Tissue interactions with lasers and liquid nitrogen: a novel cryopreservation method

The importance of the imposed cooling rate in cryopreserving native cells and tissues has been long recognized in the field of cryobiology. When biological tissues are subjected to cooling rates in excess of thousands of degree C per minute, the characteristic structural and physical manifestations of the ice formed are such that the traditional damage due to ice formation at lower cooling rates are suppressed. Hence, achieving high cooling rates in tissues and cells of biologically relevant sizes (mm’s and cm’s) has been a long standing research problem. In the present study, we present a novel technique to achieve high cooling rates (in the order of 8,000 to 10,000 °C/min) in large tissue sections by coupling pulsed laser heating and immediate exposure to cryogenic temperatures (liquid nitrogen vapor at -164 °C). Thermal gradient that exists between the laser heated tissue (at ~1000’s °C) and liquid nitrogen surrounding the tissue results in very high cooling rates, as opposed to the cooling rates experienced by the tissue without laser heating (which is in the order of a few hundreds of degree C per minute). Furthermore it is expected that the small time scales of energy deposition (6-7 ns) and localized heating due to laser focusing would lead to minimal thermal damage. To illustrate this idea we have developed a 1-D and 2-D numerical model to predict cooling rates experienced in a finite tissue section exposed to liquid nitrogen temperatures with and without laser heating. Based on the numerical results preliminary experiments were carried out in a variety of cryobiologically relevant solutions and using adipose tissue derived adult stem cells. Experimental results indicate the possibility of attaining better survival when cells were cryopreserved using the suggested protocol. The limitations and advantages of the technique are also assessed