Jessica Preciado

Isochoric preservation: a novel characterization method.

Isochoric (constant volume) preservation is an alternative to traditional cryopreservation methods because it requires less cryoprotectant and is simple to operate. In order to validate that this method automatically minimizes the pressure for a given temperature, pressure and temperature data were collected from a specially designed pressure vessel. This vessel was then used to examine the effect of an isochoric environment on freezing point nucleation in an aqueous antifreeze protein solution, and to generate pressure-temperature phase diagrams for various cryoprotectant solutions. Our results show that the isochoric pressure vessel follows the pressure-temperature phase diagram of water, thereby minimizing the pressure for the given temperature. We also show that the nucleation temperature of the antifreeze protein in an isochoric vessel is lower than that of the isobaric method. Furthermore, the nucleation temperature decreased with increasing concentration in the isochoric vessel while the isobaric nucleation temperature showed no change. These results indicate that the isochoric environment imposes additional constraints on ice formation and warrants further study as these results may change when a different type of cryoprotectant is used. Finally, all of the cryoprotectant phase diagrams exhibited a similar pressure-temperature slope indicating that, regardless of the cryoprotectant used or the mechanism by which it suppresses freezing, isochoric freezing affects the molecules in the same manner. Together, all of these results indicate that the isochoric method of preservation is a valuable tool for characterizing the thermodynamic properties of cryoprotectants and has great potential as a cryopreservation method in the field of cryobiology.

Radiative Properties of Polar Bear Hair

The polar bear’s ability to survive in the harsh arctic night fascinates scientific and lay audiences alike, giving rise to anecdotal and semi-factual stories on the radiative properties of the bear’s fur which permeate the popular literature, television programs, and textbooks [1–5]. One of the most interesting radiative properties of polar bear fur is that it is invisible in the infrared region. Some theories have attempted to explain this by claiming that the outer temperature of the fur is the same as that of the environment. However, this explanation is unsatisfactory because surface radiation depends on both the surface temperature and the surface radiative properties [6].Copyright

Utilization of Directional Freezing for the Construction of Tissue Engineering Scaffolds

Although the field of tissue engineering has advanced significantly in the past decade, the inability to easily produce structured scaffolds continues to hinder its progress. We have proposed a method to create a porous scaffold utilizing directional freezing that is fast, reproducible and can be easily mass produced. Most importantly, this method creates long parallel channels within the scaffold. This should allow cells in the scaffold to grow more easily, and may aid scientists in predicting diffusion rates of nutrients and drugs throughout the scaffold. A cross-linked alginate gel was utilized in a directional freezing apparatus which incorporated a mold based on the horizontal Bridgeman design. The apparatus is designed to allow crystallization to occur in only one direction. The gel was frozen from 0°C to −40°C at a cooling rate of −18.3°C/minute. The samples were then freeze dried (leaving pores where ice dendrites had been), sectioned and viewed under a scanning electron microscope (SEM). Visual inspection revealed clear directionality present within the scaffolds. SEM photos also showed evenly spaced pores on the order of 100 μm present. A lesser magnification photo showed that the pores extended to become parallel channels producing a structured mesh that resembled an air filter. The directional freezing method is successful when used to create porous tissue engineering scaffolds, especially those with a low amount of tortuosity. By altering the cooling rate, it may be possible to create different pore distributions, thereby producing a method which can be utilized to create directional tissue engineering scaffolds quickly and effectively.Copyright

The effect of undissolved air on isochoric freezing.

This study evaluates the effect of undissolved air on isochoric freezing of aqueous solutions. Isochoric freezing is concerned with freezing in a constant volume thermodynamic system. A possible advantage of the process is that it substantially reduces the percentage of ice in the system at every subzero temperature, relative to atmospheric freezing. At the pressures generated by isochoric freezing, or high pressure isobaric freezing, air cannot be considered an incompressible substance and the presence of undissolved air substantially increases the amount of ice that forms at any subfreezing temperature. This effect is measurable at air volumes as low as 1%. Therefore eliminating the undissolved air, or any separate gaseous phase, from the system is essential for retaining the properties of isochoric freezing.

The effect of isochoric freezing on mammalian cells in an extracellular phosphate buffered solution

Isochoric (constant volume) freezing has been recently suggested as a new method for cell and organ preservation. As a first step in studying the effect of isochoric freezing on mammalian cells, Madin-Darby canine kidney epithelial cells (MDCK), were frozen in an isochoric system, in a simple extracellular phosphate buffered solution to -10 °C (96.5 MPa), - 15 °C (162 MPa) and -20 °C (205 MPa) for 60 and 120 min. Cell membrane integrity and cell metabolism were studied with a Live/Dead cell vitality assay and flow cytometry. We found that cell survival decreases with an increase in pressure (lower temperatures) and time of exposure. For example, 60% of cells survived 60 min at - 10 °C and only 18% survived 120 min at this temperature. Negligible survival was measured at - 20 °C. This study may serve as the baseline towards further research on techniques to optimize the effects of isochoric freezing on living biological matter.

Escherichia coli viability in an isochoric system at subfreezing temperatures

In comparison with isobaric (constant pressure) freezing, isochoric (constant volume) freezing reduces potential mechanical damage from ice crystals and exposes stored biological matter to a lower extracellular concentration, at the price of increased hydrostatic pressure. This study evaluates the effects of isochoric freezing to low temperatures and high pressures on Escherichia coli (E. coli) survival. The viability of E. coli was examined after freezing to final temperatures between -5 °C and -20 °C for periods from 0.5 h to 12 h, with recovery periods from 0 h to 24 h. Freezing for up to two hours to -10 °C and -15 °C had little effect on the percentage of viable E. coli, relative to the controls. However, after two hours of exposure at -20 °C, when left to recover for 24 h, a 75% reduction in survival is observed. Furthermore, after 12 h of isochoric freezing at -15 °C and -20 °C, E. coli population is reduced by 2.5 logs while freezing to these temperatures in conventional isobaric atmospheric conditions reduces population by only one log. This suggests that the combination of low temperature and high pressure experienced during isochoric freezing close to the triple point may be more detrimental to biological matter survival than the combination of elevated concentration, low temperature, and ice crystallization experienced during conventional freezing, and that this effect may be related to the time of exposure to these conditions.