A09 Session 2: Biopreservation of biologics: Translation from stem cells to cancer : Cryobiological biophysics and freeze–thaw viability of the HL-1 cardiac muscle cell line: Implications for cryoablation studies

Abstract

Atrial fibrillation (AF) is the irregular beating of the heart affecting an estimated 2.7 million Americans and responsible for 26 billion dollars of the nation’s annual healthcare cost. Cryoablation is a clinically approved technique for the treatment of paroxysmal AF in which an ablation catheter rapidly cools the cardiac tissue. Through freezing, discrete lesions are created in the region where the pulmonary veins join with the atria in order to terminate the conduction of abnormal electrical signals responsible for AF. While the efficacy of AF cryoablation is well known, the thermal thresholds and mechanisms of cryoinjury that determine efficacy are not known thereby hindering development of the next generation of AF cryoablation systems. This study begins to address this by improving understanding of the mechanisms and thermal thresholds of cryodestruction in a cardiac muscle cell line (HL-1). Specifically, we determine biophysical parameters governing known mechanisms of injury (membrane permeability and likelihood of intracellular ice formation, IIF). Additionally we correlate these biophysics and thermal history (cooling rate and end = temperature) to post-thaw viability in vitro in HL-1s guide optimal lesion formation and thus cryoablation device design. HL-1 cells were cultured using previously published methods. Cells were harvested using trypsinization and kept as a suspension on ice until needed. A temperature stage was used in conjunction with a light microscope to observe freezing of the samples. The controlled thermal history included 3 variables including: cooling rate (0.5 to 130 °C/min), end temperature (−5 to −60 °C), and hold time (0–5 min). These were varied in an experimental matrix to determine the importance of each. Before cooling, samples were pre-nucleated by applying a chilled needle on the outer edge of the sample. Change in water content within cells (cellular dehydration) was measured based on observed changes to projected cell area during freezing. IIF events were measured based on observing a sudden change in opacity (darkening) of the cell interior. Measured data were applied to water transport and nucleation models to determine membrane permeability and IIF nucleation parameters. Additionally, post-thaw viability of samples were determined after a rapid thaw and 15 min incubation period using a fluorescence membrane dye exclusion assay. For cases when the end temperature was set as −20 °C the highest viability of around 15% was observed at a cooling rate of 5 °C/min, while slower or faster cooling rates resulted in reduced viability. Subsequent measurements with cooling rate set as 5 °C/min and varied end temperatures resulted in viability values around 94%, 72%, 15%, 6% and less than 5% at −5 °C, −10 °C, −20 °C, −40 °C, and −60 °C, respectively. Our results suggest a large difference in post-thaw cell viability dependent on cooling rate and end temperature, while difference in hold times (0, 1, and 5 min) appeared less important. The application of this work to pulmonary vein freezing numerical models, and to future work in more translational systems such as attached (monolayer, artificial constructs) cell systems and tissues will be discussed.