Biobanks around the world store many different types of biological samples. Circulating tumor cells (CTCs) are commonly stored in biobanks as these cells can be used to diagnose cancer in patients and to monitor disease progression. CTCs break off from primary cancerous tumors and enter the blood stream or lymphatic fluid where they can travel to distant tissues and develop into metastatic tumors1. As these cells come from the primary tumor, they contain important genetic and proteomic information about the cancer. This information can be accessed through liquid biopsies, where CTCs are isolated from patient blood samples and used in diagnostic tests. Researchers are also studying CTCs in the hope of finding new targets for cancer therapeutics. Biobanks are a central part of that process.
CTCs are rare. There are only between 1 and 3 CTCs in each milliliter of blood. Furthermore, CTCs are a very heterogeneous cell population. These two factors make these cells very difficult to immortalize into cell lines. Moreover, CTCs, like all biological samples, quickly degrade if left at room temperature. Therefore, to provide useful data, CTCs must be either processed and analyzed immediately after collection or stored at low temperatures in biobanks for future study. Cryogenic storage at temperatures below the glass transition temperature of water (around -135°C) can prevent degradative biochemical reactions and maintain the integrity of biological samples such as CTCs.
Before biobanking CTCs, or other biological samples at cryogenic temperatures, those samples must be cooled to at least -40°C. Ice crystals can form inside cells during the cooling process, damaging cells and potentially causing cell death. Two main methods are used to control the formation of intracellular ice crystals: controlled-rate freezing and vitrification, or flash-freezing. Cryoprotectant solutions such as DMSO can also help prevent the formation of ice crystals. Faster freezing rates require higher concentrations of cryoprotectants to protect cells. Therefore, vitrification requires much higher levels of cryoprotectants than slow-rate freezing. While cryoprotectants help minimize cell damage due to freezing, they can be toxic to cells and adversely affect cell function and viability post-thawing.
Protocols for biobanking CTCs are yet to be standardized across the industry.In a recent open-access publication, researchers from Harvard Medical School report an optimized vitrification protocol to preserve patient-derived CTC cell lines2. This work adapts a previously published ultra-fast vitrification protocol3. In the optimized method, researchers loaded CTCs into very narrow, fused silica microcapillaries and immediately lowered the microcapillaries into liquid nitrogen. They estimated the cooling rate of this method to be around 4000K/second. The authorsfound they could use much lower levels of cryoprotectants with this method than previously used in vitrification methods (1-2M versus 4-8M in standard vitrification protocols).
The researchers then tested their ultra-fast vitrification methods on five different CTC cell lines derived from metastatic breast cancer patients. All five cell lines showed high post-thaw viability (55-86%) and normal cell growth rates after thawing. Post-thawing, the CTCs also had normal expression of EpCAM, a cell surface marker commonly used to identify CTCs. Moreover, all vitrified and thawed CTC cell lines maintained their RNA integrity and showed similar mRNA levels of cancer biomarkers such as EpCAM, Her2 and EGFR to untreated control cells.
The authors believe that this method of ultra-fast vitrification could offer a standardized protocol to freezing CTCs within biobanking facilities.
- Williams. Circulating tumor cells. PNAS. 2013
- Sandlin et al. Ultra-fast vitrification of patient-derived circulating tumor cell lines. PLoS One. 2018
- Heo et al. “Universal” vitrification of cells by ultra-fast cooling. Technology (Singap World Sci). 2015.