成人及儿童胶质母细胞瘤类器官的冷冻保存技术及其对干细胞自我更新的影响

• Article
• Open access
• Published: 26 October 2026

• Sajina Shakya^1,2,
• Evan Joseph^1,2,
• Meghana A¹,
• Prakriti Shukla³,
• Tempestt Nwachukwu⁴,
• Shawn T. Ho^4,5,
• Tyler E. Miller^2,4,5 &
• …
• Christopher G. Hubert^1,2,3 

Scientific Reports (2026) Cite this article

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Abstract

Organoids are advanced three-dimensional in vitro models that faithfully recapitulate tumor architecture, cellular heterogeneity, and microenvironmental interactions, making them valuable tools for cancer research, including for the lethal primary brain tumor glioblastoma (GBM). However, organoid generation is time- and resource-intensive, necessitating reliable cryopreservation techniques to enable long-term storage, biobanking, and sample sharing. Cryopreservation of small spheroids and organoids has been reported, but such methods have not been directly compared, and preservation of larger multi-millimeter-scale organoids has remained largely unexplored. Additionally, existing cryopreservation methods containing FBS, which induces differentiation in neural-lineage culture, have not been systematically benchmarked for their ability to maintain GBM self-renewal. We evaluated multiple custom-made and commercial cryopreservation containers and media on GBM self-renewal, as well as cell viability, structure, and stem cell preservation in large matrix-based GBM organoids and organoids directly from patient tissue. Common non-commercial cryopreservation media containing 10% DMSO, either serum-free or supplemented with 80% FBS, preserved GBM stem cell viability and self-renewal as well or better than commercial cryopreservation formulations tested. Cryopreserved organoids maintained post-thaw viability, self-renewal capacity, and histological characteristics following outgrowth. These findings demonstrate feasible methods to cryopreserve large GBM organoids, supporting their broader use in biobanking, collaborative studies, and experimental workflows.

Acknowledgements

We thank the Lathia lab at the Cleveland Clinic for providing patient-derived tumor cell culture samples utilized in this study.

Funding

This work was supported by the National Institute of Neurological Disorders and Stroke (R01NS126253 to C.G.H.), Worldwide Cancer Research (grant reference number 26–0094 to C.G.H.), the National Cancer Institute (Ruth L. Kirschstein National Research Service Award T32CA059366 to S.S.), and institutional funds from University Hospitals, Case Western Reserve University, and the Case Comprehensive Cancer Center (to C.G.H. and T.E.M.)

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Authors and Affiliations

1. Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA

   Sajina Shakya, Evan Joseph, Meghana A & Christopher G. Hubert

2. Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA

   Sajina Shakya, Evan Joseph, Tyler E. Miller & Christopher G. Hubert

3. Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA

   Prakriti Shukla & Christopher G. Hubert

4. Department of Pathology, Case Western Reserve University, Cleveland, OH, USA

   Tempestt Nwachukwu, Shawn T. Ho & Tyler E. Miller

5. Division of Genomic & Molecular Pathology, University Hospitals Cleveland Medical Center, Cleveland, OH, USA

   Shawn T. Ho & Tyler E. Miller

Authors

1. Sajina Shakya
2. Evan Joseph
3. Meghana A
4. Prakriti Shukla
5. Tempestt Nwachukwu
6. Shawn T. Ho
7. Tyler E. Miller
8. Christopher G. Hubert

Corresponding author

Correspondence to Christopher G. Hubert.

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The authors declare no competing interests.

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Shakya, S., Joseph, E., A, M. et al. Cryopreservation techniques for adult and pediatric glioblastoma organoids and their effects on stem cell self-renewal. Sci Rep (2026). https://doi.org/10.1038/s41598-026-77087-3

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• Received: 08 June 2026

• Accepted: 16 October 2026

• Published: 26 October 2026

• DOI: https://doi.org/10.1038/s41598-026-77087-3

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