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STEMdiffâ„¢ Midbrain Organoid Differentiation Kit

Cell culture medium kit for robust generation of midbrain neural organoids from human pluripotent stem cells.

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Need a high-quality cell source? Use the hiPSC SCTi003-A (female) or SCTi004-A (male) control lines, manufactured with mTeSRâ„¢ Plus.

STEMdiffâ„¢ Midbrain Organoid Differentiation Kit

Cell culture medium kit for robust generation of midbrain neural organoids from human pluripotent stem cells.

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Cell culture medium kit for robust generation of midbrain neural organoids from human pluripotent stem cells.
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Product Advantages


  • Eliminate organoid embedding steps with a matrix-free system

  • Obtain consistently sized organoids with reproducible morphology between cell lines

  • Generate over 500 organoids per kit using ´¡²µ²µ°ù±ð°Â±ð±ô±ôâ„¢800

  • Achieve long-term culture survival for predictive assays, high-throughput phenotypic screening, and neurotoxicity assays

  • Combine modular region-patterned organoids to generate advanced AssemBloidsâ„¢

What's Included

  • STEMdiffâ„¢ Neural Organoid Basal Medium 1, 20 mL
  • STEMdiffâ„¢ Neural Organoid Basal Medium 2, 500 mL
  • STEMdiffâ„¢ Neural Organoid Supplement A, 10 mL
  • STEMdiffâ„¢ Neural Organoid Supplement C, 0.25 mL
  • STEMdiffâ„¢ Neural Organoid Supplement K, 0.5 mL
  • STEMdiffâ„¢ Neural Organoid Supplement L, 0.5 mL
Products for Your Protocol
To see all required products for your protocol, please consult the Protocols and Documentation.
  1. Anti-Adherence Rinsing Solution
    Anti-Adherence Rinsing Solution

    Rinsing solution for cultureware to prevent cell adhesion

  2. Trypan Blue
    Trypan Blue

    Reagent for counting viable mammalian cells

  3. Gentle Cell Dissociation Reagent
    Gentle Cell Dissociation Reagent

    cGMP, enzyme-free cell dissociation reagent

  4. STEMdiff™ Neural Organoid Maintenance Kit
    STEMdiffâ„¢ Neural Organoid Maintenance Kit

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

  5. Hausser Scientific™ Bright-Line Hemocytometer
    Hausser Scientificâ„¢ Bright-Line Hemocytomet...

    Counting chamber with 2 cover glasses

  6. Ultra-Low Adherent Plate for Suspension Culture
    Ultra-Low Adherent Plate for Suspension Cultu...

    Sterile, flat-bottom plate, with ultra-low adherent surface for suspension cultures, with lid; 6-well format

  7. 37μm Reversible Strainers|27215,27250
    Reversible Strainers

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

  8. AggreWell™800
    ´¡²µ²µ°ù±ð°Â±ð±ô±ôâ„¢800

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

  9. DMEM/F-12 with 15 mM HEPES
    DMEM/F-12 with 15 mM HEPES

    Dulbecco's Modified Eagle's Medium/Nutrient Ham's Mixture F-12 (DMEM/F-12) with 15 mM HEPES buffer

  10. D-PBS (Without Ca++ and Mg++)
    D-PBS (Without Ca++ and Mg++)

    Dulbecco’s phosphate-buffered saline without calcium and magnesium

  11. Falcon® Serological Pipettes, 10 mL
    Falcon® Serological Pipettes, 10 mL

    Sterile polystyrene pipettes for cell culture

  12. Culture Dish, Non-Treated
    Culture Dish, Non-Treated

    Sterile, flat-bottom, clear polystyrene non-treated dish with lid; 35, 60, or 100 mm formats

  13. Y-27632 (Dihydrochloride)
    Y-27632 (Dihydrochloride)

    RHO/ROCK pathway inhibitor; Inhibits ROCK1 and ROCK2

Overview

Robustly generate three-dimensional, patterned brain organoid cultures from human pluripotent stem cells without matrix embedding by using STEMdiffâ„¢ Midbrain Organoid Differentiation Kit. When paired with ´¡²µ²µ°ù±ð°Â±ð±ô±ôâ„¢800â„¢, this serum-free cell culture media can prevent organoid fusion and support the generation of over 500 organoids per kit for higher-powered statistical replicates and more detailed longitudinal study.

Adapted from protocols by Dr. Sergiu PaÅŸca, the brain-region-specific organoids generated with this kit are three-dimensional in vitro models with a cellular composition and structural organization representative of the developing human midbrain. They are consistently sized with reproducible morphology between various cell lines, and provide a reliable model for the study of dopaminergic neurons within a functional 3D system.

Midbrain organoids can be combined with striatal organoids generated using the STEMdiffâ„¢ Dorsal Forebrain Organoid Differentiation Kit with additional patterning factors to generate midbrain-striatal AssemBloidâ„¢ cultures. Organoids can be maintained with STEMdiffâ„¢ Neural Organoid Maintenance Kit to support long-term culture survival (> 50 days) for predictive assays, high-throughput phenotypic screening, and neurotoxicity assays.
Subtype
Specialized Media
Cell Type
Neural Cells, PSC-Derived, Neural Stem and Progenitor Cells, Pluripotent Stem Cells
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 Category
Serum-Free

Data Figures

Schematic protocol diagram to generate patterned midbrain organoids from iPSCs

Figure 1. Schematic for the STEMdiffâ„¢ Midbrain Organoid Differentiation Kit

hPSC-derived midbrain organoids can be generated in 43 days. Organoids are initially formed in ´¡²µ²µ°ù±ð°Â±ð±ô±ôâ„¢800 plates. After 6 days, the organoids are transferred and cultured in suspension, allowing growth and subsequent patterning to the midbrain. Long-term maintenance and further maturation of midbrain organoids can be achieved using STEMdiffâ„¢ Neural Organoid Maintenance Kit, see the Product Information Sheet (PIS) for details. hPSC = human pluripotent stem cell

Midbrain organoids generated with the STEMdiff Midbrain Organoid Kit expressing markers for midbrain floorplate progenitors and mature marginal zone dopaminergic neurons at Day 50

Figure 2. Mature STEMdiffâ„¢ Midbrain Organoids Exhibit Brain-Region-Specific Marker Expression and Organization

Representative marker expression in Day 50 organoids produced using STEMdiffâ„¢ Midbrain Organoid Kits (left panels) is compared to organoids produced using STEMdiffâ„¢ Dorsal Forebrain Organoid Kits. (A) DAPI is used for counterstaining. (B) Midbrain organoids have comparatively less PAX6 expression throughout. Dorsal forebrain organoids exhibit elevated expression of dorsal cortical progenitor marker PAX6, organized radially. (C) Midbrain organoids have elevated expression of floorplate progenitor marker FOXA2 that arranges around pseudoventricular regions (see inset) and (D) marginal zone dopaminergic neuron marker GIRK2 that expands from FOXA2 expressing progenitor zones.

Midbrain organoids generated with the STEMdiff Midbrain Organoid Kit expressing tyrosine hydroxylase at Day 50

Figure 3. STEMdiffâ„¢ Midbrain Organoids Express Catecholaminergic Protein Tyrosine Hydroxylase

Midbrain organoids were generated from human ES cell line H7 and iPS cell line STiPS-F016 using STEMdiffâ„¢ Midbrain Organoid Differentiation Kit. Organoids were further matured to Day 50 with STEMdiffâ„¢ Neural Organoid Maintenance Kit and are compared to organoids generated with STEMdiffâ„¢ Dorsal Forebrain Organoid Differentiation Kit. Both guided organoid types generate neurons that express MAP2, but only midbrain organoids express the catecholaminergic neuron specific marker TH. ES cell = embryonic stem cell; iPS cell = induced pluripotent stem cell; TH = tyrosine hydroxylase

The STEMdiff Midbrain Organoid Kit generates midbrain organoids with consistent morphology from multiple cell lines, shown at Day 25 and Day 50

Figure 4. STEMdiffâ„¢ Midbrain Organoid Differentiation Kit Supports the Generation of Homogeneous, Reproducible Neural Organoids

(A-F) Representative phase contrast images of midbrain organoids derived from WLS-1C, H7, and STiPS-F016 hPS cell lines using STEMdiff™ Midbrain Organoid Differentiation Kit show uniform size and spherical morphology at Day 25 and Day 50 in vitro. (G) Diameter measurements of midbrain organoids demonstrate reproducibility across different cell lines (mean ± SD; Day 25, n = 21; Day 50, n = 26). hPS cell = human pluripotent stem cell

Midbrain organoids generated with the STEMdiff Midbrain Organoid Kit expressing midbrain floorplate and dopaminergic marginal zone genes

Figure 5. Neural Organoids Generated with STEMdiffâ„¢ Midbrain Organoid Kit Express Key Markers of Brain-Region-Specific Patterning

Single organoids differentiated from multiple cell lines were harvested for RNA at Day 25 and Day 50. ΔΔCT (Fold Change) of midbrain organoids is normalized to TBP and respective dorsal forebrain organoid control (A & B) or respective PSC control (C); mean ± SD, n = 3 - 15. (A) Midbrain floorplate precursor markers FOXA2, LMX1A, and EN1 were elevated in midbrain organoids at Day 25. (B) More mature marginal zone dopaminergic markers NURR1, TH, GIRK2, and PITX3 were elevated at Day 50. (C) Dorsal forebrain marker PAX6 and TBR1 expression is downregulated in Day 25 midbrain organoids. PSC = pluripotent stem cell

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type
Product Name
Catalog #
Lot #
Language
Document Type
Product Name
Catalog #
100-1096
Lot #
All
Language
English
Document Type
Product Name
Catalog #
100-1096
Lot #
All
Language
English
Document Type
Product Name
Catalog #
100-1096
Lot #
All
Language
English
Document Type
Product Name
Catalog #
100-1096
Lot #
All
Language
English
Document Type
Product Name
Catalog #
100-1096
Lot #
All
Language
English
Document Type
Product Name
Catalog #
100-1096
Lot #
All
Language
English
Document Type
Product Name
Catalog #
100-1096
Lot #
All
Language
English

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.

Research Area
Workflow Stages
Workflow Stages for Organoids
View All

Resources and Publications

Educational Materials (16)

Brochure
Brochure
Brochure
Cryogenic Tissue Processing and Section Immunofluorescence of Neural Organoids
Protocol
Cryogenic Tissue Processing and Section Immunofluorescence of Neural Organoids
Measuring Neuronal Activity in Human Pluripotent Stem Cell-Derived Cerebral Organoids on 3D High-Density Multi-Electrode Arrays
Protocol
Measuring Neuronal Activity in Human Pluripotent Stem Cell-Derived Cerebral Organoids on 3D High-Den...
Building Three-Dimensional Human Brain Organoids
Wallchart
Building Three-Dimensional Human Brain Organoids
Building Brain Organoids and AssemBloidsâ„¢ to Study Human Development and Disease
1:02:03
Webinar
Building Brain Organoids and AssemBloidsâ„¢ to Study Human Development and Disease
Single-Cell Characterization of Dorsal and Ventral Forebrain Organoid Models
23:34
Webinar
Single-Cell Characterization of Dorsal and Ventral Forebrain Organoid Models
Measuring Activity in hPSC-Derived Neural Organoids
21:42
Webinar
Measuring Activity in hPSC-Derived Neural Organoids
Phenotyping Midbrain Organoids and Neurons for Studying Parkinson’s Disease and Therapeutic Discovery
54:43
Webinar
Phenotyping Midbrain Organoids and Neurons for Studying Parkinson’s Disease and Therapeutic Discov...
Using Human Pluripotent Stem Cell-Derived Neural Organoids for Disease Modeling
31:49
Webinar
Using Human Pluripotent Stem Cell-Derived Neural Organoids for Disease Modeling
Guided Neural Organoid Culture Systems: Adding Dimensions to Model Neural Circuitry and Function
38:43
Webinar
Guided Neural Organoid Culture Systems: Adding Dimensions to Model Neural Circuitry and Function
iPSCs As Models, Part 2: Modeling the Human Brain with Organoids
33:07
Webinar
iPSCs As Models, Part 2: Modeling the Human Brain with Organoids
Scientific Poster
Scientific Poster
Dr. Bas Trietsch
Interview
Dr. Bas Trietsch

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Need a high-quality cell source? Use the hiPSC SCTi003-A (female) or SCTi004-A (male) control lines, manufactured with mTeSRâ„¢ Plus.
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