Enhancement of post-thaw viability of cells in suspension via pulsed laser heating prior to immersion in liquid nitrogen

Abstract

In the present study, we compared the structure of ice formation in cryobiologically relevant solutions immersed in liquid nitrogen with solutions that were laser heated prior to immersion in a liquid-nitrogen bath. The results suggest that there is a distinct and observable difference in the type of ice formed. Furthermore, we also present data that suggest pulsed laser heating prior to immersion in liquid nitrogen enhances post-thaw survival of cells in suspension. The laser used in our experimental approach was a Q-switched Quanta-Ray DCR-3 laser (Nd:yttrium aluminum garnet laser) operating in the second harmonic at 532nm. The laser irradiance consisted of pulses with a pulse width of 5–7ns and a pulse energy stability of 3%. The pulse energy at a pulse repetition rate of 1Hz was about 360mJ and the peak power delivered was about 60MW. The samples were held in a copper- (sheet thickness of 0.5mm) molded rectangular box of 5mm thick, 25mm wide, and 15mm long. Preliminary experiments were carried out using adipose-tissue-derived adult stem cells. The pre- and postfreeze∕thaw viability of ADAS cells was assessed using membrane-excluded fluorescent dyes. ADAS cells enclosed in a copper box, laser irradiated, and exposed to liquid nitrogen had a viability of 69%±5%. Similarly, when the cells were exposed directly to liquid nitrogen (note that as before the cell suspension was enclosed and sealed in the copper box), the viability reduced significantly to 9%±10%. These results are very encouraging and demonstrate the feasibility of using lasers and liquid nitrogen to cryopreserve cells in suspension.