P03.08 * IMPROVED YIELD OF STEM-LIKE CELLS USING GLIOBLASTOMA SUSPENSIONS FROM CAVITRON ULTRASONIC SURGERY ASPIRATOR (CUSA) BAGS AS STARTING MATERIAL

Absence of serum and presence of growth factors (mostly EGF and bFGF) has been used to culture cancer stem cells (CSC). In case of GB the term neurospheres (NS) has been considered as synonymous of glioma stem-like cells (GSCs). In our laboratories we isolate and culture GSC from fresh GB specimens growing in the absence of serum and in the presence of EGF and bFGF as neurospheres after mechanical and enzymatic dissociation. Previous data and our experience suggest that NS may mirror more closely than previous, serum-based glioma cell lines, the actual biology of GB. Tumor fragments were used to start the culture. The Cavitation Ultrasonics Surgical Aspirator (CUSA) is increasingly used for GB surgery, decreasing the availability of tumor fragments. CUSA delivers an irrigating solution that converts the fragmented tissue into an emulsion and then aspirates the particles directly into a sterile bag. The bag contains some larger pieces of tissue, debris and high amounts of erythrocytes, and occasionally necrotic or reactive tissue. Here we describe our experience with CUSA bags as the staring material for GSC preparation. Tissue fragments in CUSA bags after several rounds of spinning ad washing are dissociated using the GentleMacs Dissociator (Miltenyi Biotec) that provides a closed system and reproducible results. We have optimized a gentle and effective protocol starting from appropriate gentleMACS programs, allowing to obtain a high yield of viable tumor cells. After processing, cell suspensions are cultured as neurospheres in DMEM/F12, B27 supplement, human recombinant b-FGF and EGF. During the last year we obtained from the Department of Neurosurgery of Istituto Besta a total of 54 CUSA. Primary GB were the most represented brain tumors (87%). Using tumor fragments obtained from surgery and combining mechanical dissociation with enzymatic disaggregation on a series of primary GB we previously obtained GSC in 52% of the cases. This percentage is now increased to 71% with the use of surgical material from CUSA and the optimized protocol. Proliferation kinetics was studied by plating three primary cell lines obtained in parallel from GB specimen and from CUSA material at density of 15,000-30,000 cells/cm2. Cultures were collected every 5 days and the total number of viable cells assessed at each passage by Trypan Blue exclusion. A long term proliferation of cell lines at low sub-culturing stages (3 to 10 passages), showed that proliferation increased exponentially, but the proliferation index of NS from CUSA materials was higher that cells from specimens (1.32 vs 1.18 respectively, p = 0.03). These improvements and the use of a closed system providing a wider margin of safety support the incorporation of this process in a clinical trial protocol which plans to use GSC as a source of antigens for dendritic cell loading, replacing the whole lysate from GB specimens used in current immunotherapy protocols.