Abstract 4894: Universal vitrification of circulating tumor cells (CTCs) by ultra-fast cooling
Authors: Heo Yun Seok , Nagrath Sunitha , Moore Alessandra L. , Malinowska Izabela , Zenali Mahnaz , Kwiatkowski David , Toner Mehmet 
Published: June 2012

Abstract
Circulating tumor cell (CTC) banking at various stages of disease may allow for opportunities to test the biology of tumors, biomarker analysis, and drug efficacy during the course of therapy. Building a depository of CTCs is, however, not an easy task. CTCs are very rare and precious cell source, and thus require a preservation protocol with high yield of recovery. Though high yield cryopreservation protocols have been optimized for particular cell types, none have been developed which have the capability of cryoprotecting multiple tumor geneologies and pathologies. Indeed, it is a tedious task to optimize individual protocols for each cell type of interest. As a result, there is no “universal” protocol for the cryoprotection of CTCs from different patients and stages of cancer. Here, we propose a unified preservation protocol that is capable of storing CTCs, using a low non-toxic concentration of cryoprotectants (1.5M Propane-1,2-diol + 0.5M trehalose), with both high recovery and viability, through minimizing the thermal mass of the freezing vessel.
The precisely machined microcapillary made of highly conductive fused silica results in an ice-free, vitrified solution which minimizes cellular damage caused by crystallization during freezing and thawing. Five cancer cell lines were used as CTC models for testing the efficacy of the optimized condition. The results suggested the cell lines were highly viable (&gt; 90%) after freezing and thawing: PC3 (prostate cancer, 92.60±4.96%, N=6), T24 (bladder cancer, 90.03±4.27%, N=7), SKBR-3 (Breast cancer, 90.04±3.56%, N=7), H1650 (human non-small-cell lung cancer, 90.84±3.48%, N=6), HepG2 (liver cancer, 90.10±65%, N=4). The overall yield (percentage of actual recovered cells over the estimated loaded cells into the freezing vessel) was 108.6±29.5% (N=26), indicating that most of the cells were secured during the preservation procedures. Growth rates for each cell line were also measured every 24hr for up to five days and showed no evidence of damage. Also, there were no significant differences in cytokeratin expression over the culture periods.
The freezing protocol was further evaluated by investigating disseminated cancer cells in pleural effusions from patients with lymphangioleiomyomatosis. The viability of cells from pleural effusion was 84.23±7.00% (N=10) of the viability of the control. Also, the growth rates (N = 5) and cytokeratin expression over the course of five days are comparable. The normal growth rates and cytokeratin expression, along with the high viability, prove the optimized protocol universally is effective not only for cancer cell lines, but also for clinically collected cancer cells.
Thus, we expect this new, universal cryopreservation technology might enable investigators to efficiently store CTCs for later analysis as well as create banked repositories of CTCs.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4894. doi:10.1158/1538-7445.AM2011-4894