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

Nature Protocols volume 15, pages 4000–4033 (2020)Cite this article

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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.

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Fig. 1: Overview of the procedures to generate GBOs from resected tumor tissue and to co-culture GBOs with CAR T cells.
Fig. 2: GBO success rate and growth.
Fig. 3: Establishment and maintenance of GBO culture.
Fig. 4: Immunofluorescence characterization of GBOs and parent tumors.
Fig. 5: Detection of hypoxia gradients and actively proliferating cells in GBOs.
Fig. 6: GBO co-culture with CAR T cells.

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Acknowledgements

We thank all patients who generously donated tissue. We thank members of the Ming and Song laboratories for comments and suggestions, B. Temsamrit and E. LaNoce for technical support, J. Schnoll for lab coordination, and D. O’Rourke, M. Nasrallah and others in the Departments of Neurosurgery and Pathology at the Hospital of the University of Pennsylvania for assistance with tissue acquisition. This work was supported by the Glioblastoma Translational Center of Excellence at the Abramson Cancer Center at the University of Pennsylvania, grants from the National Institutes of Health (R37NS047344 and R35NS116843 to H.S., R35NS097370 and U19AI131130 to G.-l.M.), the Sheldon G. Adelson Medical Research Foundation (to G.-l.M.) and the Pennsylvania Department of Health (to G.-l.M.).

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Authors and Affiliations

  1. Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Fadi Jacob, Guo-li Ming & Hongjun Song

  2. The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Fadi Jacob

  3. Medical Scientist Training Program, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Fadi Jacob

  4. Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Guo-li Ming & Hongjun Song

  5. Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Guo-li Ming & Hongjun Song

  6. Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Guo-li Ming

  7. Glioblastoma Translational Center of Excellence, The Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA

    Hongjun Song

  8. The Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Hongjun Song

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Contributions

F.J. developed the procedures to generate, culture and biobank GBOs and to co-culture GBOs with CAR T cells and produced the sample data. F.J., G.-l.M. and H.S. conceived the project and wrote the manuscript.

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Correspondence to Fadi Jacob, Guo-li Ming or Hongjun Song.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Lisa Ebert, Henner Farin, Guillermo Gomez, Yanhong Shi and Ali G Turhan for their contribution to the peer review of this work.

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Key reference using this protocol

Jacob, F. et al. Cell 180, 188–204.e122 (2020): https://doi.org/10.1016/j.cell.2019.11.036

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Jacob, F. et al. Cell 180, 188–204.e122 (2020): https://doi.org/10.1016/j.cell.2019.11.036

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Supplementary Video 1

Video showing procedures for processing patient glioblastoma tissue for organoid culture

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Jacob, F., Ming, Gl. & Song, H. Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR T cell testing. Nat Protoc 15, 4000–4033 (2020). https://doi.org/10.1038/s41596-020-0402-9

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