Abstract
Background: Technologies related to the establishment of primary tumor cell cultures from solid tumors, including glioblastoma, are increasingly important to oncology research and practice. However, processing of fresh tumor specimens for establishment of primary cultures on the day of surgical collection is logistically difficult. The feasibility of viable cryopreservation of glioblastoma specimens, allowing for primary culture establishment weeks to months after surgical tumor collection and freezing, was demonstrated by Mullins et al. in 2013, with a success rate of 59% that was not significantly lower than that achieved with fresh tumor tissue. However, research targeting optimization of viable glioblastoma cryopreservation protocols for establishment of primary tumor cultures has been limited.Objectives: The objective of this study was to optimize glioblastoma cryopreservation methods for viable cryobanking and to determine if two-dimensional (2D) or three-dimensional (3D) culture conditions were more supportive of glioblastoma growth after thawing of frozen tumor specimens.Methods: Portions of eight human glioblastoma specimens were cryopreserved by four different protocols differing in the time of enzymatic digestion (before or after cryopreservation), and in the type of cryopreservation media (CryoStor CS10 or 10% dimethyl sulfoxide and 90% fetal calf serum). After 1 month, frozen tissues were thawed, enzymatically digested, if not digested before, and used for initiation of 2D or 3D primary tumor cultures to determine viability.Results: Among the tested cryopreservation and culturing protocols, the most efficient combinations of cryopreservation and culture were those associated with the use of CryoStor CS10 cryopreservation medium, enzymatic digestion before freezing, and 2D culturing after thawing with a successful culture rate of 8 out of 8 cases (100%). Two-dimensional cultures were in general more efficient for the support of tumor cell growth after thawing than 3D cultures.Conclusions: This study supports development of evidence-based viable glioblastoma cryopreservation methods for use in glioblastoma biobanking and research.
Highlights
Glioblastoma is the most common malignant primary brain tumor and is the focus of extensive basic and clinical research efforts targeting improved therapies.[1,2] Technologies related to the establishment of primary low passage tumor cultures are increasingly important to oncology research and practice.[3,4,5,6,7] processing of fresh tumor specimens for the establishment of primary cultures on the day of surgical collection is logistically difficult
To tissue biorepository practices, viable cryopreservation of glioblastoma specimens allowing for primary culture establishment months to years after surgical tumor collection and freezing has been demonstrated with a success rate not significantly lower than that achieved with fresh tumor tissue.[8]
Success rate of establishment of primary glioblastoma cultures following the use of different cryopreservation protocols and 2D or 3D culture conditions
Summary
Glioblastoma is the most common malignant primary brain tumor and is the focus of extensive basic and clinical research efforts targeting improved therapies.[1,2] Technologies related to the establishment of primary low passage tumor cultures are increasingly important to oncology research and practice.[3,4,5,6,7] processing of fresh tumor specimens for the establishment of primary cultures on the day of surgical collection is logistically difficult. The feasibility of viable cryopreservation of glioblastoma specimens, allowing for primary culture establishment weeks to months after surgical tumor collection and freezing, was demonstrated by Mullins et al in 2013, with a success rate of 59% that was not significantly lower than that achieved with fresh tumor tissue. Objectives: The objective of this study was to optimize glioblastoma cryopreservation methods for viable cryobanking and to determine if two-dimensional (2D) or three-dimensional (3D) culture conditions were more supportive of glioblastoma growth after thawing of frozen tumor specimens. Conclusions: This study supports development of evidence-based viable glioblastoma cryopreservation methods for use in glioblastoma biobanking and research
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