Cryopreservation of model organisms: a great tool for biogeochemistry research. *E. Paredes1, P. Mazur1 Fundamental and applied Cryobiology group. Biochemistry, cellular and Molecular Biology . University of Tennessee-Knoxville Principles of Cryobiology Vitrification: Vitrification is the transformation of a substance into a glass. Usually, it is achieved by rapidly cooling a liquid through the glass transition. It was believed that to achieve vitrification a high concentration of permeating solutes and a high cooling rate were needed. New paradigm of vitrification: In our lab we have demonstrated that one of the key issues of survival is not the high cooling rate but the high warming rate needed to prevent the recrystallization of ice during warming. In order to achieve warming rates fast enough to achieve high survivals we are using an IR laser pulse to achieve warming rates of 10,000,000 °C min-1. We found that survivals depended on the dehydration previous to vitrification rather than the permeation of solutes inside the cells, so vitrification can be achieved by the exposure of cells to non permeating solutes. Cryopreservation can be a very powerful bio-technique allowing preservation of cells/organisms in your own lab, at your disposal for future experiments, to preserve special genomes, for out of season stock that can allow you to work all year round with your desired test specie. There are two methodologies of cryopreservation, slow cooling and Vitrification. Slow cooling: Low concentration of cryoprotective solutes, the cell will dehydrate slowly as temperature decreases, the major factor determining if the cell survives is the cooling rate.. Once the cooling starts the external media will freeze before the cells and water inside will be under its thermodynamic freezing point (supercooled) with higher vapor pressure than the ice outside the cell . As long as this difference in potential remains, water will slowly leave the cell and freeze externally. Following this process the cell will slowly dehydrate avoiding the Internal Ice Formation (IIF). Cryo-Jig designed in our lab that operates inside the laser chamber. The laser is manufactured by LaserStar (it emits at 1064 nm in the infrared), and we use carbon black (India Ink) particles to absorb energy at that wave length and transfer the resulting heat to the surrounding solution, which in turn would transfer it to the oocyte or embryo . The plot shows survival at different cooling rates usually take the form of an Inverted U ( from Mazur 1980), with low survivals at very low and very high cooling rates, and high survivals at intermediate rates. That optimal cooling rate varies among cell types, as well as the sensitivity of the cells to different cryoprotective solutes. Species CPA Cooling rate Warming Rate % Survival D. melanogaster3 8.5M EG + 10% PVP ≥ 200°C min-1 100,000°Cmin-1 68% P. lividus4 1.5M Me2SO 1.5M + 0.04M TRE 1°C min-1 ~100°C min-1 50% C. gigas6 EG 15% (v/v) 1 – 0.5°C min-1 ~2000°C min-1 60% N. crassa1 - 500°C min-1 1000°C min-1 100% N. gaditana2 Me2SO 10% (v/v) 40-60% ICR Mice6 0.33X EAFS 69,000°C min-1 107 °C min-1 95% Classical models, NIH recommended models and emerging model organisms. The fact is that we need model organisms for our tests, to probe our hypothesis but obtaining and maintaining those organisms can be a logistic problem, a costly effort and time-consuming task.. 4 5 P. lividus C. gigas 6 ICR mice 1 N. crassa 2 N. gaditana D. melanogaster 3 Future research The fields of application of cryopreservation have extensively increased along the years, from the use in breeding industry (cattle, aquaculture), conservation either of endangered species, DNA or germaplasm cryobanking of organisms (repopulation, conservation and genetic variability studies), marine water quality assessment (embryo-larval bioassays, microalgae toxicity assays), medical applications (reproductive medicine, drug testing, organ/ tissues transplants) and research ( cryopreservation of test organisms). Our future objectives : To revisit some of the slow cooling protocols using vitrification with ultra-rapid warming with IR laser in order to increase survivals and reduce cryoinjuries. Apply vitrification to cryopreserve other cells which have very poor survivals due to chilling sensitivity or have very low tolerance to the toxicity of the permeating solutions used in slow cooling. Vitrify other model organisms that haven’t been vitrified so far. 1 J.L. Leef, P. Mazur. 1978 Physiological response of Neurospora conidia to freezing in dehydrated, hydrated and germinated state. Applied and environmental microbiology 35 v1, pp: 72-83. 2 Paredes, E., Costas, D., Martinez, A. 2012 Marine microalgae cryopreservation (Nannochloropsis gaditana, Rhodomonas lens, Cylindroteca closterium, Chaetoceros gracilis, Synechoccocus sp and Isocrysis aff.galbana clone T-iso ) with Me2SO using a passive freezer and a temperature controlled freezer. FIRMA symposium, Cadiz, Spain 2012 3 Schreuders, P., Mazur, P. 1994 Vitrification-based cryopreservation of Drosophila embryos. Advances in cryogenic engineering 39,pp: 2031-2038. 4Paredes, E. Bellas, J. 2014 Sea urchin (Paracentrotus lividus) cryopreserved embryos survival and growth: effects of cryopreservation parameters and reproductive seasonality. Cryoletters 35 (6), pp: 482-494 5Paredes, E. Bellas, J. Adams, S.L. 2012 Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and blue mussel (Mytilus galloprovinciallis). Cryobiology 65, pp: 256-262. 6Jin, B., Kleinhans, F.W., Mazur, P. 2014 Survival of mouse approach 100% after vitrification in 3-fold diluted media and ultra-rapid warming by an IR laser pulse. Cryobiology 68 (3), pp: 419-430