Cryopreservation of neurospheres derived from malignant human glioma

C1

Cancer stem cells have been isolated from a variety of cancers as well as brain tumors. In this study, we develop a novel method of preserving brain tumor neurospheres in order to facilitate cryopreservation in investigative work. We focus on comparing cryopreservation results by vitrification or conventional slow-cooling methods. Isolated brain tumor neurospheres containing the tumor-initiating CD133-positive population from two glioblastoma multiforme (GBM) patients were cryopreserved. We demonstrate that vitrification yielded better viability upon thawing after long-term storage in liquid nitrogen, as well as maintained “stemness” properties exhibited by the neurospheres prior to cryopreservation as determined by real-time PCR and immunofluorescence studies. There was also no change in the multipotentiality of vitrified or non-vitrified neurospheres. The cytogenetic hallmarks of GBM neurospheres remained unaltered after vitrification, demonstrating that the cryopresevation method did not cause additional chromosomal abnormalities. Importantly, our vitrified tumor neurospheres when implanted in immune-compromised mice formed glioma xenografts that were phenocopies of the patients’ original tumors. These data support the use of the vitrification process in establishing a brain tumor neurosphere repository for facilitating further studies focused on this cellular sub-population.