26. Invariance of the glass transition temperature of plant vitrification solutions with cooling rate

Abstract

Glass, the state of matter where molecular mobility is so reduced that most physicochemical processes are virtually detained (including ice formation), is basic for cryopreservation. The glass transition temperature ( T g ), a temperature range at which supercoooled liquid becomes glass, is characterized by a change in heat capacity ( C p ), evaluated at its inflection point. Vitrification in cryopreservation protocols is achieved, without sophisticated cooling equipment, simply plunging specimens into liquid nitrogen (LN) after a set of physicochemical treatments increasing cytoplasmatic microviscosity and enhancing tissue resistance to cold and dehydration. Quick cooling is required to achieve vitrification avoiding ice formation. Both T g and C p are generally considered dependent on cooling rate (e.g. Angell et al. 82 (1978) J. Phys. Chem., 2622; Debenedetti et al. 410 (2001) Nature 259). The present work endeavors to increase knowledge in this area characterizing the calorimetric glass transition of the most common plant vitrification solutions, under a wide range of cooling rates. The solutions studied were Plant Vitrification Solutions 1, 2 and 3: (Uragami et al. 8 (1989) Plant Cell Rep. 418; Sakai et al. 9 (1990) Plant Cell Rep. 30; Nishizawa et al. 91 (1993) Plant Sci. 67). Cooling was performed using the calorimeter control (5, 10 and 20 °C min −1 ), or for higher rates, by quenching closed pans with PVS in LN, either naked (− 5580 °C min −1 ) or inside cryovials (360 °C min −1 ). Quenched pans were then transferred to the pre-cooled sample chamber. Glass transition temperature was observed by DSC with a TA 2920 instrument, upon warming pans with solution samples from −145 °C to room temperature, at standard warming rate: 10 °C min −1 . Glass transitions showed clear and consistent temperature differences among vitrification solutions, related to composition and water content. Roughly, two sets of T g values were obtained, for PVS1 and 2, at −112 °C and −114 °C, respectively, and for PSV3, at −90 °C. The observed T g did not significantly change within a wide range of cooling rates (from 5 to 20 °C min −1 ). The highest cooling rate (5580 °C min −1 ) increased glass transition temperature significantly, compared to the values at the slowest cooling rates (5–20°C min −1 ). This change in T g inflexion (by 1.2 °C min −1 ) did not influence considerably the glass transition region because the whole transition interval was, on average, 7 °C. However, no significant differences were found between T g obtained with the highest cooling rate and that with the middle cooling rate (360 °C min −1 ). In conclusion, T g of plant vitrification solutions did not significantly change when the cryopreservation methods based on either direct plunging samples into liquid nitrogen or plunging of samples in closed cryovials were used. We can conclude that the T g of commonly used PVSs did not change with the cooling rates tested.