Multi-inlet spheroid generator for modeling immune microenvironment of glioblastoma (GBM)

Using the multi-inlet spheroid generator (MSG) platform, we generated stable GBM spheroids and observed their interactions with various immune cells, including microglia and natural killer (NK) cells, at different time points. HMO6 and NK-92 cells were used for microglia and NK cells, respectively. We examined the interactions between tumor-immune cells as well as between different immune cells. The generation of tri-cultured spheroids successfully recapitulated the immunosuppressive microenvironment of the GBM tumor. Overall design: We performed gene expression profiling analysis of bulk RNA-seq data obtained from patient-derived GBM spheroids at various time points. Total RNA was isolated from these spheroids using the QIAzol lysis reagent (Qiagen,79306 )following the manufacturer's protocol. Libraries for bulk RNA-seq were prepared using the TruSeq Stranded mRNA Library Prep Kit. GBM spheroids were cultured and maintained in Dulbecco's modified Eagle's high glucose medium (SH30243.01, Hyclone) supplemented with 10% heat-inactivated fetal bovine serum (2357664RP, Gibco) and 1% antibiotic-antimycotic (15240062, Gibco). Spheroids were maintained at 37°C in a humidified atmosphere containing 5% CO2 and tested regularly for mycoplasma contamination by mycoplasma detection kit (rep-mys-20, InvivoGen).