STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit

Cell culture medium kit for robust generation of dorsal forebrain neural organoids from human pluripotent stem cells
STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit

Cell culture medium kit for robust generation of dorsal forebrain neural organoids from human pluripotent stem cells

1 Kit
Catalog # 08620
548 USD
Required Products
  1. AggreWell™ Rinsing Solution
    Anti-Adherence Rinsing Solution

    Rinsing solution for cultureware to prevent cell adhesion

  2. 37μm Reversible Strainers|27215,27250
    Reversible Strainers

    Reversible strainer for filtration of single cells and isolation of cellular aggregates, including embryoid bodies and spheroids

  3. AggreWell™800 24-well plate|34811
    AggreWell™800

    Microwell culture plates for easy and reproducible production of embryoid bodies and spheroids

  4. STEMdiff™ Neural Organoid Maintenance Kit
    STEMdiff™ Neural Organoid Maintenance Kit

    Cell culture medium kit for long-term maintenance and maturation of patterned neural organoids

Overview

Robustly generate three-dimensional, patterned brain organoid cultures from human pluripotent stem cells without matrix embedding. STEMdiff™ Dorsal and Ventral Forebrain Organoid Differentiation Kits are serum-free cell culture media that work with AggreWell™-generated embryoid bodies to prevent organoid fusion and enable the scalable generation of over 500 highly reproducible organoids per kit. Adapted from protocols by Sergiu Paşca (F Birey et al. Nature, 2017), these brain-region-specific organoids are three-dimensional in vitro models with a cellular composition and structural organization that is representative of the developing human forebrain. STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit (Catalog #08620) generates tissue of the early developing dorsal pallium, while STEMdiff™ Ventral Forebrain Organoid Differentiation Kit (Catalog #08630) generates tissue of the early developing ventral subpallium. Organoids generated with these kits can also be co-cultured as assembloids to study brain region interactions (F Birey et al. Nature, 2017). For extended periods of organoid culture (> 50 days), the components required for organoid maintenance are available as STEMdiff™ Neural Organoid Maintenance Kit (Catalog #100-0120).
Advantages
⦁ Reduce handling and media waste with fusion-free growth media
⦁ Obtain greater analytic sensitivity for disease phenotypes with reproducible morphology between lines and individual organoids
⦁ Generate over 500 organoids per kit using AggreWell™800 for higher-powered statistical replicates and more detailed longitudinal study
⦁ Eliminate matrix embedding steps with the matrix-free formulation and protocol
⦁ Easily process and section consistently sized organoids
⦁ Achieve long-term culture survival and reduced caspase-3 expression for neurotoxicity and neurodegenerative models
⦁ Combine modular region-patterned organoids to generate advanced assembloids for disease modeling and regenerative applications
Components
  • STEMdiff™ Neural Organoid Basal Medium 1, 20 mL
  • STEMdiff™ Neural Organoid Basal Medium 2, 500 mL
  • STEMdiff™ Neural Organoid Supplement A, 10 mL
  • STEMdiff™ Neural Organoid Supplement B, 0.25 mL
  • STEMdiff™ Neural Organoid Supplement C, 0.25 mL
Subtype
Specialized Media
Cell Type
Neural Cells, PSC-Derived, Neural Stem and Progenitor Cells, Pluripotent Stem Cells
Species
Human
Application
Cell Culture, Characterization, Differentiation, Functional Assay, Immunofluorescence, Organoid Culture, Phenotyping, Spheroid Culture
Brand
STEMdiff
Area of Interest
Disease Modeling, Drug Discovery and Toxicity Testing, Neuroscience, Organoids
Formulation
Serum-Free

Scientific Resources

Product Documentation

Document Type Product Name Catalog # Lot # Language
Document Type
Product Information Sheet
Product Name
STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit
Catalog #
08620
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit
Catalog #
08620
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit
Catalog #
08620
Lot #
All
Language
English
Document Type
Safety Data Sheet 3
Product Name
STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit
Catalog #
08620
Lot #
All
Language
English
Document Type
Safety Data Sheet 4
Product Name
STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit
Catalog #
08620
Lot #
All
Language
English
Document Type
Safety Data Sheet 5
Product Name
STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit
Catalog #
08620
Lot #
All
Language
English

Educational Materials (19)

Brochure
Organoids
Brochure
STEMCELL Neural Product Portfolio
Brochure
hPSC-Derived Neural Cell Research
Technical Bulletin
Cryogenic Tissue Processing and Section Immunofluorescence of Cerebral Organoids
Protocol
How to Dissociate 3D Neural Organoids into a Single-Cell Suspension
Protocol
How to Generate AssemBloids™ from hPSC-Derived Dorsal and Ventral Forebrain Organoid Co-Cultures
Wallchart
Derivation and Applications of Human Pluripotent Stem Cells
Wallchart
Cell-Reprogramming Technology and Neuroscience
Wallchart
Building Three-Dimensional Human Brain Organoids
Webinar
Single-Cell Characterization of Dorsal and Ventral Forebrain Organoid Models
23:34
Single-Cell Characterization of Dorsal and Ventral Forebrain Organoid Models
Webinar
Nature Research Round Table: The Promise of Organoid Medicine
17:17
Nature Research Round Table: The Promise of Organoid Medicine
Webinar
hPSC Quality: Essential Considerations for Gene Editing, Cloning, Maintenance and Disease Modeling
50:49
hPSC Quality: Essential Considerations for Gene Editing, Cloning, Maintenance and Disease Modeling
Webinar
iPSCs As Models, Part 2: Modeling the Human Brain with Organoids
33:07
iPSCs As Models, Part 2: Modeling the Human Brain with Organoids
Webinar
ISSCR Innovation Showcase: Advanced Brain Organoid Co-Culture Systems
1:01:37
ISSCR Innovation Showcase: Advanced Brain Organoid Co-Culture Systems
Webinar
Spotlight on Organoids: Expert Panel
Webinar
Building Brain Organoids and AssemBloids™ to Study Human Development and Disease
1:02:03
Building Brain Organoids and AssemBloids™ to Study Human Development and Disease
Webinar
Challenges in Translating iPSC Technology
41:47
Challenges in Translating iPSC Technology
Scientific Poster
Single-Cell RNA Sequencing Analysis of Regionally Patterned Human Pluripotent Stem Cell-Derived Neural Organoids
Scientific Poster
A Reliable, Efficient, and Feeder-Free Method to Generate Brain-Region-Specific Dorsal and Ventral Forebrain Organoids From Human Pluripotent Stem Cells to Model Early Human Brain Development
Load More Educational Materials

Product Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Data and Publications

Data

Schematic protocol diagram to generate patterned dorsal forebrain organoids from iPSCs

Figure 1. Schematic for the STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit

Human ES or iPS cell-derived dorsal forebrain organoids can be generated in 43 days. Embryoid bodies can be created in 6 days with AggreWell™800 plates. The EBs are then cultured in suspension, allowing growth and subsequent patterning to the dorsal forebrain. For the long-term maintenance and further maturation of dorsal forebrain organoids, see the PIS.

Figure 2. STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit Supports the Generation of Homogeneous, Reproducible Neural Organoids

(A) Neural aggregates formed in AggreWell™800 microwell plates exhibit uniform size and shape at day 6. H9-derived dorsal forebrain organoids from a single batch have spherical morphology at days (B) 15 (C) 25 (D) 50 (E) 75 and (F) 100. Scale bar = 1 mm. (G) Dorsal forebrain organoids exhibit homogeneous size over multiple cell lines (average ± SD, using 11 cell lines with 3 - 5 organoids counted per cell line and time point). (H-N) Day 25 organoids generated by a novice user with STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit show reproducibility across different cell lines.

Figure 3. STEMdiff™ Dorsal and Ventral Forebrain Organoid Differentiation Kits Reduce Unwanted Organoid Fusion

Whole-well images of dorsal forebrain organoid cultures derived from the H9 cell line in STEMdiff™ Forebrain Organoid Expansion Medium (bottom row) vs. the control published formulation (top row) show the extent of fusion typical in control media without orbital shaking. At Day 10 post-seeding in the control formulation, 12/12 organoids that were seeded are developing, but by Day 20 a single, larger organoid can be seen in the well. In STEMdiff™ Forebrain Organoid Expansion Medium, 20/20 seeded organoids are developing and 20/20 are still developing at Day 20 without shaking. While organoids in both culture conditions grow larger between 10 and 20 days, the reduction in organoids and enlarged size in the control formulation suggest a reduction in organoid yield due to organoid fusion.

Figure 4. STEMdiff™ Dorsal Forebrain Organoids Exhibit Cortical Layering and Brain-Region-Specific Marker Expression as They Mature

(A) Day 25 dorsal forebrain organoids display multiple cortical-like regions marked by radialized PAX6+ cells surrounded by MAP2 neurons. (B) Day 50 dorsal forebrain organoids continue to display multiple cortical-like regions marked by PAX6 (green) and MAP2 (magenta). (C) Dorsal forebrain organoids cultured for 100 - 200 days show increasing separation of deep-layer neurons (CTIP2, green; TBR1, cyan) from upper-layer neurons (SATB2, magenta).

Figure 5. Neural Organoids Generated with STEMdiff™ Dorsal and Ventral Forebrain Organoid Kits Express Key Markers of Brain-Region-Specific Patterning

RNA from single organoids was harvested at a series of time points and subsequently assayed using bulk RNA-seq (1 data column = 1 organoid). (A) Heat map of select genes shows that dorsal forebrain organoids (DFO) express increasing levels of cortex- and glutaminergic neuron-specific genes from day 25 to 75. (B) Day 25 ventral forebrain organoids (VFO) exhibit high expression of markers of the medial ganglionic eminence and of GABAergic neurons. Heat map scale quantifies gene expression across each row with a normalized z-score for each gene.

Figure 6. Dorsal Forebrain Organoids, Not Ventral Forebrain Organoids, Display Early Network Bursting Activity

Day 50 dorsal and ventral forebrain organoids were plated on a microelectrode array (MEA; CytoView MEA 96, Axion Biosystems) coated with 0.1% polyethyleneimine in borate buffer and 20 µg/mL CellAdhere™ Laminin-521. Activity from 8 electrodes per well was recorded once per week for 4 weeks using a Maestro MEA system (Axion Biosystems). (A) Representative bright field image of dorsal and ventral forebrain organoids on the MEA. Insets show representative spike rate heat maps for the corresponding well (red = 12 spikes/sec). Scale bar = 1 mm. (B) Raster plots of spike activity show increasing network bursting (pink lines) for dorsal forebrain organoids between week 1 and week 4, whereas no network bursting is observed in the ventral forebrain organoid (lower panels) over the same time period. (C) Mean firing rate (average ± SEM; 3 - 6 organoids per time point) increases for dorsal forebrain organoids but not ventral forebrain organoids over 4 weeks of measurements.

Figure 7. Fluorescent Imaging in BrainPhys™ Imaging Optimized Medium Improves Signal-to-Background Ratios of 3D Neural Cultures

GFP-labeled ventral forebrain organoids were co-cultured and merged with unlabeled dorsal forebrain organoids for one week prior to live imaging in Forebrain Organoid Differentiation Medium from STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit (right) or BrainPhys™ Imaging Optimized Medium (BPI, left). Interneuron migration can be visualized more clearly in BPI. Scale bar top panels = 300μm; scale bar bottom = 100μm. Adapted from Zabolocki et al. 2020, Nature Communications, available under a Creative Commons 4.0 License. For experimental details on generating AssemBloids™ from dorsal and ventral forebrain organoid co-cultures, see the protocol in our Methods Library.

Figure 8. hPSC-Derived Microglia Incorporate into Brain Organoids After 10 Days and Display an Activated Morphology upon Injury

(A, C) Representative microglia and brain organoid co-cultures after 10 days, sectioned and stained with IBA1 for microglia (green) and MAP2 for neurons (magenta). The microglia integrate among the neurons and display an unactivated morphology with extended processes. (B, D) The microglia display an activated amoeboid morphology upon injury as shown by IBA1 staining.

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