Sansiñena, M. ; Santos, M. V. ; Taminelli, G. ; Zaritky, N. Implications of glass transition in the devitrification process and storage management of vitrified oocytes and embryos Postprint del artículo publicado en Theriogenology, abril 2014 Facultad de Ciencias Agrarias Este documento está disponible en la Biblioteca Digital de la Universidad Católica Argentina, repositorio institucional desarrollado por la Biblioteca Central “San Benito Abad”. Su objetivo es difundir y preservar la producción intelectual de la Institución. La Biblioteca posee la autorización del autor para su divulgación en línea. Cómo citar el documento: Sansinena, M., Santos, M. V., Taminelli, G. y N. Zaritky. 2014. Implications of glass transition in the devitrification process and storage management of vitrified oocytes and embroyos [en línea]. Postprint del artículo publicado en Theriogenology: http//dx.doi.org/10.1016/j.theriogenology.2014.04.003 Disponible en: http://bibliotecadigital.uca.edu.ar/repositorio/investigacion/implications-glass-devitrification.pdf [Fecha de consulta: ….] 1 REVISED “CLEAN” COPY 2 3 4 5 Implications of glass transition in the devitrification process 6 and storage management of vitrified oocytes and embryos 7 8 9 M. Sansinena a,c,*, M.V. Santos b,c, G. Taminelli a and N. Zaritky b,c 10 11 12 13 aFacultad de Ciencias Agrarias, Pontificia Universidad Católica Argentina, 14 Cap. Gral. Ramón Freire 183, CABA 1426, Argentina. 15 16 bDepto. de Ingeniería Química, Facultad de Ingeniería, Universidad Nacional 17 de La Plata and Centro de Investigación y Desarrollo en Criotecnología de 18 Alimentos (CONICET-UNLP) , Calle 47 y 116, La Plata 1900, Argentina. 19 20 cConsejo Nacional de Investigaciones Científicas y Técnicas, CONICET. Av. 21 Rivadavia 1917, CABA 1033, Argentina. 22 23 Corresponding autor: [email protected] 24 25 1 26 ABSTRACT 27 Devitrification, the process of crystallization of a formerly crystal-free, 28 amorphous glass state, can lead to damage during the warming of cells. The 29 objective of this study was to determine the glass transition temperature of a 30 cryopreservation solution typically used in the vitrification, storage and 31 warming of mammalian oocytes and embryos using Differential Scanning 32 Calorimetry. A numerical model of the heat transfer process to analyze 33 warming and devitrification thresholds for a vitrification carrier (open-pulled 34 straw, OPS) was conducted and the implications on specimen storage in 35 nitrogen vapor phase were determined. The time required for initiation of 36 devitrification was determined by mathematical modeling and compared with 37 temperatures in the vapor phase of liquid nitrogen cryogenic dewars. Results 38 indicated that the glass transition ranged from -126 to -121ºC and 39 devitrification was initiated at -109ºC. Interestingly, samples entered rubbery 40 state at -121ºC and therefore could potentially initiate devitrification above this 41 value, with the consequent damaging effects to cell survival. Devitrification 42 times were mathematically modeled considering an initial temperature of 43 material immersed in liquid nitrogen (-196ºC) and two arbitrarily selected 44 temperatures (-50 and -70ºC) to which a sample could be exposed at the 45 neck of dewar. The mathematical model indicated samples could reach glass 46 transition temperatures and undergo devitrification in 30 seconds. Results of 47 the present study indicate storage of vitrified oocytes and embryos in the 48 liquid nitrogen vapor phase (as opposed to completely immersed in liquid 49 nitrogen) poses the potential risk of devitrification. Due to the reduced time- 50 handling period before samples reach critical rubbery and devitrification 51 values, caution should be exercised when handling samples in vapor phase. 52 53 Keywords: Vitrification, embryo, glass transition, devitrification, liquid nitrogen 54 55 56 57 58 59 60 61 62 63 64 65 2 66 1. INTRODUCTION 67 Low temperature preservation of oocytes and embryos is a fundamental 68 cornerstone of assisted reproductive technologies. Cryopreservation of 69 reproductive cells has been traditionally achieved by slow cooling the samples 70 at specific rates to allow cell dehydration [1,2]. However, the preservation 71 outcome of oocytes and embryos cells by slow freezing equilibrium protocols 72 is negatively affected by cryoinjury due to formation of intra and extracellular 73 ice crystals, concentration of solutes during the freezing process and 74 prolonged cell exposures to toxic cryoprotectant and chilling temperatures [2– 75 4]. 76 Storage of cryopreserved cells is conducted in specialized cryogenic, 77 thermally insulated vacuum flask dewars that hold cryogenic fluids below their 78 boiling point [5]. Smaller to medium-sized dewars (20 to 50 L) typically used 79 by veterinary practitioners and some laboratories are routinely filled with 80 cryogenic fluid to maintain adequate chamber temperatures [6]. Even though 81 the recommendation is to maintain the dewars full at all times [5, 7], 82 manufacturers provide guideline static evaporation rates for individual models 83 and suggest close monitoring of liquid nitrogen levels based on specific usage 84 conditions [7]. 85 Cells stored in these containers are kept either immersed in liquid 86 nitrogen or in the immediate vapor phase [7] . Because the temperature of the 87 vapor phase is not a constant (as opposed to liquid nitrogen, -196ºC) a lack 88 of temperature homogeneity within the chamber is observed [8]. Noteworthy, 89 storage recommendations for oocytes and embryos in vapor phase of liquid 90 nitrogen dewars were originally formulated for cells that had been 3 91 cryopreserved using equilibrium, slow freezing protocols [25,26]. Cells 92 cryopreserved under those conditions have been reported to undergo 93 sufficient dehydration and minimal cytoplasm supercooling and thus are less 94 likely to be damaged during warming. However, these recommendations may 95 not be applicable to vitrified material, which has a higher risk of devitrification 96 and irreversible cryoinjury. 97 Vitrification, the process of solidification of a sample into an 98 amorphous, glassy-state in absence of intracellular and extracellular ice 99 crystals, requires high concentrations of cryoprotectants, extremely rapid 100 cooling rates and reduced volume handling. In the last decade, vitrification 101 has progressively become the method of choice for the cryopreservation of 102 human oocytes and embryos [10, 11] and this trend is now being followed by 103 veterinary and animal science practitioners for domestic and exotic animal 104 species [7,8]. 105 Devitrification is defined as the process of crystallization in a formerly 106 crystal-free, amorphous glass solution [12, 14-16]. Early experiments to study 107 the warming behavior of vitrified aqueous solutions were conducted by Luyet 108 [14] and Luyet and Rasmusen using differential thermal analyses to detect 109 enthalpy changes associated with transition events [17,18]. Unlike melting 110 point, devitrification phenomenon has been described not as an individual 111 point but as a temperature range determined by the composition of solution 112 and presence of nucleating particles, among other factors [14, 15, 18-21]. 113 The devitrification of the intracellular solution and the surrounding 114 extracellular medium can lead to significant damage during the warming of 115 cells [20, 21]. Several authors have indicated that above the glass transition 4 116 temperature of the cytoplasm (approximately -120 to -130°C), the vitrified 117 cytoplasm of oocytes and embryos could enter a liquid transition, promoting 118 devitrification and subsequent ice nucleation and crystallization [22-24]. There 119 are limited reports on glass transition temperatures of cryopreservation 120 solutions and those available are mostly water-sugar solutions and not the 121 complex mixtures of balanced salt solutions, permeating and non-permeating 122 cryoprotectants used in current oocyte and embryo vitrification protocols [25– 123 27]. 124 To date, there are no reports on glass transition temperatures of 125 vitrification solutions used in the storage of oocytes and embryos. This 126 information would be of value to calculate critical devitrification thresholds and 127 update recommendations for the storage of vitrified oocytes and embryos. 128 Therefore, the objective of this study was to determine the glass transition 129 temperature of a cryopreservation solution typically used in the vitrification, 130 storage and warming of mammalian oocytes and embryos. In order to analyze 131 devitrification thresholds, a numerical modeling of heat transfer for a common 132 vitrification carrier (open-pulled straw, OPS) was conducted. Finally, the 133 implications of these results on specimen storage and handling conditions in 134 nitrogen vapor phase were discussed. 135 136 2. MATERIALS AND METHODS 137 2.1 Measurement of the glass transition temperature (Tg) of the 138 vitrification solution by differential scanning calorimetry (DSC). 139 Current vitrification protocols require that cells be successively moved 140 through increasing cryoprotectant concentrations (permeable and non- 5 141 permeable) prior to their vitrification by direct plunging into liquid nitrogen and 142 long-term storage. Therefore, the glass transition temperature (Tg) of the final 143 vitrification solution routinely used in our laboratory was determined by 144 differential scanning calorimetry (DSC). 145 The Tg