipped with dry ice. |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Storage Temp. | Refer to storage specifications listed in product composition table. All reagents are transported on dry ice. |
