INTRODUCTION: Physiologically relevant pre-clinical ex vivo models 
recapitulating CNS tumor micro-environmental complexity will aid development of 
biologically-targeted agents. We present comprehensive characterization of tumor 
aggregates generated using the 3D Rotary Cell Culture System (RCCS).
METHODS: CNS cancer cell lines were grown in conventional 2D cultures and the 
RCCS and comparison with a cohort of 53 pediatric high grade gliomas conducted 
by genome wide gene expression and microRNA arrays, coupled with 
immunohistochemistry, ex vivo magnetic resonance spectroscopy and drug 
sensitivity evaluation using the histone deacetylase inhibitor, Vorinostat.
RESULTS: Macroscopic RCCS aggregates recapitulated the heterogeneous morphology 
of brain tumors with a distinct proliferating rim, necrotic core and oxygen 
tension gradient. Gene expression and microRNA analyses revealed significant 
differences with 3D expression intermediate to 2D cultures and primary brain 
tumors. Metabolic profiling revealed differential profiles, with an increase in 
tumor specific metabolites in 3D. To evaluate the potential of the RCCS as a 
drug testing tool, we determined the efficacy of Vorinostat against aggregates 
of U87 and KNS42 glioblastoma cells. Both lines demonstrated markedly reduced 
sensitivity when assaying in 3D culture conditions compared to classical 2D drug 
screen approaches.
CONCLUSIONS: Our comprehensive characterization demonstrates that 3D RCCS 
culture of high grade brain tumor cells has profound effects on the genetic, 
epigenetic and metabolic profiles of cultured cells, with these cells residing 
as an intermediate phenotype between that of 2D cultures and primary tumors. 
There is a discrepancy between 2D culture and tumor molecular profiles, and RCCS 
partially re-capitulates tissue specific features, allowing drug testing in a 
more relevant ex vivo system.