Ultrasonic Surgical Aspirate is a Reliable Source For Culturing Glioblastoma Stem Cells

Abstract

Glioma stem cells (GSCs) are thought to be the source of tumor growth and therapy resistance. To understand the biology of GSCs, and target these tumors therapeutically, we need robust strategies for in vitro expansion of primary GSCs. To date, tumor core biopsies have been the main established source of GSCs. Since these samples are used for diagnostic purposes, the available tissue for cell culture and therapeutic targeting can be limited. In addition, a core biopsy is usually taken from one part of the tumor, thus would be unlikely to represent intra-tumor heterogeneity. To overcome these problems, tissue fragments from all over the tumor can be collected using an ultrasonic aspirator during surgery, thus assembling a “global tumor biopsy”. Usually, this ultrasonic aspirate (UA) sample is considered as biological waste after operations. Here, we show that UA samples offer a large and reliable source of live cells. Similar to core biopsies, UA samples enriched for GSCs that differentiated into neural lineages, showed inter-individual variation of GSC markers, and induced tumors. Molecular profiling showed that UA samples cover tumor heterogeneity better than core biopsies. These results suggest that UA samples can be used to establish large scale cultures for therapeutic applications.

The landscape of the mesenchymal signature in brain tumours

“…This finding supports our hypothesis that there are at least two subclasses within the mesenchymal subtype, one subclass induced by recruited cells and the other by tumour cells. Furthermore, in our experience, the mesenchymal subtype cells from GBM patients, enriched under neuro-sphere culture conditions, also have two subclasses depending on their in vitro growth morphology (Behnan et al , 2016, 2017 a ). One type grows as floating spheres and the other grows adherently in serum-free non-adherent conditions (Behnan et al , 2017 a ).…”

Brain tissue banking for stem cells for our future

“…In the case of the tumour tissue sample that did not yield viable cells, an ultrasonically aspirated efflux of the resection site did yield viable cells in great numbers12. So in fact all patient sources were successfully recovered using this method.…”

Identification and characterization of a new source of adult human neural progenitors

“…Single cell suspensions were prepared from these samples following the protocol developed by us for processing brain tumor tissue from UA samples. 16 Total cell count, viability and cell yields are shown in Table 1 . Cell viability in fresh samples ranged between 37-82%.…”

“…To investigate the multilineage neural differentiation properties of cells derived from UA samples under different culture conditions, we subjected them to a neural differentiation protocol for 2 weeks as previously described. 16 Under neural differentiation conditions, both neurosphere culture (UA-Sp) and adherent serum cultures (UA-AD1 and UA-AD10) gave rise to cells that express the glial marker GFAP and mature neuron marker TUBIII. Cells from UA-AD1 and UA-AD10 showed low expression for MAP2.…”

“…The collected tissue sample was divided into three fractions: one was cryopreserved in FBS/DMSO (BioChrome, VWR and Sigma, St Louis, MO, USA), one was snap frozen for protein and RNA extraction, and one was weighed and then processed into a single cell suspension following the protocol established by us for processing UA samples from brain tumor patients. 16 Cells were counted and plated in three different sets of culture conditions: (1) adherent culture expanded in 'failsafe' medium (AD1): DMEM/F12-GlutaMAX medium supplemented with 1% FBS, 2% B-27 (Gibco, Life Technologies, Paisley, UK), 10 ng/ml basic fibroblast growth factor, 20 ng/ml TGF α , 10 mm HEPES (Lonza, BioWhittaker, Cologne, Germany), 100 U/ml penicillin/streptomycin of both (LonzaBioWhittaker) and 2.5 μ g/ml heparin (Leo Pharma AS, Esbjerg, Denmark); 13 (2) traditional adherent serum culture expanded in DMEM/F12-GlutaMAX medium, 10% FBS, 10 mm HEPES, and 100 U/ml Penicillin/Streptomycin. (3) neurosphere culture conditions which is serum-free sphere culture containing DMEM/F12-GlutaMAX medium, 10 ng/ml bFGF, 20 ng/ml EGF (both R&D Inc, Minneapolis, MN, USA), 1:50 B27-supplement, 100 U/ml penicillin/streptomycin, 1 ng/ml Heparin (Leo Pharma), and HEPES 5 mM.…”