Isolation and profiling of viable tumor cells from human ex vivo glioblastoma cultures through single-cell transcriptomics

Junyi Zhang; Jakob Straehle; Kevin Joseph; Nicolas Neidert; Simon Behringer; Jonathan Göldner; Andreas Vlachos; Marco Prinz; Christian Fung; Jürgen Beck; Oliver Schnell; Dieter Henrik Heiland; Vidhya M Ravi

doi:10.1016/j.xpro.2023.102383

Isolation and profiling of viable tumor cells from human ex vivo glioblastoma cultures through single-cell transcriptomics

STAR Protoc. 2023 Sep 15;4(3):102383. doi: 10.1016/j.xpro.2023.102383. Epub 2023 Jul 1.

Authors

Affiliations

¹ 3D-Brain Models for Neurodegenerative Diseases, Medical Center, University of Freiburg, Freiburg, Germany; Microenvironment and Immunology Research Laboratory, Medical Center, University of Freiburg, Freiburg, Germany; Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany; Translational NeuroOncology Research Group, Medical Center - University of Freiburg, Freiburg, Germany.
² Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg, Germany; Center of Advanced Surgical Tissue Analysis (CAST), University of Freiburg, Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany.
³ NeuroEngineering Laboratory, Medical Centre, University of Freiburg, Freiburg, Germany; Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg, Germany; Center of Advanced Surgical Tissue Analysis (CAST), University of Freiburg, Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany; Translational NeuroOncology Research Group, Medical Center - University of Freiburg, Freiburg, Germany.
⁴ Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany.
⁵ Department of Neuroanatomy, Institute of Anatomy and Cell Biology, Faculty of Medicine, University of Freiburg, Freiburg, Germany; BrainLinks-BrainTools Center, University of Freiburg, Freiburg, Germany; Center for Basics in Neuromodulation (NeuroModulBasics), Faculty of Medicine, University of Freiburg, Freiburg, Germany; Center for NeuroModulation (NeuroModul), University of Freiburg, Freiburg, Germany.
⁶ Faculty of Medicine, University of Freiburg, Freiburg, Germany; Center for Basics in Neuromodulation (NeuroModulBasics), Faculty of Medicine, University of Freiburg, Freiburg, Germany; Center for NeuroModulation (NeuroModul), University of Freiburg, Freiburg, Germany; Institute of Neuropathology, Medical Center - University of Freiburg, Freiburg, Germany; Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany.
⁷ Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg, Germany; Center of Advanced Surgical Tissue Analysis (CAST), University of Freiburg, Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany; Center for Basics in Neuromodulation (NeuroModulBasics), Faculty of Medicine, University of Freiburg, Freiburg, Germany; Comprehensive Cancer Center Freiburg (CCCF), Faculty of Medicine and Medical Center- University of Freiburg, Freiburg, Germany.
⁸ Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg, Germany; Center of Advanced Surgical Tissue Analysis (CAST), University of Freiburg, Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany; Translational NeuroOncology Research Group, Medical Center - University of Freiburg, Freiburg, Germany.
⁹ Microenvironment and Immunology Research Laboratory, Medical Center, University of Freiburg, Freiburg, Germany; Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg, Germany; Center of Advanced Surgical Tissue Analysis (CAST), University of Freiburg, Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany; Translational NeuroOncology Research Group, Medical Center - University of Freiburg, Freiburg, Germany; Comprehensive Cancer Center Freiburg (CCCF), Faculty of Medicine and Medical Center- University of Freiburg, Freiburg, Germany; German Cancer Consortium (DKTK), Partner Site Freiburg; Department of Neurological Surgery, Lou and Jean Malnati Brain Tumor Institute, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
¹⁰ 3D-Brain Models for Neurodegenerative Diseases, Medical Center, University of Freiburg, Freiburg, Germany; Department of Neurosurgery, Medical Center, University of Freiburg, Freiburg, Germany; Center of Advanced Surgical Tissue Analysis (CAST), University of Freiburg, Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany; Translational NeuroOncology Research Group, Medical Center - University of Freiburg, Freiburg, Germany; Freiburg Institute of Advanced Studies (FRIAS), Freiburg, Germany. Electronic address: vidhya.ravi@uniklinik-freiburg.de.

Abstract

Single-cell RNA-sequencing (scRNA-seq) is becoming a ubiquitous method in profiling the cellular transcriptomes of both malignant and non-malignant cells from the human brain. Here, we present a protocol to isolate viable tumor cells from human ex vivo glioblastoma cultures for single-cell transcriptomic analysis. We describe steps including surgical tissue collection, sectioning, culturing, primary tumor cells inoculation, growth tracking, fluorescence-based cell sorting, and population-enriched scRNA-seq. This comprehensive methodology empowers in-depth understanding of brain tumor biology at the single-cell level. For complete details on the use and execution of this protocol, please refer to Ravi et al.¹.

Keywords: Cancer; Cell Biology; Cell Isolation; Flow Cytometry/Mass Cytometry; Microscopy; Model Organisms; Molecular Biology; RNAseq; Sequencing; Single Cell; Tissue Engineering.

MeSH terms

Brain
Brain Neoplasms* / genetics
Gene Expression Profiling
Glioblastoma* / genetics
Humans
Transcriptome / genetics