• Stine Skov Jensen, Charlotte Aaberg-Jessen, Claus Andersen, Henrik Daa Schrøder, and Bjarne Winther Kristensen.
• *Department of Pathology and ‡Department of Neurosurgery, Odense University Hospital, and Institute of Clinical Research, University of Southern Denmark, Odense, Denmark.
• Neurosurgery. 2013 Nov 1;73(5):868-86; discussion 886.

BackgroundUltrasonic aspirators allow safe, rapid, and accurate removal of brain tumors. However, the tissue fragments removed are used surprisingly little in research.ObjectiveTo investigate whether such tissue fragments could be cultured as organotypic multicellular spheroids because access to biopsy tissue is often limited.MethodsTissue fragments obtained by ultrasonic aspiration from 10 glioblastomas and tumor biopsy tissue from 7 of these tumors were cultured in serum-containing and serum-free medium. On culturing, the fragments formed spheroids, which were prepared for histology. Two glioblastoma cell lines from ultrasonic fragments and biopsy tissue were established as well.ResultsHematoxylin and eosin staining showed viable glioma spheroids obtained from both ultrasonic and biopsy tissue in both types of medium. Endothelial growth factor receptor and PTEN/chromosome 10 status was found to be preserved in most spheroids (7-8 of 10 tumors), together with the level of glial fibrillary acidic protein, von Willebrand factor, and Ki-67. The levels of stem cell markers CD133, Bmi-1, nestin, and Sox2 also were preserved. The ultrasonic spheroids had higher levels of glial fibrillary acidic protein and von Willebrand factor and lower levels of Bmi-1, nestin, Sox2, and Olig2 compared with conventional biopsy spheroids. For both types of spheroids, the stem cell medium seemed to favor expression of stem cell markers. The established cell lines were capable of both spheroid formation at clonal density and tumor formation in vivo.ConclusionViable organotypic and proliferating spheroids were easily obtained from ultrasonic tissue fragments. The preservation of markers and the establishment of cell lines with tumor-initiating cell properties suggest ultrasonic spheroids as a new tissue resource for glioma research.

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