Welcome guest, Sign in



This protocol has been developed for culturing primary glioblastoma cells. We have most experience in using it on rodent preparations, but it can also be used in culturing cells from other species.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.


Primary Tumor Preparation

Cancer Biology > General technique > Cell biology assays > Cell isolation and culture
Author: Liang Lei
Liang LeiAffiliation: Department of Pathology and Cell Biology, Columbia University, New York, USA
For correspondence: ll2239@columbia.edu
Bio-protocol author page: a56
Vol 2, Iss 12, 6/20/2012, 6468 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.229

[Abstract] This protocol has been developed for culturing primary glioblastoma cells. We have most experience in using it on rodent preparations, but it can also be used in culturing cells from other species.

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)


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


  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.


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


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.


  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.

How to cite: Lei, L. (2012). Primary Tumor Preparation. Bio-protocol 2(12): e229. DOI: 10.21769/BioProtoc.229; Full Text

Share Your Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Ask the Authors:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register
How to cite
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook