Volume 33, No. 5 September/October 2012 ISSN 0143-2044
	Cryopreservation of the model alga Ectocarpus (Phaeophyceae) Svenja Heesch, John G. Day, Takahiro Yamagishi, Hiroshi Kawai, Dieter G. Müller and Frithjof C. Küpper	327-336
	Differential expression of two antifreeze proteins in the desert beetle Anatolica polita (Coleoptera: Tenebriondae): seasonal variation and environmental effects Ji Ma, Jing Wang, Xinfang Mao and Yan Wang	337-348
	Cryopreservation of germinal vesicle stage porcine oocytes based on intracellular ice formation assessment Chiang-Yi Yang, Ming-Cheng Chen, Po-Ting Lee and Ta-Te Lin	349-362
	Cryopreservation of Thymus cariensis and T. vulgaris shoot tips: comparison of three vitrification-based methods Elif Aylin Ozudogru and Ergun Kaya	363-375
	Rapid isolation of leukocyte subsets from fresh and cryopreserved peripheral blood mononuclear cells in clincal research Subhashini Arimilli, Brad E. Damratoski, Peter Chen, Bobbette A. Jones and G. L. Prasad	376-384
	Cryopreservation of tambaqui (Colossoma macropomum) semen: extenders, cryoprotectants, dilution ratios and freezing methods Paulo C.F. Carneiro, Hymerson C. Azevedo, Jadson P. Santos and Alexandre N. Maria	385-393
	Changes in total soluble proteins and Ca2+ upon cryopreservation of Prunus mume pollen Y. L. Zhang, B.L. Li, H. Wang and Y. Liu	394-401
	Cryopreservation of Kalopanax septemlobus embryogenic callus using vitrification and droplet-vitrification Dong-Jin Shin, Hyunjung Kong, Elena V. Popova, Heung Kyu Moon, So Young Park, Sang-Un Park, Sheong-Chun Lee and Haeng-Hoon Kim	402-410

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CryoLetters 33 (5), 327-336 (2012)
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CRYOPRESERVATION OF THE MODEL ALGA Ectocarpus (PHAEOPHYCEAE)

Svenja Heesch^{1, 2}, John G. Day^1*, Takahiro Yamagishi³, Hiroshi Kawai³, Dieter G. Müller⁴ and Frithjof C. Küpper^{1, 5*}

¹Culture Collection of Algae and Protozoa, Scottish Association for Marine Science, Scottish Marine Institute, Oban, Argyll, PA37 1QA, UK. ^* E-mail: jgd@sams.ac.uk
²Current address: Irish Seaweed Research Group, Ryan Institute, National University of Ireland Galway, University Road, Galway, Ireland
³Kobe University Research Center for Inland Seas, Rokkodai, Nadaku, Kobe 657-8501, Japan
⁴Fachbereich Biologie, Universität Konstanz, D-78457 Konstanz, Germany
⁵Current address: Oceanlab, University of Aberdeen, Main Street, Newburgh, Aberdeenshire AB41 6AA, UK.
^*Corresponding author email: fck@sams.ac.uk

Abstract

The brown alga Ectocarpus has recently become the first fully sequenced multicellular alga and is an important biological model. Due to the large and growing number of Ectocarpus strains isolated and maintained by the research community, including increasing numbers of mutants, there is an urgent need for developing reliable, cost-effective long-term maintenance techniques. We report here that cryopreservation constitutes an attractive option in this respect, using a simple two-step protocol employing combined DMSO 10% (v/v) and sorbitol 9% (w/v) as cryoprotectants. This model organism appears to be remarkably robust and post-cryo recovery has been observed in all strains tested in this study. Cultures can be regenerated by the germination of cryopreserved zooids (spores), or the recovery of vegetative cells. In the latter case, dividing surviving cells may grow into the cell lumen of a neighbouring dead cell, eventually regenerating a phenotypically normal thalloidal structure.

Keywords: Barcoding, brown algae, cryopreservation, Ectocarpus, model organism

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CryoLetters 33 (5), 337-348 (2012)
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DIFFERENTIAL EXPRESSION OF TWO ANTIFREEZE PROTEINS IN THE DESERT BEETLE Anatolica polita (COLEOPTERA: TENEBRIONDAE): SEASONAL VARIATION AND ENVIRONMENTAL EFFECTS

Ji Ma*, Jing Wang, Xinfang Mao and Yan Wang

Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China
*Corresponding author e-mail: majibrge@yahoo.cn

Abstract

Antifreeze proteins (AFPs) can inhibit and modify the growth of ice crystals. Two antifreeze protein genes, Apafp752 and Apafp914, were cloned from the desert beetle Anatolica polita (Coleoptera: Tenebriondae), and they shared 61.3% similarity at the amino acid level. Apafp752 also contained one variation in the most conserved TCT motif of beetle AFPs. Apafp752 and Apafp914 mRNAs had similar seasonal expression pattern. Both were stimulated by cold stress, but they expressed slightly differentially with Apafp752 being more sensitive to cold stress than Apafp914, and no more sensitive to desiccation stress than Apafp914. The thermal hysteresis activity (THA) in the beetle's hemolymph followed approximately the patterns of mRNA seasonal expression and expression upon environmental stress, with a time lag. Summer adults of the desert beetle also express mRNA of Apafp752 and Apafp914, and exhibit some hemolymph THA, suggesting other likely function of these proteins beyond antifreeze.

Keywords: desert beetle; antifreeze proteins (AFPs); differential expression; seasonal variation; cold adaptation, desiccation tolerance.

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CryoLetters 33 (5), 349-362 (2012)
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CRYOPRESERVATION OF GERMINAL VESICLE STAGE PORCINE OOCYTES BASED ON INTRACELLULAR ICE FORMATION ASSESSMENT

Chiang-Yi Yang¹,3, Ming-Cheng Chen², Po-Ting Lee³ and Ta-Te Lin¹,*

¹Department of Bio-Industrial Mechatronics Engineering, National Taiwan University, Taipei, Taiwan, R.O.C.
²Department of Animal Science, National I-Lan University, I-Lan, Taiwan, R.O.C.
³Department of Bio-Mechatronics Engineering, National I-Lan University, I-Lan, Taiwan, R.O.C.
*Corresponding author e-mail:m456@ntu.edu.tw

Abstract

This study aimed at evaluating the feasibility of slow freezing for cryopreservation of germinal vesicle (GV) stage porcine oocytes. In this study, intracellular ice formation (IIF) characteristics of GV porcine oocytes were investigated by using a thermoelectric cooling (TEC) cryomicroscope system. This cryomicroscope system used a thermoelectric cooling (TEC) chip in its cold stage as a heat sink and employed a PID control algorithm to achieve accurate temperature control. The temperature was controlled to a range between 70°C and -55°C with an accuracy of ±0.5°C. Five constant cooling rates of 24, 12, 6, 3 and 1.5°C/min were tested in experiments in freezing GV porcine oocytes from 20°C to -50°C in an NCSU-23 medium plus 2.0 M DMSO. The IIF temperature of each individual oocyte was recorded and cumulative IIF probabilities were calculated for each cooling rate. The total cumulative probabilities of IIF temperature distribution were 100%, 100%, 50.0%, 54.3% and 58.6% at cooling rates of 24, 12, 6, 3 and 1.5°C/min, respectively. A Weibull distribution model was found to adequately describe the distribution of IIF temperatures of GV porcine oocytes for the cooling rates tested (R² = 0.858±0.09). The IIF experimental results indicate that cooling rates of 6, 3 and 1.5°C/min could be considered as possible cryopreservation protocols. Further experiments were performed to examine the feasibility of using these protocols to cryopreserve GV porcine oocytes. After 44 h of in-vitro maturation in NCSU-23, the survival of thawed oocytes was checked. Porcine oocytes developed from the GV stage to the MII stage by using Hoechst 33258 staining, followed by Lacmoid staining as a secondary check. Normalized survival rates of 37.7±4.6%, 45.0±4.4% and 45.4±5.9% were obtained for GV oocytes frozen at 1.5, 3 and 6°C/min, respectively. The experimental results indicate that slow freezing is a feasible approach for cryopreservation of GV porcine oocytes when cooling rate is properly selected. This study also demonstrated an efficient approach for investigating optimal cooling rates by assessing the IIF characteristics of GV porcine COCs.

Keywords: Cryopreservation, TEC cryomicroscope, intracellular ice formation, porcine oocytes

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CryoLetters 33 (5), 363-375 (2012)
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CRYOPRESERVATION OF Thymus cariensis AND T. vulgaris SHOOT TIPS: COMPARISON OF THREE VITRIFICATION-BASED METHODS

Elif Aylin Ozudogru* and Ergun Kaya

Gebze Institute of Technology, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul Caddesi, No 101, 41400, Gebze (Kocaeli), Turkey
*Corresponding author email: elifaylinozudogru@yahoo.it or elif@gyte.edu.tr

Abstract

Thymus is an important genus of the Lamiaceae family, comprising more than 400 perennial aromatic thyme species, which are used extensively for medicinal and culinary purposes. The present study focused on the development of cryopreservation procedures for Thymus vulgaris and T. cariensis, the latter being an endemic and endangered species of Turkey. For cryopreservation of T. vulgaris shoot tips, PVS2-based one-step freezing methods, i.e., PVS2 vitrification, encapsulation-vitrification and droplet-vitrification, were compared. Cold hardening and sucrose preculture were also optimized before the cryopreservation trials. For T. cariensis, a droplet-vitrification method was applied to cold-hardened shoot tips, and after sucrose preculture. In all the methods tested, PVS2 was applied for up to 120 min. The best T. vulgaris cryopreservation was achieved with a droplet-vitrification method, that involved 2-weeks cold hardening of shoot cultures, 48 h preculture of shoot tips on MS medium supplemented with 0.25 M sucrose, and a 90 min PVS2 treatment in droplets. After direct immersion in LN, thawing and plating, 80% of shoot-tips recovered. Post-thaw recovery was significantly lower when the same procedure was applied to T. cariensis shoot tips; however also here 90 min PVS2 treatment produced the highest survival (25%) and recovery (25%) levels.

Keywords: cold hardening, droplet-vitrification, encapsulation-vitrification, long-term conservation, PVS2 vitrification, sucrose preculture, thyme.

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CryoLetters 33 (5), 376-384 (2012)
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RAPID ISOLATION OF LEUKOCYTE SUBSETS FROM FRESH AND CRYOPRESERVED PERIPHERAL BLOOD MONONUCLEAR CELLS IN CLINCAL RESEARCH

Subhashini Arimilli^1*, Brad E. Damratoski¹, Peter Chen², Bobbette A. Jones² and G. L. Prasad²

¹Department of Microbiology & Immunology, Wake Forest Baptist Health, Winston Salem, NC 27157
²R&D, R.J. Reynolds Tobacco Company, Winston Salem, NC 27102
*Corresponding author e-mail: sarimill@wfubmc.edu Tel 336-716-4870

Abstract

Isolation and processing blood into leukocyte subsets are important processes in research.  Although methods have been developed to fractionate small volumes of blood, optimizing the methods and balancing the underlying costs are often necessary. The need for such optimization is particularly critical when processing larger volumes of blood. We describe a simple and reproducible method for processing larger volumes of fresh blood rapidly and consistently, which yields peripheral blood mononuclear cells (PBMCs) and leukocyte subsets with high purity (81-96%; n=13) and higher yields relative to stored blood. RNA isolated from these cells was found to be suitable for downstream applications.  Blood stored for 24 hours (n=4) before processing resulted in significantly lower yields of PBMCs (58% lower), T cells (52% lower), B cells (21% lower) and monocytes (25% lower) compared to fresh blood. However, the purity of the fractionated cells was comparable to that obtained with fresh blood.  Furthermore, we report that the yield and purity of the leukocyte subsets isolated from cryopreserved PBMCs (n=4) were not compromised.

Keywords: peripheral blood mononuclear cells, leukocyte subsets, cryopreserved.

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CryoLetters 33 (5), 385-393 (2012)
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CRYOPRESERVATION OF TAMBAQUI (Colossoma macropomum) SEMEN: EXTENDERS, CRYOPROTECTANTS, DILUTION RATIOS AND FREEZING METHODS

Paulo C.F. Carneiro^*, Hymerson C. Azevedo, Jadson P. Santos and Alexandre N. Maria

Laboratory of Biotechnology of Animal Reproduction, Embrapa Tabuleiros Costeiros, Av. Beira Mar 3250, 49025-040, Aracaju, Sergipe, Brazil.
* Corresponding author email: paulo@cpatc.embrapa.br

Abstract

The tambaqui is an Amazonian fish of great economic and environmental importance to Brazil and other South American countries. Several semen cryopreservation methodologies have been tested for different Brazilian fish species; however, there is little information on the use of this technique on tambaqui semen. The aim of the present study was to investigate the effect of osmolarity and activation solutions on sperm kinetics and, glucose solutions, cryoprotectants, dilution ratios, egg yolk and freezing methods on tambaqui semen freezing. The osmolarity of 230 mOsm was suitable for simultaneously yielding higher sperm motility (85%) and motility time (54 sec.) and osmolarities above 360 mOsm maintain immobile tambaqui sperm. The tambaqui semen can be successfully cryopreserved when diluted 1:9 in freezing medium composed of 5% glucose solution (290 mOsm) with 10% methylglycol and 5% egg yolk, and frozen directly in a dry shipper container.

Keywords: fish, sperm, motility, viability, protocol.

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CryoLetters 33 (5), 394-401 (2012)
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CHANGES IN TOTAL SOLUBLE PROTEINS AND CA²⁺ UPON CRYOPRESERVATION OF PRUNUS MUME POLLEN

Y. L. Zhang¹, B.L. Li ^2,3, H. Wang ⁴ and Y. Liu ^2,3*

¹Shanghai Botanical Garden, Shanghai, 200231, China.
²College of Landscape Architecture, Beijing Forestry University, Beijing, 100083, China
³National Floriculture Engineering Research Center, Beijing, 100083, China.
⁴Suzhou Polytechnic Institute of Agriculture, Suzhou, 215008, China.
*Corresponding author  email: chbly@sohu.com

Abstract

Mei Flowers (Prunus mume) are traditional Chinese ornamental plants. Fifty-one Mei cultivars have been conserved in a pollen cryobank since 2003. We used two-dimensional electrophoresis (2-DE) of total soluble proteins and flow cytometric detection of Ca²⁺ fluorescence to probe changes in pollen grains before and after cryopreservation. Results indicated that: (1) electrophoresis maps of total soluble proteins before and after cryostorage of pollen from three cultivars were different, even though 70% of the protein spots among these three cultivars were matched after cryopreservation. We found some protein spots that changed in all three cultivars; their molecular weights and pI were between 12.6-72.8 kDa and 5.6-7.3, respectively; (2): the geometric mean of Ca²⁺ fluorescence intensity (GMFI) value of cryopreserved pollen was significantly higher compared with that of fresh pollen in cultivar 'Beijing Yudie'. GMFI increased during pollen germination in our studied cultivars, especially after 0.5 and 1.0 h of culturing. In addition, no positive correlation was found between pollen germination rate and GMFI in the present study.

Keywords: pollen germination rate; 2-DE; flow cytometry; fluorescence intensity

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CryoLetters 33 (5), 402-410 (2012)
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CRYOPRESERVATION OF KALOPANAX SEPTEMLOBUS EMBRYOGENIC CALLUS USING VITRIFICATION AND DROPLET-VITRIFICATION

Dong-Jin Shin¹, Hyunjung Kong², Elena V. Popova³, Heung Kyu Moon³, So Young Park³, Sang-Un Park⁴, Sheong-Chun Lee² and Haeng-Hoon Kim^2*

¹Species Restoration Center, Korea National Park Service, Gurye 542-853, Korea.
²Dept. of Well-being Resources, Sunchon National University, Suncheon 540-742, Korea.
³Korea Forest Research Institute, 44-3 Omokdong, Suwon 441-350, Korea.
⁴Division of Plant Science and Resources, Chungnam National University, Daejeon 305-764, Korea.
^*Corresponding author email: cryohkim@sunchon.ac.kr

Abstract

A cryopreservation protocol has been developed for embryogenic callus cultures of castor aralia (Kalopanax septemlobus), a deciduous tree which is widely used in oriental medicine and in landscape design. Three preculture treatments, four loading and six vitrification solutions were tested in a vitrification procedure. Preculture of embryogenic callus (EC) with high sucrose concentrations (up to 0.7 M) showed no effect on regrowth after cryopreservation. Loading for 20 min at ambient temperature improved regrowth of cryopreserved EC by 70-75% compared with non-loaded samples, regardless of the composition of the loading solution. Among vitrification solutions, the highest regrowth of 95-100% after cryopreservation was obtained after incubation of EC in a vitrification solution A3-80% comprising (w/v) 33.3% glycerol + 13.3% DMSO + 13.3% EG + 20.1% sucrose for 40 min at 0°C. Profiling of crystallization and recrystallization events using differential scanning calorimetry (DSC) confirmed that freezing injury was minimized in samples after loading and cryoprotection with this vitrification solution. Unlike many other papers, the droplet-vitrification protocol did not produce higher post-cryopreservation regrowth of Kalopanax EC, compared with the vitrification procedure. When samples are sufficiently cryoprotected during VS treatment, vitrification using cryovials may be preferred, since droplet-vitrification is more complex and requires skilled personnel. Cryopreserved callus grew rapidly and produced numerous somatic embryos, which developed similarly to embryos obtained from non-cryopreserved samples.

Keywords: droplet-vitrification, embryogenic callus, Kalopanax septemlobus, vitrification, vitrification solution.

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