Sankha Bhowmick

Mouse Sperm Desiccated and Stored in Trehalose Medium Without Freezing

Abstract Mouse sperm with and without trehalose were desiccated under nitrogen gas and stored at 4°C and 22°C. After rehydration, sperm were injected into oocytes using intracytoplasmic sperm injection and embryonic development was followed. Sperm were dried for 5.0, 6.25, or 7.5 min, stored at 22°C for 1 wk with and without trehalose. The percentages of blastocysts that developed from sperm with trehalose were 51%, 31%, and 20%, respectively, which was significantly higher than sperm without trehalose (10%, 3%, and 5%, respectively). Desiccation and storage in medium with trehalose significantly increased sperm developmental potential compared to medium without trehalose. Sperm dried for 5 min produced more blastocysts than sperm dried for 6.25 or 7.5 min. When sperm were dried in trehalose for 5 min and stored for 1 wk, 2 wk, 1 mo, or 3 mo at 4°C, the percentages of blastocysts were 73%, 84%, 63%, and 39%; whereas those stored at 22°C for 1 wk, 2 wk, or 1 mo were significantly lower (53%, 17%, and 6%, respectively). Embryos from sperm partially desiccated in trehalose for 5 min and stored at 4°C for 1 or 3 mo were transferred to 10 pseudopregnant recipients. Implantation rates were 81% and 48%; live fetuses were 26% and 5%, respectively. One of the recipients delivered three live fetuses. The results show that trehalose has a significant beneficial effect in preserving the developmental potential of mouse sperm following partial desiccation and storage at temperatures above freezing.

Desiccation Tolerance of Spermatozoa Dried at Ambient Temperature: Production of Fetal Mice

Abstract Long-term preservation of mouse sperm by desiccation is economically and logistically attractive. The current investigation is a feasibility study of the preservation of mouse sperm by convective drying in an inert gas (nitrogen). Mouse sperm from the B6D2F1 strain isolated in an EGTA-supplemented Tris-HCl buffer were dried using three different drying rates and were stored for 18–24 h at 4°C. The mean final moisture content was <5% for all the protocols. After intracytoplasmic sperm injection (ICSI), the mean blastocyst formation rates were 64%, 58%, and 35% using the rapid-, moderate-, and slow-drying protocols, respectively. The slow-drying protocol resulted in a rate of development significantly lower than that observed using rapid- and moderate-drying protocols and indicated that a slower drying rate may be detrimental to the DNA integrity of mouse sperm. The transfer of 85 two- or four-cell embryos that were produced using rapidly desiccated sperm resulted in 11 fetuses (13%) on Day 15 compared with the production of 34 fetuses (40%) produced using the transfer of 86 two- or four-cell embryos that were produced using fresh sperm (P < 0.05). The results demonstrate the feasibility of using a convective drying protocol for the successful desiccation of mouse sperm and identifies some of the important parameters required for optimization of the procedure.

Supraphysiological Thermal Injury in Dunning AT-1 Prostate Tumor Cells

To investigate the potential application of thermal therapy in the treatment of prostate cancer, the effects of supraphysiological temperatures (40-70 degrees C) for clinically relevant time periods (approximately 15 minutes) were experimentally studied on attached Dunning AT-1 rat prostate cancer cells using multiple assays. The membrane and reproductive machinery were the targets of injury selected for this study. In order to assess membrane injury, the leakage of calcein was measured dynamically, and the uptake of PI was measured postheating (1-3 hours). Clonogenicity was used as a measure of injury to the reproductive machinery 7 days post-injury after comparable thermal insults. Experimental results from all three assays show a broad trend of increasing injury with an increase in temperature and time of insult. Membrane injury, as measured by the fluorescent dye assays, does not correlate with clonogenic survival for many of the thermal histories investigated. In particular, the calcein assay at temperatures of < or = 40 degrees C led to measurable injury accumulation (dye leakage), which was considered sublethal, as shown by significant survival for comparable insult in the clonogenic assay. Additionally, the PI uptake assay used to measure injury post-thermal insult shows that membrane injury continues to accumulate after thermal insult at temperatures > or = 50 degrees C and may not always correlate with clonogenicity at hyperthermic temperatures such as 45 degrees C. Last, although the clonogenic assay yields the most accurate cell survival data, it is difficult to acquire these data at temperatures > or = 50 degrees C because the thermal transients in the experimental setup are significant as compared to the time scale of the experiment. To improve prediction and understanding of thermal injury in this prostate cancer cell line, a first-order rate process model of injury accumulation (the Arrhenius model) was fit to the experimental results. The activation energy (E) obtained using the Arrhenius model for an injury criterion of 30 percent for all three assays revealed that the mechanism of thermal injury measured is likely different for each of the three assays: clonogenics (526.39 kJ/mole), PI (244.8 kJ/mole), and calcein (81.33 kJ/mole). Moreover, the sensitivity of the rate of injury accumulation (d omega/dt) to temperature was highest for the clonogenic assay, lowest for calcein leakage, and intermediate for PI uptake, indicating the strong influence of E value on d omega/dt. Since the clonogenic assay is linked to the ultimate survival of the cell and accounts for all lethal mechanisms of cellular injury, the E and A values obtained from clonogenic study are the best values to apply to predict thermal injury in cells. For higher temperatures (> or = 50 degrees C) indicative of thermal therapies, the results of PI uptake can be used as a conservative estimate of cell death (underprediction). This is useful until better experimental protocols are available to account for thermal transients at high temperature to assess clonogenic ability. These results provide further insights into the mechanisms of thermal injury in single cell systems and may be useful for designing optimal protocols for clinical thermal therapy.

Harvesting energy from asphalt pavements and reducing the heat island effect

A rise in temperature of asphalt pavements contributes towards the urban heat island effect, causes problems with air quality and increases the power requirement for cooling buildings. A high temperature would also lead to the potential of rutting failure in asphalt pavements. The concept of mining heat from asphalt pavements, utilising an appropriate fluid flowing in pipes installed within the pavement, has been proposed. Theoretical considerations and results of laboratory testing and modelling simulation have been presented. The results indicate that the concept is feasible, and that the efficiency of heat mining can be improved by selecting appropriate surface layer and aggregates for pavement materials. The use of this proposed method would lead to a significant reduction in pavement and near-surface air temperature, and extension of asphalt pavement life.

In Vitro Assessment of the Efficacy of Thermal Therapy in Human Benign Prostatic Hyperplasia

The successful management of BPH with minimally invasive thermal therapies requires a firm understanding of the temperature–time relationship for tissue destruction. In order to accomplish this objective, the present in vitro study assesses the cellular viability of human BPH tissue subjected to an experimental matrix of different temperature–time combinations. Hyperplastic prostate tissue was obtained from 10 radical prostatectomy specimens resected for adenocarcinoma. A portion of hyperplastic tissue from the lateral lobe of each prostate was sectioned into multiple 1 mm thick tissue strips, placed on a coverslip and thermally treated on a controlled temperature copper block with various temperatures (45–70°C) for various times (1–60 min). After heat treatment, the tissue slices were cultured for 72 h and viability was assessed using two independent assays: histology and dye uptake for stromal tissue and using histology alone for the glandular tissue. The hyperplastic human prostate tissue showed a progressive histological increase in irreversible injury with increasing temperature–time severity. The dye uptake and histology results for stromal viability were similar for all temperature–time combinations. In vitro thermal injury showed 85–90% stromal destruction (raw data) of human BPH for temperature–time combinations of 45°C for 60 min, 50°C for 30 min, 55°C for 5 min, 60°C for 2 min and 70°C for 1 min. Apoptosis was also identified in the control and milder treated tissues with the degree of glandular apoptosis (about 20%) more than that seen in the stromal regions (<5%). The Arrhenius model of injury was fitted to the data for conditions leading to a 90% drop in viability (normalized to control) obtained for stromal tissue. The activation energies (E) were 40.1 and 38.4 kcal/mole for the dye uptake study and histology, respectively, and the corresponding frequency factors (A) were 1.1 × 1024 and 7.78 × 1022/s. This study presents the first temperature–time versus tissue destruction relation for human BPH tissue. Moreover, it supports the concept that higher temperatures can be used for shorter durations to induce tissue injury comparable with the current clinically recommended lower temperature–longer time treatments (i.e. 45°C for 60 min) for transurethral microwave thermotherapy of the prostate.

In vitro thermal therapy of AT-1 Dunning prostate tumours

To advance the utility of prostate thermal therapy, this study investigated the thermal thresholds (temperature-time) for prostate tissue destructionin vitro. The AT-1 Dunning prostate tumour model was chosen for the study. Three hundred micron thick sections were subjected to controlled temperature-time heating, which ranged from low (40°C, 15 min) to high thermal exposures (70°C, 2 min) (n = 6). After subsequent tissue culture at 37°C, the sections were evaluated for tissue injury at 3, 24 and 72 h by two independent methods: histology and dye uptake. A graded increase in injury was identified between the low and high thermal exposures. Maximum histologic injury occurred above 70°C, 1 min with >95% of the tissue area undergoing significant cell injury and coagulative necrosis. The control and 40°C, 15 min sections showed histologic evidence of apoptosis following 24 and 72 h in culture. Similar signs of apoptosis were minimal or absent at higher thermal histories. Vital-dye uptake quantitatively confirmed complete cell death after 70°C, 2 min. Using the dye data, Arrhenius analysis showed an apparent breakpoint at 50°C, with activation energies of 135.8 kcal/mole below and 4.7 kcal/mole above the threshold after 3 h in culture. These results can be used as a conservative benchmark for thermal injury in the cancerous prostate. Further characterization of the response to thermal therapy in an animal model and in human tissues will be important in establishing the efficacy of the procedure

Thermal Therapy in Urologic Systems: A Comparison of Arrhenius and Thermal Isoeffective Dose Models in Predicting Hyperthermic Injury

The Arrhenius and thermal isoeffective dose (TID) models are the two most commonly used models for predicting hyperthermic injury. The TID model is essentially derived from the Arrhenius model, but due to a variety of assumptions and simplifications now leads to different predictions, particularly at temperatures higher than 50 degrees C. In the present study, the two models are compared and their appropriateness tested for predicting hyperthermic injury in both the traditional hyperthermia (usually, 43-50 degrees C) and thermal surgery (or thermal therapy/thermal ablation, usually, >50 degrees C) regime. The kinetic parameters of thermal injury in both models were obtained from the literature (or literature data), tabulated, and analyzed for various prostate and kidney systems. It was found that the kinetic parameters vary widely, and were particularly dependent on the cell or tissue type, injury assay used, and the time when the injury assessment was performed. In order to compare the capability of the two models for thermal injury prediction, thermal thresholds for complete killing (i.e., 99% cell or tissue injury) were predicted using the models in two important urologic systems, viz., the benign prostatic hyperplasia tissue and the normal porcine kidney tissue. The predictions of the two models matched well at temperatures below 50 degrees C. At higher temperatures, however, the thermal thresholds predicted using the TID model with a constant R value of 0.5, the value commonly used in the traditional hyperthermia literature, are much lower than those predicted using the Arrhenius model. This suggests that traditional use of the TID model (i.e., R=0.5) is inappropriate for predicting hyperthermic injury in the thermal surgery regime (>50 degrees C). Finally, the time-temperature relationships for complete killing (i.e., 99% injury) were calculated and analyzed using the Arrhenius model for the various prostate and kidney systems.

The glass transition temperature of mixtures of trehalose and hydroxyethyl starch

Although mixtures of HES and sugars are used to preserve cells during freezing or drying, little is known about the glass transition of HES, or how mixtures of HES and sugars vitrify. These difficulties may be due to the polydispersity between HES samples or differences in preparation techniques, as well as problems in measuring the glass transition temperature (T(g)) using differential scanning calorimetry (DSC). In this report, we examine the T(g) of mixtures of HES and trehalose sugar with <1% moisture content using DSC measurements. By extrapolating these measurements to pure HES using the Gordon-Taylor and Fox equations, we were able to estimate the T(g) of our HES sample at 44 degrees C. These results were additionally confirmed by using mixtures of glucose-HES which yielded a similar extrapolated T(g) value. Our approach to estimating the glass transition temperature of HES may be useful in other cases where glass transitions are not easily identified.

OPTIMIZATION OF ELECTROSPINNING PROCESS PARAMETERS FOR TISSUE ENGINEERING SCAFFOLDS

The goal of the current study was to optimize important process parameters for electrospinning polycaprolactone (PCL) for growing 3T3 fibroblasts. We hypothesized that the smallest obtainable fiber diameter would provide the best cell growth kinetics and we tested this hypothesis for three different process parameters: solution concentration, voltage and collector screen distance. Beaded structures were formed when using low concentration electrospinning solutions (8 wt% to 13 wt%), in which the viscosity ranged from 16.0 cP to 340.0 cP. In this concentration range, cell growth kinetics was impeded when using a high concentration of cells (8–10 × 105). Higher PCL concentration led to an increase in the average fiber diameter from 400 nm to 1600 nm when PCL solution concentration changed from 15 wt% to 20 wt%. Although, the mean values indicated that cell growth kinetics were higher at the lower end of the concentration (15% as opposed to 20%) and this correlated with lower average fiber diameter, the results in this range were not statistically significant (p > 0.05). The average fiber diameter of scaffolds first decreased and then increased when electrospinning voltage was increased. The cell growth kinetics demonstrated that smaller average diameter PCL fiber scaffolds had higher growth kinetics than larger average diameter scaffolds with the best conditions obtained at 15 KV. By increasing the screen distance, the average fiber diameter decreased but had no significant impact on cell growth kinetics. In summary, the optimal parametric space for 3T3 fibroblast growth for our studies was electrospinning a 15 wt% PCL solution using 15 kV voltage and a 25 cm collector distance.

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.