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  • Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR T cell testing.

Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR T cell testing.

Nature protocols (2020-11-11)
Fadi Jacob, Guo-Li Ming, Hongjun Song
ABSTRACT

Glioblastoma tumors exhibit extensive inter- and intratumoral heterogeneity, which has contributed to the poor outcomes of numerous clinical trials and continues to complicate the development of effective therapeutic strategies. Most in vitro models do not preserve the cellular and mutational diversity of parent tumors and often require a lengthy generation time with variable efficiency. Here, we describe detailed procedures for generating glioblastoma organoids (GBOs) from surgically resected patient tumor tissue using a chemically defined medium without cell dissociation. By preserving cell-cell interactions and minimizing clonal selection, GBOs maintain the cellular heterogeneity of parent tumors. We include details of how to passage and cryopreserve GBOs for continued use, biobanking and long-term recovery. In addition, we describe procedures for investigating patient-specific responses to immunotherapies by co-culturing GBOs with chimeric antigen receptor (CAR) T cells. It takes ~2-4 weeks to generate GBOs and 5-7 d to perform CAR T cell co-culture using this protocol. Competence with human cell culture, tissue processing, immunohistology and microscopy is required for optimal results.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Insulin solution human, sterile-filtered, BioXtra, suitable for cell culture
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Sucrose, for molecular biology, ≥99.5% (GC)
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Dimethyl sulfoxide, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%
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Roche
DAPI, 4′,6-Diamidine-2′-phenylindole dihydrochloride
Pricing and availability is not currently available.