title: A standardized and reproducible protocol for serum-free monolayer culturing of primary paediatric brain tumours to be utilized for therapeutic assays.
abstract: In vitro cultured brain tumour cells are indispensable tools for drug screening and therapeutic development. Serum-free culture conditions tentatively preserve the features of the original tumour, but commonly comprise neurosphere propagation, which is a technically challenging procedure. Here, we define a simple, non-expensive and reproducible serum-free cell culture protocol for establishment and propagation of primary paediatric brain tumour cultures as adherent monolayers. The success rates for establishment of primary cultures (including medulloblastomas, atypical rhabdoid tumour, ependymomas and astrocytomas) were 65% (11/17) and 78% (14/18) for sphere cultures and monolayers respectively. Monolayer culturing was particularly feasible for less aggressive tumour subsets, where neurosphere cultures could not be generated. We show by immunofluorescent labelling that monolayers display phenotypic similarities with corresponding sphere cultures and primary tumours, and secrete clinically relevant inflammatory factors, including PGE2, VEGF, IL-6, IL-8 and IL-15. Moreover, secretion of PGE2 was considerably reduced by treatment with the COX-2 inhibitor Valdecoxib, demonstrating the functional utility of our newly established monolayer for preclinical therapeutic assays. Our findings suggest that this culture method could increase the availability and comparability of clinically representative in vitro models of paediatric brain tumours, and encourages further molecular evaluation of serum-free monolayer cultures.
authors.affiliation: Glioma Immunotherapy Group, Division of Neurosurgery, Department of Clinical Sciences Lund, Lund University, Lund, Sweden.
authors.forename: Emma
authors.initials: E
authors.lastname: Sandén
authors.affiliation: Glioma Immunotherapy Group, Division of Neurosurgery, Department of Clinical Sciences Lund, Lund University, Lund, Sweden.
authors.forename: Sofia
authors.initials: S
authors.lastname: Eberstål
authors.affiliation: Glioma Immunotherapy Group, Division of Neurosurgery, Department of Clinical Sciences Lund, Lund University, Lund, Sweden.
authors.forename: Edward
authors.initials: E
authors.lastname: Visse
authors.affiliation: 1] Glioma Immunotherapy Group, Division of Neurosurgery, Department of Clinical Sciences Lund, Lund University, Lund, Sweden [2] Division of Neurosurgery, Department of Clinical Sciences Lund, Skåne University Hospital, Lund, Sweden.
authors.forename: Peter
authors.initials: P
authors.lastname: Siesjö
authors.affiliation: Glioma Immunotherapy Group, Division of Neurosurgery, Department of Clinical Sciences Lund, Lund University, Lund, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Darabi
mesh_terms: D000293:Adolescent; D015703:Antigens, CD / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D015415:Biomarkers; D001932:Brain Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D002448:Cell Adhesion; D049109:Cell Proliferation / Q000187:drug effects; D002648:Child; D002675:Child, Preschool; D016895:Culture Media, Serum-Free; D051546:Cyclooxygenase 2 / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D005260:Female; D006801:Humans; D008297:Male; D060787:Neoplasm Grading; D010641:Phenotype; D061251:Primary Cell Culture / Q000379:methods; D018874:Spheroids, Cellular; D014407:Tumor Cells, Cultured
chemical_list: D015703:Antigens, CD; D000970:Antineoplastic Agents; D015415:Biomarkers; D016895:Culture Media, Serum-Free; D016207:Cytokines; D051546:Cyclooxygenase 2; D015232:Dinoprostone