Heidi Elmoazzen

Ultra-rapid vitrification of mouse oocytes in low cryoprotectant concentrations

The ideal cryopreservation protocol would combine the benefits of slow freezing with the benefits of vitrification. This report describes a method for the ultra-rapid vitrification of oocytes using slush nitrogen in quartz capillaries. The approach minimizes the thermal mass of the vitrification vessel by using open microcapillaries made of highly conductive quartz and achieves cooling rates of 250,000 degrees C/min. The process of vitrification can be optimized by maximizing the rate at which the sample is cooled, which allows for the use of lower cryoprotectant concentrations. Mouse oocytes can be successfully vitrified using 1.5 mol/l 1,2-propanediol and 0.5 mol/l trehalose and achieve survival rates of 90.0%(36/40). Fertilization and blastocyst formation rates of vitrified-warmed and fresh oocytes were not significantly different. A total of 120 blastocysts from each of the vitrified-warmed and fresh oocytes were transferred to surrogate mothers and 23 and 27 offspring were born respectively. All offspring in both groups were healthy, grew and bred normally and gave rise to a second generation of pups. Thus, an ultra-rapid vitrification technique has been developed for mouse oocytes that uses low concentrations of cryoprotectants and slush nitrogen in quartz capillaries, which combines the benefits of slow freezing and vitrification.

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.

Controlled loading of cryoprotectants (CPAs) to oocyte with linear and complex CPA profiles on a microfluidic platform

Oocyte cryopreservation has become an essential tool in the treatment of infertility by preserving oocytes for women undergoing chemotherapy. However, despite recent advances, pregnancy rates from all cryopreserved oocytes remain low. The inevitable use of the cryoprotectants (CPAs) during preservation affects the viability of the preserved oocytes and pregnancy rates either through CPA toxicity or osmotic injury. Current protocols attempt to reduce CPA toxicity by minimizing CPA concentrations, or by minimizing the volume changes via the step-wise addition of CPAs to the cells. Although the step-wise addition decreases osmotic shock to oocytes, it unfortunately increases toxic injuries due to the long exposure times to CPAs. To address limitations of current protocols and to rationally design protocols that minimize the exposure to CPAs, we developed a microfluidic device for the quantitative measurements of oocyte volume during various CPA loading protocols. We spatially secured a single oocyte on the microfluidic device, created precisely controlled continuous CPA profiles (step-wise, linear and complex) for the addition of CPAs to the oocyte and measured the oocyte volumetric response to each profile. With both linear and complex profiles, we were able to load 1.5 M propanediol to oocytes in less than 15 min and with a volumetric change of less than 10%. Thus, we believe this single oocyte analysis technology will eventually help future advances in assisted reproductive technologies and fertility preservation.

Desiccation tolerance in bovine sperm: a study of the effect of intracellular sugars and the supplemental roles of an antioxidant and a chelator.

Desiccation preservation holds promise as a simplified alternative to cryopreservation for the long term storage of cells. We report a study on the protective effects of intracellular and extracellular sugars during bovine sperm desiccation and the supplemental effects of the addition of an antioxidant (catalase) or a chelator (desferal). The goal of the study was to preserve mammalian sperm in a partially or completely desiccated state. Sperm loaded intracellularly with two different types of sugars, trehalose or sucrose, were dried with and without catalase and desferal and evaluated for motility and membrane integrity immediately after rehydration. Intracellular sugars were loaded using ATP induced poration. Drying was performed in desiccator boxes maintained at 11% relative humidity (RH). Results indicated that sperm exhibited improved desiccation tolerance if they were loaded with either intracellular trehalose or sucrose. Survival was further enhanced by the addition of 1mM desferal to the desiccation buffer. Though sperm motility after drying to low dry basis water fractions (DBWF) did not show significant improvement under any of the tested conditions, there was an increase in the sperm membrane integrity that could be retained after partial desiccation through the use of intracellular sugars and desferal.

Trehalose Transporter from African Chironomid Larvae Improves Desiccation Tolerance of Chinese Hamster Ovary Cells

Dry preservation has been explored as an energy-efficient alternative to cryopreservation, but the high sensitivity of mammalian cells to desiccation stress has been one of the major hurdles in storing cells in the desiccated state. An important strategy to reduce desiccation sensitivity involves use of the disaccharide trehalose. Trehalose is known to improve desiccation tolerance in mammalian cells when present on both sides of the cell membrane. Because trehalose is membrane impermeant the development of desiccation strategies involving this promising sugar is hindered. We explored the potential of using a high-capacity trehalose transporter (TRET1) from the African chironomid Polypedilum vanderplanki[21] to introduce trehalose into the cytoplasm of mammalian cells and thereby increase desiccation tolerance. When Chinese hamster ovary cells (CHO) were stably transfected with TRET1 (CHO-TRET1 cells) and incubated with 0.4M trehalose for 4h at 37°C, a sevenfold increase in trehalose uptake was observed compared to the wild-type CHO cells. Following trehalose loading, desiccation tolerance was investigated by evaporative drying of cells at 14% relative humidity. After desiccation to 2.60g of water per gram dry weight, a 170% increase in viability and a 400% increase in growth (after 7days) was observed for CHO-TRET1 relative to control CHO cells. Our results demonstrate the beneficial effect of intracellular trehalose for imparting tolerance to partial desiccation.

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.

Choline Chloride Improves the Desiccation Tolerance of Chinese Hamster Ovary Cells

Recently there has been much interest in using sugars such as trehalose to preserve mammalian cells in a dry state as an alternative to cryopreservation (1–5). However, some studies indicate that sugars alone may not be sufficient to prevent cell injury during drying. Other factors like sodium toxicity, ionic imbalance and pH excursions during dehydration are a few of the mechanisms that have been hypothesized to decrease the viability of mammalian cells. In the present study, we investigated whether or not substituting sodium chloride with choline chloride (2-hydroxy-N, N,N-trimethylethanaminium chloride) in the preservation medium improves desiccation tolerance of Chinese Hamster Ovary (CHO) cells.© 2010 ASME