Ultra-Rapid Laser Calorimetry for the Assessment of Crystallization in Low-Concentration Cryoprotectants

Cryoprotective agents (CPAs) are routinely used to vitrify, attain an amorphous glass state void of crystallization, and thereby cryopreserve biomaterials. Two vital characteristics of a CPA loaded system are the critical cooling and warming rates (CCR and CWR), the temperature rates needed to achieve and return from a vitrified state respectively. Due to the toxicity associated with CPAs, it is often desirable to use the lowest concentrations possible, driving up CWR and making it increasingly difficult to measure. This paper describes a novel method for assessing CWR between the 0.4×105-107 °C/min in microliter CPA loaded droplet systems with a new ultra-rapid laser calorimetric approach. Cooling was achieved by direct quenching in liquid nitrogen, while warming was achieved by the irradiation of plasmonic gold nanoparticle-loaded vitrified droplets by a high-power 1064 nm millisecond pulsed laser. We assume "apparent" vitrification is achieved provided ice is not visually apparent (i.e. opacity) upon imaging with a camera during cooling or highspeed camera during warming. Using this approach, we were able to investigate CWR in single CPA systems such as glycerol, PG, and Trehalose in water, and mixtures of glycerol-trehalose-water and propylene glycol-trehalose-water CPA at low concentrations (20-40 wt%). Further, an phenomenological model for determining the CCRs and CWRs of CPA was developed which allowed for predictions of CCR or CWR of single component CPA and mixtures, providing an avenue for optimizing CCR and CWR and perhaps future CPA cocktail discovery.