TMOD-23. Investigation of factors contributing to the establishment of brain tumor cell lines using a semi-automated dissociator

Abstract BACKGROUND In brain tumor research, numerous patient-derived tumor cell lines have been established and utilized to develop new therapies and understand disease pathology. However, conventional methods for establishing cell lines face significant limitations in terms of procedures, equipment, and operation time/timing. Furthermore, the biological factors essential for establishing a successful cell line remain unclear. Therefore, in this study, we investigated various aspects of the conditions required for the establishment of long-term surviving stable brain tumor cell lines by elective use of a semi-automated tissue dissociation device, aiming to identify key factors that contribute to successful cell line establishment. METHODS Brain tumor specimens resected at our institution were processed within 48 hours post-excision using the gentleMACS dissociator (Miltenyi Biotec) with a protocol uniquely modified by our team. The dissociated cells were then cultured under various conditions. We defined successful stable cell line establishment as the ability to stably maintain the cell phenotype over 50 passages in vitro. We analyzed the success rate and contributing factors of cell line establishment. RESULTS A total of 55 tumor specimens were cultured, out of which 5 (9 %) were successfully established as stable cell lines. The pathological diagnoses included one case of embryonal tumor, one case of pilocytic astrocytoma, one case of high-grade glioma (HGG), and two cases of glioblastoma (GBM). All six stable cell lines were maintained by DMEM/F12-based cell culture medium and type I collagen-coated cell culture dishes in common. Genetic analysis of these five established lines revealed TERT promoter mutations in three cases (one HGG case and two GBM cases). CONCLUSION The elective use of a semi-automated tissue dissociation device may enable more efficient establishment of patient-derived brain tumor cell lines. Additionally, it is suggested that the presence of TERT promoter mutations may be associated with successful stable cell line establishment.