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ABSTRACT ARCHIVE |
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CryoLetters is a bimonthly, international journal for low temperature science and technology |
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CryoLetters 37 (5), 303-307 (2016) CHARACTERIZATION OF NORMAL AND FREEZE-THAWED TISSUES IN VITRO THROUGH THE ULTRASONIC INTEGRATED BACKSCATTER Lei Sheng1#, Pengfei Yang2#, Zhizhu He3, Gubing Wang4, 1 Department of Biomedical Engineering, Tsinghua University, Beijing, China; Abstract BACKGROUND: The ultrasound guidance of cryotherapy could be improved if changes of tissue characterization parameters during freezing could be used to monitor freezing patterns and estimate the postoperative effects after surgery. OBJECTIVE: The ultrasonic integrated backscatter analysis holds promise as an effective method for signal analysis and characterization of thawed tissues. MATERIALS AND METHODS: The ultrasonic integrated backscatter has been found to be an important parameter for describing the ultrasonic scattering and characterization of biological tissues. However, its potential values in the evaluation of cryosurgical effects of tissues reserved unclear so far. Here, we investigated the power spectrum of acoustic signal to estimate the ultrasound integrated backscatter in normal and freeze-thawed tissues on porcine livers in vitro. RESULTS: The experimental results carried out at 10 MHz using weakly focused pulse-echo signal element transducer indicated that the ultrasonic integrated backscatter in normal liver tissues is 31.3±4.6 whereas it is 44.2±6.5 in several pre-frozen and then thawed tissues. CONCLUSION: These results disclosed the good correlation between the ultrasonic integrated backscatter and microstructures of the normal or thawed tissues, and hence demonstrated that the power spectrum holds promise to be used as an effective method for the characterization of thawed tissues ultrasonic integrated backscatter, which might offer a potential pragmatic prospect for the practice of monitoring transition zone between frozen and unfrozen tissues during the surgical therapy, and evaluating postoperative effects. Keywords: cryosurgery, freeze-thawed tissue, ultrasonic integrated backscatter.
CryoLetters 37 (5), 308-317 (2016) OPTIMIZATION OF WATER CONTENT FOR THE CRYOPRESERVATION OF Allium sativum IN VITRO CULTURES BY ENCAPSULATION-DEHYDRATION Paul T Lynch1, Graham R Souch1, Jiří Zámečník2 and Keith Harding3* 1 Environmental Sustainability Research Centre, University of Derby, Kedleston Road, Derby, DE22 1GB, UK Abstract BACKGROUND: There is a general requirement to determine and correlate water content to viability for the standardization of conservation protocols to facilitate effective cryostorage of plant germplasm. OBJECTIVE: This study examined water content as a critical factor to optimize the cryostorage of Allium sativum. MATERIALS AND METHODS: Stem discs were excised from post-harvest, stored bulbs prior to cryopreservation by encapsulation-dehydration and water content was determined gravimetrically. RESULTS: Survival of cryopreserved stem discs was 42.5%, with 22.5% exhibiting shoot regrowth following 6 h desiccation. Gravimetric data demonstrated a correlation between water content corresponding with survival / regrowth from desiccated, cryopreserved stem discs. For encapsulated stem discs a 25% residual moisture and corresponding water content of 0.36 g H2O g-1 d.wt correlated with maximal survival following ~6.5 h of desiccation. CONCLUSION: The data concurs with the literature suggesting the formation of a stable vitrified state and a ‘window’ for optimal survival and regrowth that is between 6 – 10 h desiccation. Further studies using differential scanning calorimetry (DSC) are suggested to substantiate these findings. Keywords: Cryopreservation, garlic, regrowth, stem discs, survival, vitrification
CryoLetters 37 (5), 318-329 (2016) THE POTATO CRYOBANK AT THE INTERNATIONAL POTATO CENTER (CIP): A MODEL FOR LONG TERM CONSERVATION OF CLONAL PLANT GENETIC RESOURCES COLLECTIONS OF THE FUTURE R. Vollmer*, R. Villagaray, V. Egúsquiza, J. Espirilla, M. García, International Potato Center, Av. La Molina 1895, La Molina, Lima, Peru Abstract BACKGROUND: Cryobanks are a secure, efficient and low cost method for the long-term conservation of plant genetic resources for theoretically centuries or millennia with minimal maintenance. OBJECTIVE: The present manuscript describes CIP’s modified protocol for potato cryopreservation, its large-scale application, and the establishment of quality and operational standards, which included a viability reassessment of material entering the cryobank. MATERIALS AND METHODS: In 2013, CIP established stricter quality and operational standards under which 1,028 potato accessions were cryopreserved with an improved PVS2-droplet protocol. In 2014 the viability of 114 accessions cryopreserved in 2013 accessions were reassessed. RESULTS: The average recovery rate (full plant recovery after LN exposure) of 1028 cryopreserved Solanum species ranged from 34 to 59%, and 70% of the processed accessions showed a minimum recovery rate of ≥20% and were considered as successfully cryopreserved. CONCLUSION: CIP has established a new high quality management system for cryobanking. Periodic viability reassessment, strict and clear recovery criteria and the monitoring of the percent of successful accessions meeting the criteria as well as contamination rates are metrics that need to be considered in cryobanks. Keywords: cryopreservation, potato, PVS2-droplet, cryobank, Solanum
CryoLetters 37 (5), 330-334 (2016) OPTIMIZATION OF ARTIFICIAL PROPAGATION IN PIRACANJUBA FISH Brycon orbignyanus USING CRYOPRESERVED SEMEN Viviane de Oliveira Felizardo1, Carlos Cicinato Vieira Melo2, 1 Veterinary Medicine Department, Abstract BACKGROUND: Cryopreserved semen could facilitate procedures during the artificial reproduction in fish. Factors affecting cryopreservation efficiency are important to define efficient protocols. OBJECTIVE: This study investigated the application of cryoprotectants on the quality of piracanjuba fish semen, the sperm concentration required for oocyte fertilization and spermatic activation. MATERIALS AND METHODS: We evaluated two intracellular cryoprotectant solutions (DMSO and methanol) and two extracellular cryoprotectant solutions (egg yolk and lactose) to cryopreserved piracanjuba semen. Sperm motility rate, motility duration and spermatic alterations were assessed. CONCLUSION: The protocol for piracanjuba semen cryopreservation can use solutions including either DMSO or methanol as intracellular cryoprotectant and egg yolk or lactose as extracellular cryoprotectants. Keywords: Fertilization, fish, gametes, motility, water
CryoLetters 37 (5), 335-345 (2016) DEHYDRATION PREPARATION OF MOUSE SPERM FOR VITRIFICATION AND RAPID LASER WARMING *Estefania Paredes1 and † Peter. Mazur1 1 Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee 37996-0840, USA Abstract BACKGROUND: Mice are fundamental models of study due to their ease of breeding, manipulation, and the well-studied genome. There has been extensive research focused on the cryopreservation of mouse germaplasm, as a way to help maintain the different transgenic mouse breeds. The first protocols for mouse sperm were developed in the 90’s using slow cooling and a mixture of raffinose and glycerol. Since then, the rate of success reported remains highly variable. OBJECTIVE: The Aim of this work is to study factors that are key for developing vitrification protocols for ultra-rapid laser warming of mouse sperm. RESULTS: Our results show that due to the exquisite sensitivity of sperm cells to osmotic excursions, our target levels of dehydration (~85% water content) cannot be achieved without causing a significant decrease in sperm motility and membrane fusion. CONCLUSION: It seems likely that mouse sperm vitrification is going to be difficult to develop due to the exquisite sensitivity of mouse sperm cells to handling and dehydration. Keywords: Sperm, Mouse, Dehydration, Volume changes, Vitrification, Laser warming
CryoLetters 37 (5), 346-356 (2016) A NOVEL ADJUVANT-SOLUTION LAYER STRATEGY FOR IMPROVING THE EFFICACY OF CRYOSURGERY K.K. Ramajayam1, A.Kumar2, S.K. Sarangi2 and A.Thirugnanam1* 1 Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha-769008, India. Abstract BACKGROUND: During cryosurgery, studies reported earlier suggest increased destruction inside the tumour due to adjuvants or the prevention of damage to neighbouring healthy tissue through different methods. OBJECTIVE: This study advocates a novel strategy that increases the freezing inside agarose gel phantoms by addition of glycine and limits the freezing to the desired location using a perfluorohexane layer during cryosurgery. MATERIALS AND METHODS: Cryosurgery of glycine containing gels is carried out with and without perfluorohexane layer and the thermal history is measured using K-type thermocouples connected to a data acquisition system. RESULTS: The presence of glycine causes increased freezing during cryosurgery with an ice ball depth of 16 mm, while with a perfluorohexane layer at this gel interface, this depth is 13 mm, indicating the ability of this layer to limit freezing. CONCLUSION: It can be concluded that glycine addition results in substantial temperature decrease and perfluorohexane layer insulates the glycine gel effectively during cryosurgical cooling. Keywords: Cryosurgery, temperature, glycine, gel phantom, perfluorohexane layer
CryoLetters 37 (5), 357-364 (2016) IMPAIRMENT OF BARRIER PROPERTIES OF ERYTHROCYTE MEMBRANES CAUSED BY LOW TEMPERATURES IS A RESULT OF DISORGANIZATION OF HEMOGLOBIN SUPRAMOLECULAR STRUCTURE Gulevskyy А. К.*, Repin N.V. and Schenyavsky I.I. Institute of Problems for Cryobiology and Cryomedicine of NAS of Ukraine, Kharkiv, Abstract BACKGROUND: The antecedence of impairment of plasmatic membrane structure and functions forms the basis of the dominative concept about mechanisms of cell cryoinjuries. A role of alterations of hemoglobin supramolecular structure in erythrocytes remains unclear. OBJECTIVE: Comparison of continuity of membranes of native erythrocytes and resealed ghosts after freeze-thawing with a cryoprotectant at a low concentration (4%). MATERIALS AND METHODS: Cryoresistance of native erythrocytes and resealed ghosts with and without low concentrations of cryoprotectants (4% glycerol) was compared according to egress of the following markers: hemoglobin, 14С-sucrose and K+ as well as by scanning electron microscopy. RESULTS: It was found that resealed erythrocyte ghosts, where hemoglobin content was 4-5 times lower than in erythrocytes, were much more cryoresistant than native erythrocytes, which was especially noticeable when a low concentration of cryoprotectant (4% glycerol) was used. CONCLUSION: These data confirm an earlier proposed hypothesis on the role of supramolecular hemoglobin structure in cryoinjury mechanisms of erythrocytes. Keywords: erythrocytes, resealed ghosts, cryoresistance, supramolecular hemoglobin structure
CryoLetters 37 (5), 365-378 (2016) APPROPRIATE OSMOTIC BALANCE DURATION FOR DIFFERENT VOLUMES OF OVARIAN TISSUE IN VITRIFICATION SOLUTION: A STUDY OF OVARY TISSUE VITRIFICATION AND TRANSPLANTATION IN SHEEP Dang Ling1#, Zheng Xiaomin1#, Chang Qing1,Yang Yanzhou1, 1 Key Laboratory of Fertility Preservation and Maintenance, Ministry of Education, Key Laboratory of Reproduction and Genetics in Ningxia, Department of Histology and
Embryology, Nursing school, Ningxia Medical University, Ningxia, 75004, P.R. China; Abstract BACKGROUND: Auto-transplantation of cryopreserved ovarian tissue has become a promising method for fertility preservation and standardization of the process is crucial for practical applications. OBJECTIVE: Here we used different size of large sheep ovarian cortex to study the most suitable osmotic balance durations in the vitrification solution for large piece ovary cortex. MATERIALS AND METHODS: The ovarian cortices from six-month old female sheep were divided into 40 mm3, 80 mm3 and 160 mm3 volume, A two-step osmotic balance method was used based on the results from morphological and histological study, we detected the expression of VEGF after thawing, the percentage of follicles that survived and serum E2 levels,together with apoptosis test by TUNEL. RESULTS: the morphology of follicles and stromal cells were the best when the osmotic balance duration was 7 min, 11 min and 19 min, respectively. Osmotic balance time(T) is proportional to the area(S), we deduced that areas(S) of large pieces of ovarian cortex in vitrification fluid conformed to the standardized osmotic balance time(T) formula; i.e., T=ø¨S+15)/5 ( 22~24˚C, sample thickness 1-2 mm), In vitro culture of vitrification-thawed large cortex ovaries and xenogenic heterotopic transplantation by using the standardized osmotic balance duration formula were both successful. CONCLUSION: Formula T=ø¨S+15)/5 can be used to calculate optimal osmotic balance duration for different size of ovarian cortexes during vitrification. Keywords: osmotic balance durations, ovary, vitrification, stromal cells, large ovarian cortex |
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For Abstracts published from meetings, such as SLTB meetings, go to the
relevant Volume Year of the journal (above). For Full text Free Access Content (from 2000 onwards) go to CryoLetters at Ingenta and look for the blue symbol. |
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