Home › Cell Biology › A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)
Cell Biology JoVE (Open Access) Citable · DOI

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

DOI: 10.3791/55360-v
What you'll learn
  • ✓ Perform mechanical and enzymatic dissociation of post-mortem DIPG tissue
  • ✓ Isolate viable tumor cells using sucrose gradient centrifugation
  • ✓ Establish and maintain primary patient-derived DIPG cell cultures
  • ✓ Characterize tumor and microenvironmental cells from clinical samples
Protocol

Biopharma Insights This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

Difficulty
advanced
Total time
~3-4 hours per sample
Biosafety
BSL-2

Steps

1
Mechanically dissociate post-mortem DIPG tissue

Prepare and mechanically fragment post-mortem diffuse intrinsic pontine glioma tissue samples using manual dissociation techniques to increase surface area for enzymatic processing.

▶ 01:12
2
Enzymatically digest tumor tissue into single cells

Treat mechanically dissociated tissue with enzymatic digestion solution to break down extracellular matrix and generate a single-cell suspension.

▶ 03:25
3
Purify viable cells via sucrose gradient centrifugation

Separate viable tumor cells from debris and dead cells by centrifuging the cell suspension through a sucrose density gradient.

▶ 04:50
4
Lyse red blood cells and initiate culture

Remove contaminating red blood cells using ACK lysis buffer, then resuspend viable cells in culture medium for establishment of primary cell cultures.

▶ 05:49
5
Evaluate primary culture viability and morphology

Visualize and document morphology of primary cultures and early-passage cells to confirm successful isolation and culture establishment.

▶ 06:50
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