117. Cryopreservation of vanilla (Vanilla planifolia A.) apices. A comparison of vitrification and droplet-vitrification procedures

Abstracts / Cryobiology 57 (2008) 315–340 tion is a critical requirement that underlines the need for efficient utilization of all suitable available pancreata. Optimizing pancreas preservation is a key factor in the success of islet isolation and transplantation. Considering that we have already proved the feasibility of hypothermic machine perfusion of large animal pancreases, this study was focused on optimization of pancreas perfusate through the selection and testing of a panel of cytoprotective agents known to enhance tissue tolerance to cold ischemia. High throughput screening was performed using two beta-cell lines, hamster (HIT-T15) and mouse (bTC3). The cells were stored, using three baseline solutions (Unisol-I, Unisol-E, and Belzer’s machine perfusion solution, BMPS), at 4 °C for up to 72 h in the presence of various cytoprotective agents of different doses. Antioxidants, anti-apoptotic agents and trophic factors, both individually and in combination were used. Post storage the cells were monitored for metabolic status and cell death during 7 days of recovery. Results: Both cell lines showed approximately 25–50% metabolic activity after 24 h of cold ischemia, longer time points demonstrated minimal if any cell viability. Further experimentation using 24 h storage and employing cytoprotective agents demonstrated statistically significant (P < 0.05) benefits of the Q-VD-OPH, a-tocopherol and trolox agents, including both immediate cell survival and re-growth upon return to physiological culture conditions. It is anticipated that combinations of one or more of these cytoprotective agents will provide improved organ preservation as additives to the intracellular-type baseline solutions (Unisol-I or BMPS). (Conflicts of interest: None declared. Source of funding: Funded in part by NIH grant 5R44DK076326-03.)

339

and the survival rate was about 80% in the regrowth rate basis. We investigated whether M. polymorpha cultured cells with preculture treatment were vitrified under desiccated conditions by differential scanning calorimetry (DSC). At 0.07 gH2O/gDW, the precultured cells showed a glass transition at about 50 °C on a DSC chart, and the cultured cells without preculture treatment showed no glass transition. We confirmed that the accumulation of a large amount of sucrose and de novo synthesis of several boiling-stable proteins occurred in the cultured cells during preculture. This cellular composition is similar to that of some anhydrobiotes. We also confirmed that a very high level of desiccation tolerance was maintained in protoplasts isolated from precultured M. polymorpha cells. Therefore, we concluded that cultured cells of M. polymorpha are useful material for studying cellular mechanisms of desiccation tolerance and vitrification. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.117

117. Cryopreservation of vanilla (Vanilla planifolia A.) apices. A comparison of vitrification and droplet-vitrification procedures. Maria T. Gonzalez Arnao a, Claudia Lazaro Vallejo a, Roberto Gamez Pastrana a, Yolanda Martinez Ocampo a, Miriam Pastelin Solano a, Florent Engelmann b, a Universidad Veracruzana, Orizaba, Mexico, b IRD, Montpellier, France

doi:10.1016/j.cryobiol.2008.10.115

115. Fast online algorithm to monitor tissue freezing during cryoablation of prostate. Vadim L. Stakhursky, Thomas J. Polascik, Janice M. Mayes, Nathan Richards, Vladimir Mouraviev, Duke University Medical Center, Durham, NC, USA Cryoablation of prostate is a low morbidity option in the treatment of localized prostate cancer. However, a parallel insertion and orientation of the cryoprobes is rarely achievable. In this study the algorithm for cryodose analysis was based on the real intraoperative room prostate geometry that was built, based on 3D ultrasound imaging of the target volume. The prostate volume was segmented, and accurate 3D orientation and position of the cryoneedles, thermosensors and urethra were reconstructed, based on US greyscale images. The time-dependent equations for temperature distribution were then solved in the volume of treatment target (prostate, SV) using finite element software (ePhysics, Ansoft Co). At first, Pennes’ bio-heat framework with blood perfusion was used to calculate time-dependent temperature distribution due to all inserted cryo-needles. Alternatively, the temperature distribution in the prostate volume was evaluated as due to the closest needle neighboring a given point (method 2), and a simple function of temperature due to individual needles (method 3). The platform was evaluated using data obtained from the analysis of salvage cryosurgery performed on 3 patients, with cryo-dose volume histograms (CDVH) compared for all 3 computational methods. Method 2 showed reasonable accuracy with a tendency to underestimate freezing volume. The target isotherm ( 20 °C) coverage was at 64% of the volume, compared to 81% in the full bioheat model. However our functional method (#3) allowed approximation of the prostate dose coverage with only 2.8% error. We conclude that the proposed thermal dose computational schemes have good time performance, as the solution of Pennes’ equations can be parameterized and tabulated for the highly symmetrical data used in Methods 2–3. With its apparent accuracy, method 3 is a great candidate for fast on-board evaluation of prostate cryo treatment. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.116

Poster presentations 3. Plant systems

116. Development of desiccation tolerance and vitrification in cultured cells of Marchantia polymorpha. Rie Hatanaka, Yasutake Sugawara, Saitama University, Saitama, Japan Plant cells and tissues have the ability to induce tolerance to various stresses such as freezing and desiccation when cultured under controlled conditions. The extent of desiccation tolerance in M. polymorpha cultured cells is low. The water content of cultured cells was approximately 9.0 gH2O/gDW and decreased rapidly to less than 0.1 gH2O/gDW after desiccation on silica gel for 6 hours. The survival rate of cultured cells after desiccation for 6 hours was less than 10%. After desiccation for 24 hours, the cultured cells, which had a water content of 0.03 gH2O/ gDW, were not able to grow on the agar plate. For the development of desiccation tolerance, the cultured cells were precultured on a medium with 0.3 M sucrose for 12 h and then transferred to a medium containing 0.5 M sucrose and cultured for 1 day. Water content of these precultured cells after desiccation for 24 h was 0.07 gH2O/ gDW. The desiccated cells with preculture treatment actively grew on the agar plate

Apices isolated from in vitro plantlets of Vanilla planifolia Andrews were subjected to cryopreservation using vitrification and droplet-vitrification approaches. Apices were precultured on solid MS medium supplemented with 0.3 M sucrose for 1, 7 or 10 days, then loaded in different solutions, including 2 M glycerol with 0.4 M sucrose, 0.4 M trehalose or both sugars at 0.2 M. After loading, tissues were exposed to the vitrification solutions PVS3 or PVS2 for 0, 30 or 60 minutes at room temperature, and then frozen directly in liquid nitrogen either within cryovials containing 1 ml PVS solution or in droplets of the same solution placed on aluminium foil strips. With the vitrification procedure, samples were rewarmed by immersing the cryovials in a water-bath at +40 °C for 1–2 minutes, while with the droplet-vitrification method, the foil strips were immersed in liquid culture medium supplemented with 1.2 M sucrose at room temperature. Survival (30%) and further regeneration (10%) of new multiple shoots were achieved only using droplet-vitrification, with the following experimental conditions: 1 day pregrowth of apices on solid medium with 0.3 M sucrose, loading with 0.4 M sucrose + 2 M glycerol solution for 20–30 minutes and exposure to PVS3 for 30 minutes at room temperature. Even though the cryogenic protocol needs to be optimized to improve results, this is the first successful report of cryopreservation of vanilla apices. (Conflicts of interest: None declared. Source of funding: This work was supported by a FOMIX project 37551 sponsored by CONACyTGobierno del Estado de Veracruz, Mexico.) doi:10.1016/j.cryobiol.2008.10.118

118. Cryopreservation of Musa acuminata AAA cv. Pisang Nangka. Philip Sipen, Paul Anthony, Michael R. Davey, University of Nottingham, Loughborough, United Kingdom This study was conducted to investigate the effects of sucrose on cryopreservation of meristem scalps of Musa acuminata AAA cv. Pisang Nangka. Scalps (2 mm2) were cultured on medium containing elevated concentrations of sucrose for different durations of incubation. Meristem scalp survival decreased with increasing sucrose concentration and incubation duration. Survival of 100% was consistent for 0.1 M sucrose (normal sucrose concentration for banana tissue cultures) during the timecourse of the incubation. However, survival decreased for all concentrations of sucrose after 3 days to 0% (0.5–0.75 M) and 20–70% (0.2–0.4 M) at 14 days. This result shows that higher sucrose concentrations and prolonged incubation durations were deleterious for meristem scalp survival. To investigate the effects of sucrose preculture on meristem scalp cryopreservation, scalps were precultured on medium with the same sucrose concentration for 14 days, plunged into liquid nitrogen, thawed rapidly and recovered on regeneration medium. Before cryopreservation, survival rates were similar to those obtained in 14-day sucrose incubation studies. This result shows that survivability remains constant upon transfer to regeneration medium, regardless of the sucrose concentration pretreatment. However, freezing in liquid nitrogen was deleterious to the meristem scalps as demonstrated by the 100% fatality after cryopreservation. The results of this study show that sucrose was ineffective as a cryoprotectant for cryopreservation of cv. Pisang Nangka meristem scalps using the protocol described here. Further optimisation of cryopreservation steps are necessary to develop a successful cryopreservation protocol for the cv. meristem scalps. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.119