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Method Article
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July 8th, 2025
Source: Lin, G. L. et al., A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG). J. Vis. Exp. (2017)
This video demonstrates the process of isolating and culturing glial progenitor cells from diffuse intrinsic pontine glioma, or DIPG-affected brainstem tissue. Glial cells isolated from tumor tissue via an enzymatic digestion are cultured in a neurobasal medium, which promotes their growth and the development of free-floating neurospheres.
All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Obtaining Tissue Samples
NOTE: A critical component of this protocol requires proper handling of the tissue donation starting immediately at the time of autopsy. The overall sample viability depends significantly on minimizing the Post-Mortem Interval (PMI), immediately transferring the tissue into the appropriate cold medium supplemented with antibiotics/anti....
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Figure 1. Autopsy Sample of a Pontine Tumor. Immediate post-mortem appearance of a diffuse intrinsic pontine glioma. The tumor appears as a large, myelin-rich mass on the ventral surface of the pons.
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Hibernate-A | Gibco | A12475-01 | |
| HBSS with Calcium/Magnesium | Gibco | 24020-117 | |
| HBSS | Corning | 21-022-CV | |
| HEPES | Gibco | 15630-080 | |
| Liberase DH (Collagenase/Dispase) | Roche | 5401054001 | |
| DNAse I | Worthington Biochemical | LS002007 | |
| Sucrose | Sigma | S0389 | |
| Antibiotic/Antimycotic | Gibco | 15240-096 | |
| Neurobasal-A | Gibco | 10888-022 | |
| DMEM/F12 | Gibco | 11330-032 | |
| GlutaMAX-I (100X) | Gibco | 35050-061 | |
| Sodium Pyruvate (100mM) | Gibco | 11360-070 | |
| B27 Supplement Minus Vitamin A | Gibco | 12587-010 | |
| MEM Non-Essential Amino Acids (100X) | Gibco | 11140-050 | |
| Human PDGF-AA | Shenandoah Biotechnology | 100-16 | |
| Human PDGF-BB | Shenandoah Biotechnology | 100-18 | |
| Human EGF | Shenandoah Biotechnology | 100-26 | |
| Human FGF | Shenandoah Biotechnology | 100-146 | |
| Sterile scalpel blades | Bard Paker | 372610 | |
| Curved hemostats | Fine Science Tools | 13013-14 | |
| Razor blades | VWR | 55411-050 | |
| 100 x 20 mm cell culture dish | Corning | 353003 | |
| Sterile forceps | TWD Tradewinds | DF8988-5 | |
| Sterile drapes | 3M | 2037 | |
| Sterile gloves | Kimberly Clark | 1182307 | |
| ACK Lysis Buffer | Gibco | A1049201 | |
| 100 micron mesh filter | Fisher | 352360 | |
| 50 mL conical tube | Fisher | 1495949A |
