Cryopreservation techniques for adult and pediatric glioblastoma organoids and their effects on stem cell self-renewal

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.