Jens O.M. Karlsson

Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide.

A three-part, coupled model of cell dehydration, nucleation, and crystal growth was used to study intracellular ice formation (IIF) in cultured hepatocytes frozen in the presence of dimethyl sulfoxide (DMSO). Heterogeneous nucleation temperatures were predicted as a function of DMSO concentration and were in good agreement with experimental data. Simulated freezing protocols correctly predicted and explained experimentally observed effects of cooling rate, warming rate, and storage temperature on hepatocyte function. For cells cooled to -40 degrees C, no IIF occurred for cooling rates less than 10 degrees C/min. IIF did occur at faster cooling rates, and the predicted volume of intracellular ice increased with increasing cooling rate. Cells cooled at 5 degrees C/min to -80 degrees C were shown to undergo nucleation at -46.8 degrees C, with the consequence that storage temperatures above this value resulted in high viability independent of warming rate, whereas colder storage temperatures resulted in cell injury for slow warming rates. Cell damage correlated positively with predicted intracellular ice volume, and an upper limit for the critical ice content was estimated to be 3.7% of the isotonic water content. The power of the model was limited by difficulties in estimating the cytosol viscosity and membrane permeability as functions of DMSO concentration at low temperatures.

A MODEL OF DIFFUSION-LIMITED ICE GROWTH INSIDE BIOLOGICAL CELLS DURING FREEZING

A theoretical model for predicting the kinetics of ice crystallization inside cells during cryopreservation was developed, and applied to mouse oocytes, by coupling separate models of (1) water transport across the cell membrane, (2) ice nucleation, and (3) crystal growth. The instantaneous cell volume and cytosol composition during continuous cooling in the presence of glycerol were predicted using the water transport model. Classical nucleation theory was used to predict ice nucleation rates, and a nonisothermal diffusion‐limited crystal‐growth model was used to compute the resulting crystallization kinetics. The model requires knowledge of the nucleation rate parameters Ω and κ, as well as the viscosity η of a water‐NaCl‐glycerol solution as a function of both the composition and temperature of the solution. These dependences were estimated from data available in the literature. Cell‐specific biophysical parameters were obtained from previous studies on mouse oocytes. A sensitivity analysis showed that...

Permeability of the rhesus monkey oocyte membrane to water and common cryoprotectants

Successful cryopreservation of oocytes of the rhesus monkey (Macaca mulatta) would facilitate the use of this valuable animal model in research on reproduction and development, while providing a stepping stone towards human oocyte cryopreservation and the conservation of endangered primate species. To enable rational design of cryopreservation techniques for rhesus monkey oocytes, we have determined their osmotic and permeability characteristics in the presence of dimethylsulfoxide (DMSO), ethylene glycol (EG), and propylene glycol (PROH), three widely used cryoprotectants. Using nonlinear regression to fit a membrane transport model to measurements of dynamic cell volume changes, we estimated the hydraulic conductivity (Lp) and cryoprotectant permeability (Ps) of mature and immature oocytes at 23.5°C. Mature oocyte membranes were most permeable to PROH (Ps = 0.56 ± 0.05 µm/sec) and least permeable to DMSO (Ps = 0.24 ± 0.02 µm/sec); the permeability to EG was 0.34 ± 0.07 µm/sec. In the absence of penetrating cryoprotectants, mature oocytes had Lp = 0.55 ± 0.05 µm/min/atm, whereas the hydraulic conductivity increased to 1.01 ± 0.10, 0.61 ± 0.07, or 0.86 ± 0.06 µm/min/atm when mature oocytes were exposed to DMSO, EG, or PROH, respectively. The osmotically inactive volume (Vb) in mature oocytes was 19.7 ± 2.4% of the isotonic cell volume. The only statistically significant difference between mature and immature oocytes was a larger hydraulic conductivity in immature oocytes that were exposed to DMSO. The biophysical parameters measured in this study were used to demonstrate the design of cryoprotectant loading and dilution protocols by computer‐aided optimization. Mol. Reprod. Dev. 76: 321–333, 2009.

Effects of solution composition on the theoretical prediction of ice nucleation kinetics and thermodynamics

Predictions by various mathematical models of intracellular ice formation (proposed by Mazur, Pitt, Toner, and Karlsson, respectively) were compared to the known thermodynamic and kinetic behavior of ice formation in supercooled aqueous systems. The older models (Mazur, Pitt, and Toner) significantly underestimated the magnitude of colligative nonequilibrium freezing point depression in response to increased concentration of solutes, such as salts or cryoprotectants. Furthermore, kinetics predicted using phenomenological models (by Mazur and Pitt) exhibited implausible temperature-dependence, with the probability of intracellular ice formation being allowed to increase even at temperatures below the glass transition point. The Toner model, on the other hand, produced invalid results at temperatures below -48 degrees C. The Karlsson model was the only model that consistently yielded realistic predictions over a wide range of temperatures and solute concentrations, especially in the presence of cryoprotectant additives. To facilitate adoption of the Karlsson model of intracellular ice nucleation, the complete set of model equations has been collected and described in detail.

Optimization of cryoprotectant loading into murine and human oocytes

Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethyl sulfoxide (Me(2)SO) into mouse oocytes at 23°C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me(2)SO exposure time, revealing that neither shrinkage nor Me(2)SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me(2)SO addition appears to result from interactions between the effects of Me(2)SO toxicity and osmotic stress. We also investigated Me(2)SO loading into mouse oocytes at 30°C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me(2)SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me(2)SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach.

Effects of Intercellular Junction Protein Expression on Intracellular Ice Formation in Mouse Insulinoma Cells

The development of cryopreservation procedures for tissues has proven to be difficult in part because cells within tissue are more susceptible to intracellular ice formation (IIF) than are isolated cells. In particular, previous studies suggest that cell-cell interactions increase the likelihood of IIF by enabling propagation of ice between neighboring cells, a process thought to be mediated by gap junction channels. In this study, we investigated the effects of cell-cell interactions on IIF using three genetically modified strains of the mouse insulinoma cell line MIN6, each of which expressed key intercellular junction proteins (connexin-36, E-cadherin, and occludin) at different levels. High-speed video cryomicroscopy was used to visualize the freezing process in pairs of adherent cells, revealing that the initial IIF event in a given cell pair was correlated with a hitherto unrecognized precursor phenomenon: penetration of extracellular ice into paracellular spaces at the cell-cell interface. Such paracellular ice penetration occurred in the majority of cell pairs observed, and typically preceded and colocalized with the IIF initiation events. Paracellular ice penetration was generally not observed at temperatures >-5.65°C, which is consistent with a penetration mechanism via defects in tight-junction barriers at the cell-cell interface. Although the maximum temperature of paracellular penetration was similar for all four cell strains, genetically modified cells exhibited a significantly higher frequency of ice penetration and a higher mean IIF temperature than did wild-type cells. A four-state Markov chain model was used to quantify the rate constants of the paracellular ice penetration process, the penetration-associated IIF initiation process, and the intercellular ice propagation process. In the initial stages of freezing (>-15°C), junction protein expression appeared to only have a modest effect on the kinetics of propagative IIF, and even cell strains lacking the gap junction protein connexin-36 exhibited nonnegligible ice propagation rates.

Curve fitting approach for measurement of cellular osmotic properties by the electrical sensing zone method. II. Membrane water permeability.

In a companion paper, we demonstrated that dynamic range limitations can confound measurement of the osmotically inactive volume using electrical sensing zone instruments (e.g., Coulter counters), and presented an improved parameter estimation method in which a lognormal function was fit to the cell volume distribution to allow extrapolation beyond the bounds of the data. Presently, we have investigated the effect of dynamic range limitations on measurement of the cell membrane water permeability (L(p)), and adapted the lognormal extrapolation method for estimation of L(p) from transient volume data. An alternative strategy (the volume limit adjustment method, in which the measured isotonic volume distribution is used to generate model predictions for curve fitting, and the bounds of the dynamic range are adjusted such that extrapolation is not required) was also developed. The performance of these new algorithms was compared to that of a conventional parameter estimation method. The best-fit L(p) values from in vitro experiments with mouse insulinoma (MIN6) cells differed significantly for the different parameter estimation techniques (p<0.001). Using in silico experiments, the volume limit adjustment method was shown to be the most accurate (relative error 0.4+/-3.2%), whereas the conventional method underestimated L(p) by 19+/-2% for MIN6 cells. Parametric analysis revealed that the error associated with the conventional method was sensitive to the dynamic range and the width of the volume distribution. Our initial implementation of the lognormal extrapolation method also yielded significant errors, whereas accuracy of this algorithm improved after including a normalization scheme.

Measurement of Intracellular Ice Formation Kinetics by High-Speed Video Cryomicroscopy

Quantitative information about the kinetics and cumulative probability of intracellular ice formation is necessary to develop minimally damaging freezing procedures for the cryopreservation of cells and tissue. Conventional cryomicroscopic assays, which rely on indirect evidence of intracellular freezing (e.g., opacity changes in the cell cytoplasm), can yield significant errors in the estimated kinetics. In contrast, the formation and growth of intracellular ice crystals can be accurately detected using temporally resolved imaging methods (i.e., video recording at sub-millisecond resolution). Here, detailed methods for the setup and operation of a high-speed video cryomicroscope system are described, including protocols for imaging of intracellular ice crystallization events, and stochastic analysis of the ice formation kinetics in a cell population. Recommendations are provided for temperature profile design, sample preparation, and configuration of the video acquisition parameters. Throughout this chapter, the protocols incorporate best practices that have been drawn from over a decade of experience with high-speed video cryomicroscopy in our laboratory.

Effect of intercellular junction protein expression on water transport during freezing of MIN6 cells.

A mouse insulinoma (MIN6) strain in which connexin expression has been inhibited by antisense technology holds promise as an experimental model system for investigating the role of gap junctions in intercellular ice propagation. However, to properly interpret measurements of intracellular ice formation kinetics, the effects of cell dehydration on cytoplasmic supercooling must be determined. Thus, the cell membrane water permeability in monolayer cultures of the antisense-transfected MIN6 strain was measured using a fluorescence quenching method. By repeating the experiments at 4°C, 12°C, 21°C, and 37°C, the activation energy for water transport was determined to be E(a) = 51 ± 3 k J/mol. Although differences between membrane permeability measurements in theantisense and wild-type strains were not statistically significant, simulation of water transport during rapid freezing (130°C/min) predicted that intracellular supercooling in the genetically modified MIN6 strain may become significantly larger than the supercooling in wild-type cells at temperatures below -15°C.

Effect of water content on the glass transition temperature of mixtures of sugars, polymers, and penetrating cryoprotectants in physiological buffer

Long-term storage of viable mammalian cells is important for applications ranging from in vitro fertilization to cell therapy. Cryopreservation is currently the most common approach, but storage in liquid nitrogen is relatively costly and the requirement for low temperatures during shipping is inconvenient. Desiccation is an alternative strategy with the potential to enable viable cell preservation at more convenient storage temperatures without the need for liquid nitrogen. To achieve stability during storage in the dried state it is necessary to remove enough water that the remaining matrix forms a non-crystalline glassy solid. Thus, the glass transition temperature is a key parameter for design of cell desiccation procedures. In this study, we have investigated the effects of moisture content on the glass transition temperature (Tg) of mixtures of sugars (trehalose or raffinose), polymers (polyvinylpyrrolidone or Ficoll), penetrating cryoprotectants (ethylene glycol, propylene glycol, or dimethyl sulfoxide), and phosphate buffered saline (PBS) solutes. Aqueous solutions were dried to different moisture contents by equilibration with saturated salt solutions, or by baking at 95°C. The glass transition temperatures of the dehydrated samples were then measured by differential scanning calorimetry. As expected, Tg increased with decreasing moisture content. For example, in a desiccation medium containing 0.1 M trehalose in PBS, Tg ranged from about 360 K for a completely dry sample to about 220 K at a water mass fraction of 0.4. Addition of polymers to the solutions increased Tg, while addition of penetrating cryoprotectants decreased Tg. Our results provide insight into the relationship between relative humidity, moisture content and glass transition temperature for cell desiccation solutions containing sugars, polymers and penetrating cryoprotectants.