Volume 34, No. 1 January/February 2013 ISSN 0143-2044
	Cryopreservation of Galanthus elwesii Hook. apical meristems by droplet vitrification M. Maślanka, B. Panis and A. Bach	1-9
	Cryopreservation of osteoblasts by use of a programmed freezer with a magnetic field Hiroyuki Koseki, Masato Kaku, DDS, Toshitsugu Kawata, Shunich Kojima, Hiromi Sumi, Hanaka Shikata, Masahide Motokawa, Tadashi Fujita, Junji Ohtani and Kazuo Tanne	10-19
	Fertilizing ability of cryopreserved pollinia of Luisia macrantha, an endemic orchid of western ghats S. Ajeeshkumar and S. William Decruse	20-29
	A life cycle model to enable research of cryostorage recalcitrance in temperate woody species: the case of sitka spruce (Picea sitchensis) Samantha Gale, Erica E Benson and Keith Harding	30-39
	Numerical simulation of water transport and intracellular ice formation for freezing of endothelial cells Gang Zhao, Yi Xu, Wei-ping Ding and Mao-bin Hu	40-51
	Development of soya milk extender for semen cryopreservation of karan fries (crossbreed cattle) VK Singh, AK Singh, R Kumar and SK Atreja	52-61
	Molecular and genetic aspects of protein cold denaturation Gulevsky A.K. and Relina L.I.	62-82
	Identification of differentially regulated micrornas in cold-hardy insects Pierre J. Lyons, Julie J. Poitras, Lynn A. Courteau, Kenneth B. Storey and Pier Jr Morin	83-89
	Silkworm (Bombyx mori) cryopreservation: embryonic development as revealed by microscopic studies Anuradha H. Jingade*, G.K. Srinivasa Babu, G. Lekha, C.V. Nair, A. Ananda Rao and A. Manjula	90-99
	Evaluation of adipocytic changes after a simil-lipocryolysis stimulus Hernán Pinto, Estefan Arredondo and David Ricart-Jané	100-105
	Announcements	106

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CryoLetters 34 (1), 1-9 (2013)
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CRYOPRESERVATION OF Galanthus elwesii Hook. APICAL MERISTEMS BY DROPLET VITRIFICATION

M. Maślanka¹*, B. Panis² and A. Bach¹

¹Department of Ornamental Plants, University of Agriculture in Kraków, Al. 29 Listopada 54, 31-425 Kraków, Poland
²Laboratory of Tropical Crop Improvement, KU Leuven, Kasteelpark Arenberg 13, 3001 Leuven, Belgium
*Corresponding author email: maslankam@ogr.ar.krakow.pl

Abstract

The aim of this study was to develop an efficient cryopreservation protocol for the geophyte giant snowdrop (Galanthus elwesii Hook.) that guarantees a high rate of survival and plant regeneration after cryopreservation. The excised apical meristems were obtained from cultures of in vitro grown bulb scales. Using a vitrification procedure and optimizing the duration of the exposure to the loading solution (LS), meristem post-rewarm survival rates higher than 90% were achieved. Also regrowth percentages were very high, ranging from 87 to 91%. After optimizing the time of exposure to the plant vitrification solution (PVS2), the survival rate was between 83 and 97%. During post-rewarm regeneration, good growth recovery was as high as 76%; however, hyperhydration and callusing were also observed. The results demonstrate that cryopreservation of Galanthus elwesii germplasm seems to be feasible.

Keywords: cryopreservation, droplet vitrification, meristem, Galanthus elwesii

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CryoLetters 34 (1), 10-19 (2013)
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CRYOPRESERVATION OF OSTEOBLASTS BY USE OF A PROGRAMMED FREEZER WITH A MAGNETIC FIELD

Hiroyuki Koseki, *Masato Kaku, DDS, Toshitsugu Kawata,
Shunich Kojima, Hiromi Sumi, Hanaka Shikata, Masahide Motokawa,
Tadashi Fujita, Junji Ohtani and Kazuo Tanne

Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan  TEL: 082-257-5686   FAX: 082-257-5687
Corresponding author e-mail: mkaku@hiroshima-u.ac.jp

Abstract

In order to determine a suitable condition for osteoblasts cryopreservation, murine osteoblasts were freezed by programmed freezer with a magnetic field (CAS freezer). After 7 days cryopreservation at -150°, the number of survival cells immediately after thawing and the growth rate of cultured cells for 48 hours were examined. Gene and protein expression of alkaline phosphatase (ALP), osteopontin (OPN) and bone sialoprotein (BSP) were compared between cryopreserved and non-cryopreserved groups. As a result, a plunging temperature of -30°, a hold-time at -5° for 15 minutes and a 0.1 mT of magnetic field led to the largest survival and growth rate. Moreover, there was no significant difference in ALP, OPN and BSP mRNA and protein expression between cryopreserved and control groups. From these results, it was suggested that the CAS freezer is available for osteoblast cryopreservation and bone tissue banking can be established in the future.

Keywords: cryopreservation, a magnetic field, osteoblasts, bone tissue banking

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CryoLetters 34 (1), 20-29 (2013)
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FERTILIZING ABILITY OF CRYOPRESERVED POLLINIA OF Luisia macrantha, AN ENDEMIC ORCHID OF WESTERN GHATS

S. Ajeeshkumar and S. William Decruse*

Biotechnology and Bioinformatics Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram, Kerala, PIN 695562, India.
*Corresponding author email: willdic@rediffmail.com

Abstract

A successful protocol for long-term preservation of pollinia of Luisia macrantha Blatter & McCann., an endemic and endangered orchid of Western Ghats has been devised through different pollen cryopreservation methods and by confirming fertilizing ability. Pollinia subjected to 0-30 min dehydration at 27 ± 2°C within a laminar air flow cabinet showed maximum germination of 65 - 67% in desiccated controls and 54% in LN treated samples. The treated pollinia retained fertilizing ability, giving 100% fruit set upon sib-mating. Pollinia dried under charged silica gel for 120 min gave 51 - 52% pollen germination, in LN treated and desiccated control samples. Exposure to vitrification solution (PVS2) was optimized at 10 min to achieve 57% and 56% germination in control and LN treated samples, respectively. These pollinia exhibited 51% pollen germination after 668 days storage in LN. Cryopreserved pollinia (10 min PVS2) used for hybridization with Vanda tessellata gave 87% fruit set and 21% viable seeds. The viable seeds germinated and developed into healthy seedlings. Thus cryopreservation has been proved useful for the successful storage of L. macrantha germplasm and their utilization in breeding.

Keywords: cryopreservation, Western Ghats, orchids, pollinia, plant vitrification solution (PVS2), orchid breeding

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CryoLetters 34 (1), 30-39 (2013)
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A LIFE CYCLE MODEL TO ENABLE RESEARCH OF CRYOSTORAGE RECALCITRANCE IN TEMPERATE WOODY SPECIES: THE CASE OF SITKA SPRUCE (Picea sitchensis)

 Samantha Gale¹, Erica E Benson² and Keith Harding^2*

¹Cawthron Institute, 98 Halifax Street East, Private Bag 2, Nelson 7042, New Zealand
²Damar Research Scientists, Damar, Drum Road, Cuparmuir, Fife, KY15 5RJ, UK
^*Corresponding author e-mail: k.harding-damar@tiscali.co.uk

Abstract

Empirical testing of protocols and fundamental investigations are the approaches usually applied to study germplasm storage recalcitrance in temperate plants. However, they can fall short of practicable solutions, even after exhaustive experimentation, and the generation of negative survival data makes it difficult to plan further investigations. Picea sitchensis somatic embryos are amenable to cryopreservation whereas in vitro shoot meristems, although able to survive, are incapable of sustained recovery. Differential Scanning Calorimetry (DSC) revealed that these disparate responses could not be attributed to biophysical factors. A model is presented hypothesising that in some cases life cycle adaptations (cold hardening, dormancy) may have opposing influences on survival causing delayed-onset, cryogenically-induced loss of viability in temperate tree species.

Keywords: vitrification, glassy state, differential scanning calorimetry, recalcitrance

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CryoLetters 34 (1), 40-51 (2013)
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NUMERICAL SIMULATION OF WATER TRANSPORT AND INTRACELLULAR ICE FORMATION FOR FREEZING OF ENDOTHELIAL CELLS

Gang Zhao^1,*, Yi Xu², Wei-ping Ding^1,#, Mao-bin Hu³

¹Department of Electronic Science and Technology, University of Science and Technology of China, Hefei 230027, P. R. China.
²Institute of Bio-thermo Science and Technology, University of Shanghai for Science and Technology, Shanghai 200093, China.
³Department of Thermal Science and Energy Engineering, University of Science and Technology of China, Hefei 230027, P. R. China.
*Corresponding author e-mail: zhaog@ustc.edu.cn
#Co-correspondence author e-mail: wpdings@ustc.edu.cn

Abstract

Endothelial cell detachment may cause failure of blood vessel and corneal cryopreservation, and thus successful cryopreservation of endothelial cells is regarded to be the first step to optimize cryopreservation of endothelial cells containing tissues. In this study, the pre-determined biophysical parameters were incorporated into the model for intracellular ice formation (IIF) and the growth of intracellular ice crystals (ICG) to calculate cell water loss, supercooling of intracellular solution, intracellular ice formation and the growth of intracellular ice crystals. The optimal protocols were determined according to the combination effect of both "solution injury" and "IIF injury".

Keywords: endothelial cells, vitrification, cell dehydration, intracellular ice formation

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CryoLetters 34 (1), 52-61 (2013)
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DEVELOPMENT OF SOYA MILK EXTENDER FOR SEMEN CRYOPRESERVATION OF KARAN FRIES (CROSSBREED CATTLE)

VK Singh, AK Singh, R Kumar and SK Atreja*

Reproductive Biochemistry Laboratory, Animal Biochemistry Division, National Dairy Research Institute, Haryana, India.
*Corresponding author email: skatreja@rediffmail.com

Abstract

Egg yolk based semen extenders are used widely, with the potential risk of xenobiotic contamination. This study was designed to develop a soya milk based extender to substitute egg yolk based extender for bovine semen cryopreservation. In the first experiment soya milk was prepared from fresh soya bean (Glycine max). Concentration of soya milk in tris based extender was standardized based on quality parameters of spermatozoa during liquid preservation at 5°C up to 72 h and compared with egg yolk tris (EYT) extender. Sperm in soya milk tris (SMT) extender with 25% soya milk showed no significant (P > 0.05) differences in all the quality parameters like motility, viability, membrane integrity and acrosome integrity, as compared to sperm in EYT extender up to 72h in liquid dilution. In the second experiment the Karan Fries semen was cryopreserved in SMT extender with 25% soya milk (selected from the first experiment) using different concentration of glycerol, as cryoprotectant, ranging from 6-7% with a difference of 0.2% to standardize optimum concentration based on post thaw motility of spermatozoa. Glycerol at a final concentration of 6.4% was found to be the best among all. Further, semen samples were split and cryopreserved in newly developed SMT extender containing 6.4% glycerol and compared with conventional EYT extender for post thaw sperm quality parameters and degree of cryocapacitation. There were no significant (P > 0.05) differences between sperm in EYT extender and SMT extender for post thaw motility, viability, membrane integrity, acrosome integrity and cryocapacitation. In conclusion, the newly developed SMT extender maintained comparable semen quality as compared to EYT extender hence it can be used to substitute egg yolk extender for cattle (Karan Fries) semen cryopreservation.

Keywords: Karan Fries; Cattle; Soya milk extender; Spermatozoa; Cryopreservation.

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CryoLetters 34 (1), 62-82 (2013)
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MOLECULAR AND GENETIC ASPECTS OF PROTEIN COLD DENATURATION

Gulevsky A.K. and Relina L.I.

Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine, Kharkov, Ukraine.
Corrresponding author email: lianaisaakovna@rambler.ru

Abstract

The phenomenon of cold-induced denaturation of proteins is reviewed. Examples of low temperature-induced changes in quaternary, tertiary and secondary structure of protein molecules are cited. Genetic aspects of cold-stability and functioning of structural proteins and enzymes from cold-tolerant organisms under low temperature conditions are discussed. Disorganisation of supramolecular assemblies under low-temperature influence is considered as one of the key factors contributing to cell cold-induced injuries.

Keywords: cold denaturation, cold-stable and cold-labile proteins.

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CryoLetters 34 (1), 83-89 (2013)
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IDENTIFICATION OF DIFFERENTIALLY REGULATED MICRORNAS IN COLD-HARDY INSECTS

Pierre J. Lyons¹, Julie J. Poitras¹, Lynn A. Courteau¹,
Kenneth B. Storey² and Pier Jr Morin^1*

¹Department of Chemistry and Biochemistry, Université de Moncton, New Brunswick, Canada.
²Institute of Biochemistry, Carleton University, Ontario, Canada
*Corresponding author email: pier.morin@umoncton.ca

Abstract

Freeze tolerance in insects is associated with cryoprotectant synthesis and strong metabolic suppression. Freeze avoidance, an alternative strategy in cold-hardy insects, is also characterized by hypometabolism, but possesses significant cellular and physiological differences when compared with freeze tolerance. We hypothesized that microRNAs, non-coding transcripts that bind to mRNA, could play a role in the regulation of energy-expensive mRNA translation in insects exposed to low temperatures. Expression levels of microRNA species were evaluated during cold acclimation of freeze tolerant Eurosta solidaginis and freeze-avoiding Epiblema scudderiana, comparing control (5°C) conditions with larvae given sequential exposures to -5°C and -15°C. MiR-1 levels were significantly elevated in frozen E. solidaginis larvae at -15°C, whereas miR-34 levels were unchanged. MiR-1 and miR-34 levels remained stable in E. scudderiana. These data demonstrate differential microRNA expression in frozen versus control insect larvae and highlight contrasting microRNA signatures between freeze tolerant and freeze avoiding species.

Keywords: microRNA, freeze tolerance, freeze avoidance, Eurosta solidaginis, Epiblema scudderiana, reversible control of translation

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CryoLetters 34 (1), 90-99 (2013)
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SILKWORM (Bombyx mori) CRYOPRESERVATION: EMBRYONIC DEVELOPMENT AS REVEALED BY MICROSCOPIC STUDIES

Anuradha H. Jingade*, G.K. Srinivasa Babu, G. Lekha,
C.V. Nair, A. Ananda Rao and A. Manjula

Central Sericultural Germplasm Resources Centre, P.B No. 44, Thally Road, Tamil Nadu-635 109, INDIA.
*Corresponding author email: anujnh@yahoo.co.in

Abstract

Embryo cryopreservation offers a way to safeguard against unwelcome mutations and inadvertent selection that can change its unique genetic makeup. Having a genetic repository of the silkworm genetic resources would ensure preservation of original genetic makeup and will permit to study what genes may have been lost in the selection process. For cryopreservation of eggs and embryos of silkworm, the determination of embryonic stages is a prerequisite. This study reports microscopic observations on embryonic development. The embryonic stages in the dechorionated eggs were determined in parallel comparison with the embryos isolated from intact eggs of different developmental ages.

Keywords: silkworm eggs, embryonic stage, cryopreservation, and mulberry silkworm.

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CryoLetters 34 (1), 100-105 (2013)
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EVALUATION OF ADIPOCYTIC CHANGES AFTER A SIMIL-LIPOCRYOLYSIS STIMULUS

Hernán Pinto ^{1 *}, Estefan Arredondo ², David Ricart-Jané ³

¹Instituto de Investigaciones para las Especialidades Estéticas y del Envejecimiento, Barcelona;
²Donation and Transplantation Insitute, Barcelona;
³Centre de Recerca del Metabolisme, University of Barcelona, Barcelona, Spain.
*Corresponding author email: portatil@ageback.es

Abstract

Lipocryolysis is considered as an effective, well-tolerated non-invasive procedure to reduce local adiposities.  However there is little information about its mechanism of action by the procedure. It is proposed that lipid phase transition or crystallization may be an unleashed apoptotic stimulus.  Yet, the post-lipocryolysis apoptosis is not easily confirmed, least of all is its correlation with crystallization.  In this study adipocytes from rat fat tissue were exposed to a lipocryolysis-session-like stimulus.  Lipid changes were observed in all test sample.

Keywords: Lipocryolysis; adipocytes; crystallization; fat tissue; temperature.

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