Papain based brain tumor dissociation kit

Adult patients at the University of Washington provided pre-operative informed consent to take part in the study in all cases after approved IRB protocol (no. STUDY00002162). Fresh tumors were collected directly from the operating room at the time of surgery and either taken fresh or snap-frozen immediately after removal in liquid nitrogen. Histopathologic diagnosis was confirmed by a board-certified neuropathologist. Fresh tissue was enzymatically dissociated using a papain-based brain tumor dissociation kit (Miltenyi Biotec) as per the manufacturer’s protocol. Cells were then cultured on laminin-coated plates in DMEM/F12 supplemented with 1× N2/B27 and 1% penicillin–streptomycin. Cultures were passaged as needed when confluent and considered stable after three serial passages. Cell line UW7gsc was used for this study at passage 3. Autopsy tissue was collected with a post-mortem interval of approximately 8.75 h after informed consent with a waiver from the University of Washington IRB. Tissue was snap-frozen in liquid nitrogen-cooled isopentane. Tumor regions were sampled based on gross examination of brain sections and processed as outlined below.

All mice were perfused with ice-cold PBS after euthanasia. The collected brains were mechanically and enzymatically dissociated using a papain-based brain tumor dissociation kit (Miltenyi Biotec) supplemented with 0.1% typeI collagenase (Thermo Fisher Scientific) / PBS. Cells were first stained with calcein Blue AM (Life Technologies) and Zombie NIR (BioLegend) for 30min at 4 °C, and with anti-mouse CD16/32 (BD Biosciences) for 5min. After washing cells with ice-cold 2% FBS / PBS, cells were then stained with anti-mouse CD45-PerCP (clone 30-F11, BD Biosciences) for 30min at 4 °C. Sorting was performed with The Becton-Dickinson Influx™ cytometer (Becton Dickinson) using 640nm (Zombie NIR, 750LP filter), 355nm (calcein Blue AM, 460/50 filter), 488nm (CD45-PerCP, 692/40 filter) and 488nm (GFP, 530/40 filter) lasers. Side scatter (SSC) width versus forward scatter (FSC) area, and Trigger Pulse Width versus FSC criteria were used to discriminate doublets and gate only singleton cells. Viable single cells were identified as calcein Blue AM positive and Zombine NIR negative. We sorted viable CD45 negative/ GFP positive single-cells into 96-well plates containing 5uL of TCL buffer (Qiagen) with 1% beta-mercaptoethanol. Plates were frozen immediately after sorting and stored at −80°C prior to whole transcriptome amplification, library preparation and sequencing.

Patients at Massachusetts General Hospital gave consent preoperatively in all cases according to Institutional Review Board Protocol 1999P008145. Fresh tumors were collected at the time of resection, and presence of malignant cells was confirmed by frozen section. Fresh tumor tissue was mechanically and enzymatically dissociated using a papain-based brain tumor dissociation kit (Miltenyi Biotec). Large pieces of debris were removed with a 100-μm strainer, and dissociated cells were layered onto a 5-ml density gradient (Lympholyte-H, Cedar Lane Labs), which was centrifuged at 2000 rpm for 10 min at room temperature to pellet dead cells and red blood cells. The interface containing live cells was saved and used for staining and flow cytometry. Viability was measured using trypan blue exclusion.