Osmotic tolerance and freezability of isolated caprine early-staged follicles

Isolated caprine early-staged follicles were submitted to osmotic tolerance tests in the presence of sucrose, ethylene glycol (EG), or NaCl solutions and were exposed to and cryopreserved (by slow or rapid cooling) in MEM alone or MEM supplemented with sucrose, EG (1.0 or 4.0 M), or both. When follicles were exposed to 1.5 M NaCl, only 2% of the follicles were viable, whereas 87% of the follicles were viable after exposure to 4.0 M EG. Regarding exposure time, the highest percentage of viable follicles was obtained when follicles were exposed for 10 min to 1.0 M EG + 0.5 M sucrose; exposure for 60 s to 4.0 M EG + 0.5 M sucrose also maintained high percentage viability in follicles. Slow cooling in the presence of 1.0 M EG + 0.5 M sucrose (75%) or rapid cooling in the presence of 4.0 M EG + 0.5 M sucrose (71%) resulted in a significantly higher proportion of viable follicles than all other treatments (P < 0.05). A 24-h culture of frozen-thawed follicles was used to assess survival; only slow-frozen follicles showed viability rates similar to control follicles (64% vs. 69% respectively; P > 0.05). Interestingly, the percentage of viable rapid-cooled follicles (59%) was similar to that obtained after in vitro culture of conventional slow-cooled follicles but was significantly lower than that in controls. Thus, in addition to determining improved procedures for the exposure of follicles to EG and sucrose before and after freezing of caprine early-staged follicles, we report the development of rapid- and slow-cooling protocols

Modelling the Cryopreservation Process of a Suspension of Cells: The Effect of a Size-Distributed Cell Population

Cryopreservation of biological material is a crucial step of tissue engineering, but biological material can be damaged by the cryopreservation process itself. Depending on some bio-physical properties that change from cell to cell lineages, an optimum cryopreservation protocol needs to be identified for any cell type to maximise post-thaw cell viability. Since a prohibitively large set of operating conditions has to be determined to avoid the principal origins of cell damage (i.e., ice formation and solution injuries), mathematical modelling represents a valuable alternative to experimental optimisation. The theoretical analysis traditionally adopted for the cryopreservation of a cell suspension addresses only a single, average cell size and ascribes the experimental evidence of different ice formation temperatures to statistical variations. In this chapter our efforts to develop a novel mathematical model based on the population balance approach that comprehensively takes into account the size distribution of a cell population are reviewed. According to this novel approach, a sound explanation for the experimental evidence of different ice formation temperatures may now be given by adopting a fully deterministic criterion based on the size distribution of the cell population. In this regard, the proposed model represents a clear novelty for the cryopreservation field and provides an original perspective to interpret system behaviour as experimentally measured so far. First our efforts to successfully validate the proposed model by comparison with suitable experimental data taken from the literature are reported. Then, in absence of suitable experimental data, the model is used to theoretically investigate system behaviour at various operating conditions. This is done both in absence or presence of a cryo-protectant agent, as well as when the extra-cellular ice is assumed to form under thermodynamic equilibrium or its dynamics is taken into account consistently by means of an additional population balance. More specifically, the effect of the cell size distribution on system behaviour when varying cooling rate and cryo-protectant content within practicable values for a standard cryopreservation protocol is investigated. It is demonstrated that, cell survival due to intra-cellular ice formation depends on the initial cell size distribution and its osmotic parameters. At practicable operating conditions in terms of cooling rate and cryo-protectant concentration, intra-cellular ice formation may be lethal for the fraction of larger size classes of the cell population whilst it may not reach a dangerous level for the intermediate size class cells and it will not even take place for the smaller ones

Long-term preservation of Lotus tenuis adventitious buds

Encapsulation-dehydration, encapsulation-vitrification, and vitrification were tested for cryopreservation of Lotus tenuis (Fagaceae) adventitious buds clusters (ABCs) obtained by a direct regeneration system from leaves cultures. Among them, the PVS3-based vitrification procedure was found to be useful for survival and regrowth of the preserved explants. For vitrification, the ABCs were dehydrated in a solution containing 2 M glycerol + 0.4 M sucrose for 25 min at room temperature, submerged in PVS3 solution for 1 h at 0 °C, then immersed in liquid nitrogen for 48 h and rapidly rewarmed. Afterword, the explants were unloaded in MS liquid medium with 1.2 M sucrose for 30 min. The washed tissues were dried superficially on filter paper and cultured in semisolid hormone-free MS medium containing 0.1 M sucrose. All cultures were maintained at 25 °C in the dark for 10 days and transferred to the light conditions. With this procedure, 79 ± 5.3% survival and more than 80% of the plantlets displaying a phenotype similar to the non-treated control after acclimatization. The data settled from ISSR showed no genetic dissimilarities between in vitro regenerants derived from cryopreserved tissues and the non-treated plants. Thus, our results indicate that the use of vitrification-based PVS3 solution offers a simple, accurate, and appropriate procedure for the cryopreservation of L. tenuis adventitious buds.Instituto de Fisiología y Recursos Genéticos VegetalesFil: Espasandin, Fabiana Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; ArgentinaFil: Brugnoli, Elsa Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; ArgentinaFil: Ayala, Paula G. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; ArgentinaFil: Ayala, Lilian P. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; ArgentinaFil: Ruiz, Oscar Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales; ArgentinaFil: Sansberro, Pedro Alfonso. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentin