Primary Tumor Preparation

Materials and Reagents

1. Phosphate buffered saline (PBS)
   
2. Fetal bovine serum (FBS)
   
3. Trypsin (Corning, Cellgro^®, catalog number: 25-054-CI )
   
4. Minimum essential medium (MEM)
   
5. HEPES
   
6. Sucrose
   
7. B104 conditioned media
   
8. N2 supplement (Life Technologies, Invitrogen™, catalog number: 17502-048 )
   
9. T3 (Sigma-Aldrich, catalog number: T2877 )
   
10. Penicillin/streptomycin/amphotericin (Life Technologies, Invitrogen™, catalog number: 15240-062 )
    
11. DMEM (Life Technologies, Invitrogen™, catalog number: 11965-167 )
    
12. Poly-L-lysine
    
13. PDGF-AA (Sigma-Aldrich)
    
14. FGFb (Life Technologies, Gibco^®)
    
15. Basal media (in DMEM) (see Recipes)
    

Equipment

1. Mesh (BD Biosciences, Falcon^®)
   
2. Shaking bath
   
3. Centrifuges
   
4. 6-well tissue culture plates
   

Procedure

1. Perform ex vivo gross total resection of the tumor, shred and mince the tissue in a small amount of PBS.
   Note: Enzymatic and mechanical dissociation followed a modified protocol (Gensert and Goldman., 2001).
   
2. Treat the shredded tissue with 11 ml per sample digestive enzyme (1:1,000 dilution of Trypsin 2.5% in MEM containing 20 mM HEPES) for 30 min in a 37 °C shaking bath. After the digestion, filter the dissociated cells through a 70 µm mesh.
   
3. Add 5 ml of 10% heat-inactivated FBS to inactivate the trypsin. Centrifuge cells for 10 min at 450 x g and re-suspend in MEM containing 20 mM HEPES and 30% sucrose.
   
4. Centrifuge cells for 20 min at 770 g and re-suspend in media.
   
5. Plate tumor cells onto 6-well tissue culture plates coated with poly-L-lysine (concentration) at a concentration of 2 million cells per well.
   
6. Grow the cells in culture media containing a 2:1 mixture of basal media and B104 conditioned media (Canoll et al., 1996), further supplemented with 10 ng/ml PDGF-AA and 10 ng/ml FGFb.
   
7. Basal media contained N2 supplement, 20 ng/ml T3, 0.5% FBS, and penicillin/streptomycin/amphotericin in DMEM.
   
8. Collect B104 conditioned media from confluent cultures of the B104 neuroblastoma cell line maintained in basal media for 48 h.
   

Recipes

1. Basal media (in DMEM)
   N2 supplement
   20 ng/ml T3
   0.5% FBS
   Penicillin/streptomycin/amphotericin
   

Acknowledgments

This protocol was developed in Dr. Peter Canoll’s lab at Columbia University, NY, USA. Please cite (Lei et al., 2011) if you use this protocol in your publications.

References

1. Canoll, P. D., Musacchio, J. M., Hardy, R., Reynolds, R., Marchionni, M. A. and Salzer, J. L. (1996). GGF/neuregulin is a neuronal signal that promotes the proliferation and survival and inhibits the differentiation of oligodendrocyte progenitors. Neuron 17(2): 229-243.
   
2. Gensert, J. M. and Goldman, J. E. (2001). Heterogeneity of cycling glial progenitors in the adult mammalian cortex and white matter. J Neurobiol 48(2): 75-86.
   
3. Lei, L., Sonabend, A. M., Guarnieri, P., Soderquist, C., Ludwig, T., Rosenfeld, S., Bruce, J. N. and Canoll, P. (2011). Glioblastoma models reveal the connection between adult glial progenitors and the proneural phenotype. PLoS One 6(5): e20041.