Abstract
Emerging technologies have enabled the isolation and characterization of rare circulating tumor cells (CTCs) from the blood of metastatic cancer patients. CTCs represent a non-invasive opportunity to gain information regarding the primary tumor and recent reports suggest CTCs have value as an indicator of disease status. CTCs are fragile and difficult to expand in vitro, so typically molecular characterization must be performed immediately following isolation. To ease experimental timelines and enable biobanking, cryopreservation methods are needed. However, extensive cellular heterogeneity and the rarity of CTCs complicates the optimization of cryopreservation methods based upon cell type, necessitating a standardized protocol. Here, we optimized a previously reported vitrification protocol to preserve patient-derived CTC cell lines using highly conductive silica microcapillaries to achieve ultra-fast cooling rates with low cryoprotectant concentrations. Using this vitrification protocol, five CTC cell lines were cooled to cryogenic temperatures. Thawed CTCs exhibited high cell viability and expanded under in vitro cell culture conditions. EpCAM biomarker expression was maintained for each CTC cell line. One CTC cell line was selected for molecular characterization, revealing that RNA integrity was maintained after storage. A qPCR panel showed no significant difference in thawed CTCs compared to fresh controls. The data presented here suggests vitrification may enable the standardization of cryopreservation methods for CTCs.
Highlights
Culture-adapted Circulating tumor cells (CTCs) cell lines obtained from women with metastatic breast cancer were selected.[16]
CTCs are a rich source of molecular information that may provide clinical information regarding the status of the primary tumor.[6,7,8,9,10]
This method was validated using five culture-adapted CTC cell lines obtained from metastatic breast cancer patients
Summary
Vitrification is a method of “ice-free” cryopreservation where high concentrations of CPAs (~4–8 M) are added to cells followed by rapid cooling through the glass transition temperature to achieve glass formation. As this high concentration of CPA is toxic to most cell types, protocols for loading and unloading CPAs must be carefully developed to avoid cell mortality.[19,20,21] Because both slow cooling and vitrification require optimization for each cell line, CTC heterogeneity is a significant challenge to the successful development of standardized cryopreservation protocols. Vitrification of circulating tumor cell lines evaluation of the BRx142 cell line by qPCR confirmed that common breast cancer biomarkers tested were unaffected by vitrification and RNA integrity was maintained
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