Preservation of Mouse Sperm by Convective Drying and Storing in 3-O-Methyl-D-Glucose

With the fast advancement in the genetics and bio-medical fields, the vast number of valuable transgenic and rare genetic mouse models need to be preserved. Preservation of mouse sperm by convective drying and subsequent storing at above freezing temperatures could dramatically reduce the cost and facilitate shipping. Mouse sperm were convectively dried under nitrogen gas in the Na-EGTA solution containing 100 mmol/L 3-O-methyl-D-glucose and stored in LiCl sorption jars (Relative Humidity, RH, 12%) at 4°C and 22°C for up to one year. The functionality of these sperm samples after storage was tested by intracytoplasmic injection into mouse oocytes. The percentages of blastocysts produced from sperm stored at 4°C for 1, 2, 3, 6, and 12 months were 62.6%, 53.4%, 39.6%, 33.3%, and 30.4%, respectively, while those stored at 22°C for 1, 2, and 3 months were 28.8%, 26.6%, and 12.2%, respectively. Transfer of 38 two- to four-cell embryos from sperm stored at 4°C for 1 year produced two live pups while 59 two- to four-cell embryos from sperm stored at 22°C for 3 months also produced two live pups. Although all the pups looked healthy at 3 weeks of age, normality of offspring produced using convectively dried sperm needs further investigation. The percentages of blastocyst from sperm stored in the higher relative humidity conditions of NaBr and MgCl2 jars and driest condition of P2O5 jars at 4°C and 22°C were all lower. A simple method of mouse sperm preservation is demonstrated. Three-O-methyl-D-glucose, a metabolically inactive derivative of glucose, offers significant protection for dried mouse sperm at above freezing temperatures without the need for poration of cell membrane

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

Biophysical Characteristics of Successful Oilseed Embryo Cryoprotection and Cryopreservation Using Vacuum Infiltration Vitrification: An Innovation in Plant Cell Preservation

Heterogeneity in morphology, physiology and cellular chemistry of plant tissues can compromise successful cryoprotection and cryopreservation. Cryoprotection is a function of exposure time × temperature × permeability for the chosen protectant and diffusion pathway length, as determined by specimen geometry, to provide sufficient dehydration whilst avoiding excessive chemical toxicity. We have developed an innovative method of vacuum infiltration vitrification (VIV) at 381 mm (15 in) Hg (50 kPa) that ensures the rapid (5 min), uniform permeation of Plant Vitrification Solution 2 (PVS2) cryoprotectant into plant embryos and their successful cryopreservation, as judged by regrowth in vitro. This method was validated on zygotic embryos/embryonic axes of three species (Carica papaya, Passiflora edulis and Laurus nobilis) up to 1.6 mg dry mass and 5.6 mm in length, with varying physiology (desiccation tolerances) and 80 °C variation in lipid thermal profiles, i.e., visco-elasticity properties, as determined by differential scanning calorimetry. Comparisons between the melting features of cryoprotected embryos and embryo regrowth indicated an optimal internal PVS2 concentration of about 60% of full strength. The physiological vigour of surviving embryos was directly related to the proportion of survivors. Compared with conventional vitrification, VIV-cryopreservation offered a ∼ 10-fold reduction in PVS2 exposure times, higher embryo viability and regrowth and greater effectiveness at two pre-treatment temperatures (0 °C and 25 °C). VIV-cryopreservation may form the basis of a generic, high throughput technology for the ex situ conservation of plant genetic resources, aiding food security and protection of species from diverse habitats and at risk of extinction