title: Optimization of high grade glioma cell culture from surgical specimens for use in clinically relevant animal models and 3D immunochemistry.
abstract: Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor. Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.
authors.affiliation: Department of Neurosurgery, Henry Ford Hospital.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Hasselbach
authors.forename: Susan M
authors.initials: SM
authors.lastname: Irtenkauf
authors.forename: Nancy W
authors.initials: NW
authors.lastname: Lemke
authors.forename: Kevin K
authors.initials: KK
authors.lastname: Nelson
authors.forename: Artem D
authors.initials: AD
authors.lastname: Berezovsky
authors.forename: Enoch T
authors.initials: ET
authors.lastname: Carlton
authors.forename: Andrea D
authors.initials: AD
authors.lastname: Transou
authors.forename: Tom
authors.initials: T
authors.lastname: Mikkelsen
authors.forename: Ana C
authors.initials: AC
authors.lastname: deCarvalho
mesh_terms: D000818:Animals; D001932:Brain Neoplasms / Q000378:metabolism / Q000473:pathology*; D018929:Cell Culture Techniques / Q000379:methods*; D016895:Culture Media, Serum-Free; D005909:Glioblastoma / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D060787:Neoplasm Grading; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology*; D058953:Neural Stem Cells / Q000378:metabolism / Q000473:pathology*; D016612:Paraffin Embedding / Q000379:methods; D018874:Spheroids, Cellular
chemical_list: D016895:Culture Media, Serum-Free