Osmotic transport across cell membranes in nondilute solutions: a new nondilute solute transport equation.

The fundamental physical mechanisms of water and solute transport across cell membranes have long been studied in the field of cell membrane biophysics. Cryobiology is a discipline that requires an understanding of osmotic transport across cell membranes under nondilute solution conditions, yet many of the currently-used transport formalisms make limiting dilute solution assumptions. While dilute solution assumptions are often appropriate under physiological conditions, they are rarely appropriate in cryobiology. The first objective of this article is to review commonly-used transport equations, and the explicit and implicit assumptions made when using the two-parameter and the Kedem-Katchalsky formalisms. The second objective of this article is to describe a set of transport equations that do not make the previous dilute solution or near-equilibrium assumptions. Specifically, a new nondilute solute transport equation is presented. Such nondilute equations are applicable to many fields including cryobiology where dilute solution conditions are not often met. An illustrative example is provided. Utilizing suitable transport equations that fit for two permeability coefficients, fits were as good as with the previous three-parameter model (which includes the reflection coefficient, sigma). There is less unexpected concentration dependence with the nondilute transport equations, suggesting that some of the unexpected concentration dependence of permeability is due to the use of inappropriate transport equations.

Further optimization of mouse spermatozoa evaporative drying techniques.

It has been shown in the past that mouse spermatozoa could be dried under a stream of nitrogen gas at ambient temperature and stored at 4 degrees C or 22 degrees C for up to 3 months and was capable of generating live-born offspring. In previous desiccation work, dried sperm were stored in a vacuum-sealed plastic bag placed in a vacuum-packed Mylar bag. However, dried specimens stored in this way often lost moisture, particularly in samples stored at higher temperatures (22 degrees C) compared to lower temperatures (4 degrees C). The present report describes a method which minimizes this water loss from the dried sperm samples. Its use is described in a preliminary study on the effect of supplementing the trehalose with glycerol. The results have demonstrated that mouse sperm can be stored at 4 degrees C over saturated NaBr without the uptake of water which occurs when they are stored in Mylar packages. In addition, we were able to get some survival of sperm (9-15%) at room temperature storage after 3 months. The addition of glycerol to trehalose had little effect on the survival of dried mouse sperm stored over NaBr for 1 and 3 months.

Long-term storage of mouse spermatozoa after evaporative drying.

To determine if mouse spermatozoa could be preserved long-term without using liquid nitrogen, mouse spermatozoa in trehalose-EGTA solution were partially evaporatively dried under nitrogen gas (5 min at flow rate10 l/min) and stored for 1 week and 5 months at 4, -20, and -80 degrees C before intracytoplasmic sperm injection. Fertilization rates were neither different with spermatozoa stored at 4, -20, or -80 degrees C for 1 week or 1, 3, and 5 months respectively, nor blastocyst formation rates with spermatozoa stored for 1 week and 1 month. However, spermatozoa stored at 4 and -20 degrees C for 3 months resulted in fewer blastocysts (35.1 and 54.3% respectively) when compared with spermatozoa stored at -80 degrees C (74.4%). Blastocyst formation rates using spermatozoa stored for 5 months at -20 degrees C (57.4%) or -80 degrees C (74.5%) were not significantly different from those stored for 3 months at the same temperatures respectively, but were significantly better than those stored for 5 months at 4 degrees C (10.2%). Blastocysts derived from spermatozoa stored for 3 and 5 months at -20 and -80 degrees C respectively, were then transferred to pseudopregnant mothers to develop into healthy liveborn offspring. No significant differences were found in embryo transfer rates (number of pups born/number of embryos transferred), weaning rates, or sex ratios of resultant pups, which were healthy and reproductively sound. These results demonstrate for the first time that partially evaporatively dried mouse spermatozoa in trehalose-EGTA solution can be preserved for long term at -20 and -80 degrees C. The possibility that the storage temperature must be less than the glass transition temperature is discussed.

Dimethyl sulfoxide toxicity kinetics in intact articular cartilage.

Osteochondral defects can degenerate into osteoarthritis and currently there are no good treatment alternatives available to most Orthopaedic surgeons. Osteochondral allografting can restore damaged joint surfaces but its clinical use is limited by poor access to high quality tissue. Vitrification of osteochondral tissue would allow the banking of this tissue but requires high concentrations of cryoprotective agents. This study was designed to ascertain dimethyl sulfoxide (DMSO) toxicity kinetics to chondrocytes in situ after exposure to DMSO at different temperatures recorded as a function of time. Porcine osteochondral dowels were exposed to 1, 3, 5, and 6M DMSO at 4, 22, and 37 degrees C for 0.5 min to 120 min. Chondrocyte recovery was determined by membrane integrity (Syto 13 and ethidium bromide) and mitochondrial (WST-1) assays. Results demonstrated that cell recovery was concentration, temperature and time dependent. At higher concentrations and temperatures, significant cell loss occurred within minutes. A rate constant calculated for chondrocyte death was dependent on temperature. 1 M DMSO appeared relatively non-toxic. This experiment established a method to examine systematically toxicity parameters for chondrocytes in situ and this data can be used to tailor vitrification protocols by limiting exposure temperature and time or lowering DMSO concentrations below toxic levels recorded.