A Non-Parametric Method for Estimating Rates of Intracellular Ice Formation Due to Independent Competing Mechanisms

The probability of intracellular ice formation (IIF) has conventionally been analyzed by counting the cumulative number of IIF events observed in a cell population, and normalizing to the total cell count to estimate the cumulative IIF probability. However, this method is invalid when attempting to distinguish among multiple, independent IIF mechanisms, because of confounding effects due to competition for a finite pool of unfrozen cells. Therefore, an alternative approach for analyzing IIF data is proposed, based on treating IIF as a marked point process, in which the points represent IIF events and the marks represent different mechanisms of IIF. Using the new method, it is possible to quantify the kinetics associated with any IIF mechanism for which corresponding events can be marked (experimentally distinguished from competing IIF mechanisms). The proposed approach is non-parametric, making possible characterization of IIF mechanisms that have not yet been fully elucidated. The new analytical approach was compared to the conventional method of IIF analysis using data from a simulated experiment, demonstrating that the new method yielded superior estimates of the cumulative distribution function of IIF times when two competing mechanisms of IIF were active. The proposed algorithm was also applied to cryomicroscopic IIF observations in adherent endothelial cells, yielding rate estimates for two concurrent IIF processes. Furthermore, a proof is presented to demonstrate that when the proposed data analysis algorithm is applied to IIF data from a single mechanism of IIF, the results are equivalent to those obtained by the conventional method of analysis.

Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20–40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 μL of EFS10 (a mixture of 10% ethylene glycol, 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2,613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18,467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.

Molecular mechanisms of cell cryopreservation with polyampholytes studied by solid-state NMR

Polyampholytes are emerging macromolecular membrane non-penetrating cryoprotectants; however, the mechanism behind their cryopreservation remains unclear. Here, we investigated the mechanism using solid-state NMR spectroscopy. The polymer-chain dynamics and the water and ion mobilities in the presence of various membrane penetrating and non-penetrating cryoprotectants were monitored at low temperatures to mimic cryopreservation conditions. NMR experiments revealed that the water, Sodium-ion, and polymer-chain signals in a carboxylated poly-ʟ-lysine (COOH-PLL) solution broadened upon cooling, indicating increasingly restricted mobility and increased solution viscosity. Moreover, strong intermolecular interactions facilitated the COOH-PLL glass transition, trapping water and salt in the gaps of the reversible matrix, preventing intracellular ice formation and osmotic shock during freezing; this reduced cell stress is responsible for cryoprotection. This simple NMR technique enabled the correlation of the cryoprotective properties of polymers that operate through mechanisms different from those of current cryoprotectants, and will facilitate the future molecular design of cryoprotectants.

n-Octyl (Thio)glycosides as Potential Cryoprotectants: Glass Transition Behaviour, Membrane Permeability, and Ice Recrystallization Inhibition Studies

A series of eight n-octyl (thio)glycosides (1α, β–4α, β) with d-glucose or d-galactose-configured head groups and varying anomeric configuration were synthesized and evaluated for glass transition behaviour, membrane permeability, and ice recrystallization inhibition (IRI) activity. Of these, n-octyl β-d-glucopyranoside (2β) exhibited a high glass transition temperatures (Tg), both as a neat sample and 20 wt-% aqueous solution. Membrane permeability studies of this compound revealed cellular uptake to concentrations relevant to the inhibition of intracellular ice formation, thus presenting a promising lead candidate for further biophysical and cryopreservation studies. Compounds were also evaluated as ice recrystallization inhibitors; however, no detectable activity was observed for the newly tested compounds.

Scanning electron microscopic study on freezing behaviour of tissue cells in dormant bud of mulberry (Morus sp.)

The freezing behaviour studies of dormant buds, were examined, employing scanning electron microscopy (SEM) and light microscopy. The differences and effect of freezing behaviour on dormant buds were observed. The dormant bud primordia of several woody plant species avoid freezing injury by deep supercooling. By slow cooling (5°C/day) of dormant buds to –30°C, all living cells in bud tissues exhibited distinct shrinkage without intracellular ice formation detectable by SEM. However, the recrystallization experiment of these slowly cooled tissue cells, which was done by further freezing of slowly cooled buds with liquid nitrogen (LN) and then rewarming to –10°C, confirmed that some of the cells in the apical meristem, area in which cells had thin walls and in which no extracellular ice accumulated, lost freezable water with slow cooling to –30°C, indicating adaptation of these cells by deep super cooling. Water in plant tissues will not supercool unless heterogeneous ice nucleating substances are absent and the spread of ice from adjacent tissue can be prevented. Deep supercooling could not occur in dormant bud primordia if xylem vessels formed a continuous conduit connecting the dormant bud primordia with the remainder of the plant. If xylem continuity was established, ice could propagate via the vascular system and nucleate the water within the primordia. It is concluded that no extracellular ice crystals accumulated in such tissues containing deep supercooling cells with thin cell walls.