Cerebral Organoids
A Simpler, Easier Method for Cerebral Organoid Generation.
Cerebral Organoids
Consider a simple and easy method for iPSC derived organoid generation, published by Lindborg et al. in Stem Cell Translational Medicine that uses only Cell-Mate3D™ matrix and maintenance media.1 In this study, cerebral organoids are generated in which forebrain, hindbrain, and cortical markers are present in 4 week old cultures.1
Does your system generate cerebral organoids in 3 weeks? If not, consider Cell-Mate3D™!
iPSCs are embedded into the matrix as whole colonies, cut into several pieces, and then cultured in maintenance media.1 After 3 weeks, cerebral organoids bud out of the surfaces of the matrix and continue to grow for several weeks.1
Major advantages over other protocols include
- Use of chemically defined reagents, increasing translatability to clinical applications.
- Setup is fast and simple, saving time.
- Requires no additional neural induction factors.
- Overall cost is 30% lower compared to Lancaster method.2
Researchers benefit because
- Neural Tubes and Neural Rosette structures are present.1
- Disease modeling of brain disorders can be achieved.
- Cerebral Organoids can be utilized for pharmaceutical and toxicology screening.3
Consider These Results!
For detailed information, see the article “Rapid Induction of Cerebral Organoids From Human Induced Pluripotent Stem Cells Using a Chemically Defined Hydrogel and Defined Cell Culture Medium” published in Stem Cells Translational Medicine in July 2016.
Organoid Formation*
4 weeks in culture
Pax6*
Immunohistochemical Staining
Beta-3 tubulin*
Immunohistochemical Staining
Figure 1. Microscopic and histological analysis of cerebral organoids.
Using phase contrast microscopy, cerebral organoids 2mm in diameter are observed budding out of the matrix surface at 4 weeks (a). Moderate Pax6 (b) and strong beta-3 tubulin (c) histologic staining demonstrates the presence of neural development.
*Images courtesy of Dr. Timothy O’Brien at the University of Minnesota.
Neural Tube-Like Structures*
Sox1 (red)/Nestin (green)
Neural Rosettes*
Sox2 (green)/Nestin (red)
Organoid Organization*
Neurite Outgrowth Stain
Figure 2. Formation of organized structures within organoids.
Cerebral Organoids were cryosectioned and stained (a, b) or stained live using a whole mount method (c). Sox1 (red)/Nestin (green) staining and organization suggests formation of neural tube-like structures (a). Sox2 (green)/Nestin (red) staining and organization suggests formation of neural rosettes (b). Staining using a neurite outgrowth staining kit (Thermo Fisher Scientific) demonstrates the overall complexity and organization of the organoids (c).
*Images courtesy of Dr. Timothy O’Brien at the University of Minnesota.
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Protocol for the Generation of Cerebral Organoids using Cell-Mate3D™
Required Reagents
1. Cell-Mate3D™ matrix (BRTI Life Sciences product # CM-1001 or CM-1002).
2. E8 culture media (Life Technologies product # A1517001).
3. 6 well culture dish.
4. Sterile blade.
Protocol
1. Refer to BRTI Life Sciences protocol for creating the Cell-Mate3D matrix.
2. Harvest iPSCs from 2D culture maintaining the iPSCs as whole colonies.
3. Following the Cell-Mate3D™ protocol, embed iPSC colonies into the Cell-Mate3D™ matrix:
• For a 500µL matrix, embed 19M cells.
• For a 250µL matrix, embed 9.5M cells.
4. Prepare a 6 well culture plate by transferring E8 media into 5 wells. Use 5mL of media per well.
5. Cut the matrix into approximately 100µL sections.
6. Organoids will begin to appear on the surface of the matrix after 3 weeks.
7. Organoids can be analyzed by:
• Histological methods.
• Cryosectioning and staining methods.
• Protein isolation and expression analysis.
• RNA isolation and expression analysis.
Organoid Generation Workflow
Expand iPSCs.
• Embed in Cell-Mate3D™ Hydrogel.
• Culture in Maintenance Medium.
Organoids (white circles) Emerge
from Hydrogel Surfaces (purple).
References
1.) Lindborg BA, Brekke JH, Vegoe AL, et al. Rapid Induction of Cerebral Organoids From Human Induced Pluripotent Stem Cells Using a Chemically Defined Hydrogel and Defined Cell Culture Medium. Stem Cells Transl Med. 2016 Jul;5(7):970-9. 2.) Cost of organoid generation using Lancaster method is approximately $150 per organoid and cost of O’Brien method is approximately $100 per organoid. Cost of organoid generation is reduced because neural induction factors are not needed. The Scientist. September 1, 2015. http://www.the-scientist.com/?articles.view/articleNo/43842/title/Orchestrating-Organoids/ accessed Feb 22 2017. 3.) Schwartz MP, Hou Z, Propson NE, et al. Human pluripotent stem cell-derived neural constructs for predicting neural toxicity. Proceedings of the National Academy of Sciences of the United States of America. 2015;112(40):12516-12521. doi:10.1073/pnas.1516645112.
