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

• Published in: STAR protocols 2023-09, Vol.4 (3), p.102383-102383, Article 102383
• Main Authors: Zhang, Junyi, Straehle, Jakob, Joseph, Kevin, Neidert, Nicolas, Behringer, Simon, Göldner, Jonathan, Vlachos, Andreas, Prinz, Marco, Fung, Christian, Beck, Jürgen, Schnell, Oliver, Heiland, Dieter Henrik, Ravi, Vidhya M.
• Format: Article
• Language: English
• Subjects: Brain; Brain Neoplasms - genetics; Cancer; Cell Biology; Cell Isolation; Flow Cytometry/Mass Cytometry; Gene Expression Profiling; Glioblastoma - genetics; Humans; Microscopy; Model Organisms; Molecular Biology; Protocol; RNAseq; Sequencing; Single Cell; Tissue Engineering; Transcriptome - genetics
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102383

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 [Display omitted] •Describes methods to re-purpose therapeutically discarded human cortical tissue•Protocol is adaptable to brain tissue from different ages of human or rodent subjects•Outlines steps for isolating single-tumor cells from human ex vivo cortical cultures•Details quality control measures and library preparation for Illumina sequencing Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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, GBM 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.