Optimization of Viable Glioblastoma Cryopreservation for Establishment of Primary Tumor Cell Cultures

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.

Assessment of the Impact of Post-Thaw Stress Pathway Modulation on Cell Recovery following Cryopreservation in a Hematopoietic Progenitor Cell Model

“…The development and use of complex cell-based products in clinical and discovery science continues to grow at an unprecedented pace. While playing a critical enabling role, cryopreservation also presents numerous challenges to the process [ 42 , 49 , 54 , 56 , 79 ]. To this end, there is no greater issue than the extensive cell death following cryopreservation, which results in compromised cell product quality.…”

“…Over the last decade, numerous studies have investigated the impact of CIDOCD on various cell systems, as well as the benefit of controlling the activation of this molecular response through the use of specialized intracellular-type cryopreservation media [ 41 , 43 , 44 , 46 , 47 , 48 , 55 , 56 , 57 , 92 , 93 ]. Furthermore, studies have also demonstrated that the incorporation of molecular pathway modulators such as caspase, ROCK, calpain inhibitors, among others, into cryopreservation media can also increase overall viability in select systems [ 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ].…”

“…Functionally, through their multi component formulation, intracellular-like solutions buffer the environment a cell is exposed to during the freeze−thaw process, modulating stress response activation, thereby reducing CIDOCD [ 23 , 25 , 41 , 42 , 50 ]. Numerous reports have detailed the benefits of using intracellular-like solutions in comparison to traditional extracellular-like cell culture media with DMSO [ 22 , 46 , 47 , 51 , 52 , 53 , 54 , 55 , 56 ]. Today, 100’s of studies have demonstrated >50% improvement in cell recovery and function through the use of intracellular-like solutions in an equal number of unique cell and tissue systems, ranging from stem cells to CAR-T cells to engineered tissues.…”

“…While the integration of cellular−molecular control and the introduction of intracellular-like cryopreservation solutions has had a transformative impact providing for significant increases in the recovery, repopulation, function, and structural integrity of cells and tissues (native and engineered) post-thaw, several challenges remain to enable an even greater impact. These include (1) continued cell loss (ranging from 10 to >50% depending on the cell type), (2) compromised function in complex cells and tissues, (3) spontaneous differentiation in native stem cell populations, (4) dependence on DMSO, and (5) lack of/limited utility of equipment used in the freezing and thawing processes [ 1 , 2 , 41 , 54 , 55 , 56 , 57 , 58 ]. Furthermore, recognition of the critical role cryopreservation plays in the success of the field of regenerative medicine remains a hurdle.…”

Effect of cryopreservation on A172 and U251 glioma cells infected with lentiviral vectors designed for CRISPR/Cas9-mediated aquaporin-8 knock-out

“…The clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR/ Cas9) gene editing technology, which is widely used, is a convenient and efficient strategy for precisely and site-specifically modifying specific gene sequences of interest [1][2][3]. Additionally, cryopreservation is a means by which cells can be temporarily stored at low temperatures to prevent sustained growth, and reportedly, various types of cells transfected with specific plasmids or infected with specific viruses can be cryopreserved for later use [4]. The initial objective of this study was to infect glioma cell lines with AQP8 knock out so as to explore the effect of AQP8 on their proliferation.…”