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Abstracts: CryoLetters 35 (5), 2014



Volume 35, No. 5 September/October 2014

ISSN 0143-2044



Effect of fasudil hydrochloride on the post-thaw viability of cryopreserved porcine adipose-derived stem cells
Yuntao Ji, Deyu Chen, Shuanglin Jiang and Changqing Qu




Efficacy of fish embryo vitrification protocols in terms of embryo morphology — a systematic review
Aline Ferreira Souza de Carvalho,
Shayenne Elizianne Ramos,
Thatijanne Santos Gonzaga de Carvalho,
Yasmin Chalfoun Pomárico de Souza,
Márcio Gilberto Zangeronimo, Luciano José Pereira and
Luis David Solis Murgas




Effect of ultrasonic waves on the freezing rates of potatoes in degassed coolant and untreated coolant
Deyang Yu and Baolin Liu




Development of an encapsulation-vitrification protocol for Rubia akane (Nakai) hairy roots: a comparison with non-encapsulation
Dong-Jin Shin, Hyo-Eun Lee, Chang-Hwan Bae,
Sang-Un Park, Ho-Nam Kang and Haeng-Hoon Kim




Cryopreservation of dormant in vivo-buds of hybrid aspen: timing as critical factor
Tuija Aronen and Leena Ryynänen




Extracellular ice phase transitions in insects
Timothy C. Hawes




Production of organ extracellular matrix using a freeze-thaw cycle employing extracellular cryoprotectants
Pulver, A. Shevtsov, B. Leybovich, I. Artyuhov, Y. Maleev,
and A. Peregudov




Coconut (Cocos Nucifera L.) Pollen cryopreservation
Anitha Karun, K.K. Sajini, V. Niral, C.H. Amarnath,
P. Remya, M.K. Rajesh, K. Samsudeen, B. A. Jerard
and Florent Engelmann




Low-temperature storage and cryopreservation of grapefruit (Citrus paradisi Macfad.) seeds
Nan Zhang, Bin Wen, Mingyue Ji and Qi Yan




Evaluation of cryoprotective effect ofturkish pine honey on common carp (Cyprinus carpio) spermatozoa
Fatih Ögretmen and Burak E. İnanan




Characterization and expression analysis of six chitinase Genes from the desert beetle Microdera punctipennis in Response to low temperature
Xueying Lu, Jieqiong Li, Xiaoning Liu, Xiaobo Li and Ji Ma




Letter to Editor: AMORPH 2014.
The Felix Franks Symposium: A Celebration
Harry Levine and Tony Auffret






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CryoLetters 35 (5), 356-360 (2014)
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Yuntao Ji, Deyu Chen, Shuanglin Jiang and Changqing Qu*

School of Life Science, FuYang Teachers College, Fuyang, Anhui 236041, P. R. China.


BACKGROUND: Adipose-derived stem cells (ADSCs) are of interest for regenerative medicine as they are isolated easily and can differentiate into multiple cell lineages. Recently, it was reported that a Rho-associated kinase (ROCK) inhibitor Y-27632 could enhance the post-thaw viability and physiological function of cryopreserved BMSCOBJECTIVE: The present study  is to investigate whether Fasudil hydrochloride  (FH,a selective Rho-kinase inhibitor like Y-27632) can exert a similar beneficial effect on the post-thaw viability of cryopreserved ADSCsMETHODS: ADSCs were allotted in three cryopreservation solutions [I:10% (v/v) (DMSO), 30% (v/v) FBS (as a negative control);II:10µM FH, 10% (v/v) DMSO, 30% (v/v) FBS;III:10µM Y-27632, 10% (v/v) DMSO, 30% (v/v) fetal bovine serum. All groups were frozen using a rapid freezing method and stored at -196° C in liquid nitrogen for 30 days.After thawing and being cultured 24h,viability of ADSCs were detected by MTT assay.  RESULT: The MTT assay showed significant differences in the proportion of adherent viable cells over the concentration of Y-27632 and FH, Additionally, FH did not induce morphological changes in the frozen-thawed ADSCs like Y-27632 did CONCLUSIONS: FH might represent a promising cryopreservation solution in enhancing the post-thaw viability and physiological function of cryopreserved ADSCs and did not chang the cell appearance.

Keywords: Porcine, Adipose-derived stem cells (ADSCs), Fasudil hydrochloride, Y27632




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CryoLetters 35 (5), 361-370 (2014)
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Aline Ferreira Souza de Carvalho1*, Shayenne Elizianne Ramos1,
Thatijanne Santos Gonzaga de Carvalho1,
Yasmin Chalfoun Pomárico de Souza2, Márcio Gilberto Zangeronimo1,
Luciano José Pereira1 and Luis David Solis Murgas1

1Department of Veterinary Medicine, Federal University of Lavras, Lavras, Minas Gerais, Brazil.
2Departament of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Pampulha, Belo Horizonte, Minas Gerais, Brazil.
*Corresponding author email:


BACKGROUND: Embryo cryopreservation has been used for the creation of genetic banks with diploid resources, and among different techniques, vitrification is considered as the most promising method. OBJECTIVE: The goal is to evaluate the major aspects of the existing vitrification techniques and to evaluate their efficacy in terms of embryo morphology.  METHODS: Electronic searches in the PubMed and ScienceDirect databases were performed with the keyword conbination: fish, embryo and vitrification. Pubmed retrieved 26 articles and Science Direct resulted in 464 articles. For this review, only studies that developed and tested vitrification protocols in fish embryos were included. Research regarding cryoprotectant toxicity and permeability were excluded. There were no restrictions on publication date or language. With these criteria, a total of ten articles were evaluated. RESULTS: In these articles, the major aspects to be considered for the development of new vitrification protocols are: the cryoprotectants' toxicity, the embryos' development stage, the exposure to and the permeability of the cryoprotectants, vitrification devices and vitrification-warning cycle. CONCLUSION: The survival were limited, however, the preservation of embryonic morphology after thawing indicates the possibility of preserving fish embryos via the vitrification technique.

Keywords: ultra-rapid freezing, embryo morphology, aquaculture, vitrificant solutions.




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CryoLetters 35 (5), 371-376 (2014)
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Deyang Yu1 and Baolin Liu2*

1Yancheng Institute of Technology, Yancheng, China;
2University of Shanghai for Science and Technology, Shanghai, China.
*Corresponding author email:


BACKGROUND: Ultrasonic waves are shown to enhance the rate of freezing.  OBJECTIVE: To elucidate the mechanism of immersion freezing of potatos with ultrasonic waves.  MATERIALS AND METHODS: Ultrasound is applied to potato samples immersed in degassed coolant and untreated coolant. Sonic waves were intermittently applied at temperature below -1°C. The freezing rates were measured under different experimental conditions. RESULTS: The use of ultrasonic waves increased the freezing rates of potatoes immersed in both degassed coolant and untreated coolant. However, the freezing rate in the degassed coolant was less than that in the untreated coolant. CONCLUSION: Heat transfer on the interface between the potato sample and sonicated degassed coolant appears to be less than that within the sample in the absence of cavitation. The interface heat transfer between the potato sample and untreated coolant is likely improved due to ultrasonic cavitation.

Keywords: ultrasonic wave, cavitation, acoustic streaming, immersion freezing




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CryoLetters 35 (5), 377-384 (2014)
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Dong-Jin Shin1, Hyo-Eun Lee2, Chang-Hwan Bae3,
Sang-Un Park4, Ho-Nam Kang1 and Haeng-Hoon Kim2*

1Species Restoration Center, Korea National Park Service, Gurye 542-853, Korea.
2Department of Well-being Resources, Sunchon National University, Suncheon, 540-742, Korea.
3National Institute of Biological Resources, 42 Nanji-ro, Seo-gu, Incheon 404-708, Korea.
4Division of Plant Science and Resources, Chungnam National University, Daejeon 305-764, Korea.
*Corresponding author e-mail:


BACKGROUND: A comparison of different cryopreservation techniques should be based on the characteristics of both the methodology and the material in question using an optimized procedure.  OBJECTIVE: This study aimed at developing an encapsulation-vitrification procedure for hairy roots of Rubia akane using alternative loading and vitrification solutions, based on the existing optimized droplet-vitrification procedure. MATERIALS AND METHODS: Encapsulated roots were first precultured in liquid medium with 10% sucrose for 3 days, then with 17.5% sucrose for 1 day, after which they were osmoprotected with solution C6-40% (20% glycerol + 20% sucrose) for 50 min, cryoprotected with solution A3-90% (37.5% glycerol + 15% DMSO + 15% EG + 22.5% sucrose, w/v) on ice for 40 min, cooled and warmed in 2 ml cryovials, and unloaded in 35% sucrose solution for 60 min. RESULTS: Through the application of this procedure to aged-clustered roots, up to 97.5% post-cryopreservation regeneration was observed. In our previous study, droplet-vitrification of hairy roots of R. akane resulted in 83.8% post-rewarming regeneration following preculture with 10% sucrose for 2 days and 17.5% sucrose for 4-5 h, and osmoprotection with solution C4-35% (17.5% glycerol + 17.5% sucrose) for 30 min, and cryoprotection with solution A3-70% (29.2% glycerol + 11.7% DMSO + 11.7% EG + 17.4% sucrose, w/v) on ice for 20 min. In the present study, higher post-cryopreservation regeneration was observed by using a higher concentration of vitrification solution (A3-70% → A3-90%, B5-80% → B1-100%) and/or a longer cryoprotection duration (A3-70% at room temperature (RT) for 8 min → 15-30 min, on ice for 20 min → 40-80 min; B5-80% for 15 min → 30-60 min).  CONCLUSION: Even though encapsulation provided some degree of protection from the cytotoxicity of vitrification solutions to cytotoxicity-sensitive R. akane hairy roots, an overall higher post-cryopreservation regrowth was obtained using the droplet-vitrification procedure under optimized conditions. This result implies that this sensitive material was not sufficiently cryoprotected, and thus, rapid cooling and warming using foil strips was more efficient than cryopreservation of encapsulated samples.

Keywords: cryoprotection, droplet-vitrification, osmoprotection, preculture.




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CryoLetters 35 (5), 385-394 (2014)
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Tuija Aronen* and Leena Ryynänen

Finnish Forest Research Institute, Finlandiantie 18, FI-58450 Punkaharju, Finland.
*Corresponding author email:


BACKGROUND: For the conservation of hybrid aspen germplasm, cryostorage of dormant in vivo buds is a convenient back-up method for field collections. In practice in Finland, bud collection is performed from February to March. OBJECTIVE: The aim of this study was to assess how this time schedule can be extended without compromising regeneration. In addition, an easily measurable marker for successful cryopreservation was examined. MATERIALS AND METHODS: Timing of cryopreservation was tested from August to February, using dormant buds from both outdoor and indoor plants. To find a marker, water content and gene expression of hydrid aspens, as well as environmental factors such as temperature, temperature sum, and light period were followed. RESULTS: Cryopreservation was successful from October to February, when, on an average, at least 75% of the buds regenerated through micropropagation, and there was no difference to non-frozen controls. Significant genotypic variation was observed in October and February, with regeneration rates of 61-100% and 37-98%, respectively. No marker for successful cryopreservation was found among the studied factors. CONCLUSION: The results provide flexibility for the undertaking of practical work, with a recommendation that cryopreservation can be carried out from November to January – earlier than the current practice.

Keywords: cryopreservation, dormant bud, micropropagation, Populus, timing




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CryoLetters 35 (5), 395-399 (2014)
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Timothy C. Hawes

Asian University, Banglamung, Chonburi 20260, Thailand.
Corresponding author email:


At temperatures below their temperature of crystallization (Tc) the extracellular body fluids of insects undergo a phase transition from liquid to solid. Insects that survive the transition to equilibrium (complete freezing of the body fluids) are designated as freeze tolerant. Although this phenomenon has been reported and described in many Insecta, current nomenclature and theory does not clearly delineate between the process of transition (freezing) and the final solid phase itself (the frozen state). Thus freeze tolerant insects are currently, by convention, described in terms of the temperature at which the crystallization of their body fluids is initiated, Tc. In fact, the correct descriptor for insects that tolerate freezing is the temperature of equilibrium freezing, Tef. The process of freezing is itself a separate physical event with unique physiological stresses that are associated with ice growth. Correspondingly there are a number of insects whose physiological cryo-limits are very specifically delineated by this transitional envelope. The distinction also has considerable significance for our understanding of insect cryobiology: firstly, because the ability to manage endogenous ice growth is a fundamental segregator of cryotype; and secondly, because our understanding of internal ice management is still largely nascent.

Keywords: crystallization, equilibrium freezing, freeze tolerance, Insecta




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CryoLetters 35 (5), 400-406 (2014)
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Pulver1*, A. Shevtsov2, B. Leybovich3, I. Artyuhov1,
Y. Maleev2 and A. Peregudov1

1Institute of Biology of Aging, LLC, 3, Begovaya alley, Moscow, 125284, Russia.
2Department of Operative Surgery and Clinical Anatomy, Burdenko Voronezh State Medical Academy, 10, Studencheskaya str., Voronezh, 394036, Russia
3Department of Pathology, Voronezh Railway Hospital, per. Zdorovja, 2, Voronezh, 394024, Russia
*Corresponding author email:


Biologic scaffold materials composed of the extracellular matrix (ECM) are typically derived by processes that involve decellularization of tissues or organs. All decellularization methods result in the ECM architecture disruption and a potential loss of surface structure and composition. Freeze-thaw processing effectively lyses cells and permits to diminish amounts of chemical lysing agents henceforth. However, it also causes certain disruptions of the ECM ultrastructure. In order to diminish these adverse effects we suggested using extracellular cryoprotectants (namely 5% trehalose) to preserve the ECM molecular network without impeding the cell lysis. The original optimization of the perfusion-mediated decellularization method to comprise the single freeze-thaw processing cycle and subsequent perfusion with chemical agents' solution is presented here.

Keywords: Tissue Engineering, Extracellular Matrix, Decellularization, Freeze-Thaw Cycle, Extracellular Cryoprotectants, Trehalose.




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CryoLetters 35 (5), 407-417 (2014)
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Anitha Karun1, K.K. Sajini 1*, V. Niral1 , C.H. Amarnath1,
P. Remya1, M.K. Rajesh1, K. Samsudeen1,
B. A. Jerard1 and Florent Engelmann2

1Central Plantation Crops Research Institute, Kasaragod, Kerala – 671 124, India.
2IRD, UMR DIADE, BP 64501, 34394 Montpellier cedex 05, France.
*Corresponding author email:


BACKGROUND: Coconut genetic resources are threatened by pests and pathogens, natural hazards and human activities. Cryopreservation is the only method allowing the safe and cost-effective long-term conservation of recalcitrant seed species such as coconut. OBJECTIVE: The objective of this work was to test the effect of cryopreservation and of cryostorage duration on coconut pollen germination and fertility. MATERIALS AND METHODS: Pollen of two coconut varieties (West Coast Tall WCT and Chowghat Orange Dwarf COD) was collected in March-May over three successive years, desiccated to 7.5 % moisture content (FW) and cryopreserved by direct immersion in liquid nitrogen. RESULTS: Germination and pollen tube length (PTL) of desiccated and cryopreserved pollen were not significantly different for both WCT and COD over the three harvest months of the three consecutive years of study. Pollen germination ranged from 24 to 32% in desiccated pollen whereas it was between 26 and 29% in cryopreserved COD pollen. In the case of WCT, germination ranged from 30 to 31% in desiccated pollen, while it was between 28 and 32% in cryopreserved pollen. PTL of cryopreserved pollen ranged between 224-390 m and 226-396 m for COD and WCT, respectively. Germination of COD pollen varied between 29.0 and 44.1% after 4 years and 1.0/1.5 years cryostorage, respectively. Germination of WCT pollen did not change significantly between 0 and 6 years cryostorage, being comprised between 32 (24 h) and 40 % (1.5 years). Germination and vigour of cryopreserved pollen were generally higher compared to that of pollen dried in oven and non-cryopreserved. Normal seed set was observed in COD and WCT palms using pollen cryostored for 6 months and 4 years. Cryopreserved pollen of five Tall and five Dwarf accessions displayed 24-31 % and 25-49 % germination, respectively.  CONCLUSION: These results show that it is now possible to establish pollen cryobanks to contribute to coconut germplasm long-term conservation.

Keywords: coconut, pollen, cryopreservation, germination, vigour, fertility




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CryoLetters 35 (5), 418-426 (2014)
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Nan Zhang1&2, Bin Wen1*, Mingyue Ji1&2 and Qi Yan1&2

1Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, Yunnan, 666303, China.
2University of Chinese Academy of Sciences, Beijing, 100049, China.
*Corresponding author email:


BACKGROUND: Grapefruit is an economically important fruit worldwide, but our knowledge of its seed biology is rather poor. OBJECTIVE: The present study aimed to develop techniques for banking and cryopreservation of grapefruit seeds. MATERIALS AND METHODS: Grapefruit seeds with the exotesta removed were used. Seeds were desiccated to three moisture levels between 5-9% and stored at 15şC, 4şC and -20şC for up to 24, months to investigate seed lifespan in conventional seed bank. Meanwhile seeds desiccated by silica gel or saturated salt solution and embryonic axes by flash drying were employed to develop cryopreservation protocols. RESULTS: It was confirmed that grapefruit seeds have some intermediate properties, being able to withstand removal of type II water up to 7% MC, but sensitive to -20°C storage. For cryopreservation, the excised embryonic axes had a wider moisture window between 5% and 15%, with a maximum past-thaw emergence of 95%, while seeds survived only with a maximum past-thaw emergence of 50% or 70% from a much narrow moisture window. CONCLUSION: In contrast to previous reports on another type II seed, coffee, we found that citrus seeds desiccated by silica gel had better post-thaw viability than those subjected to equilibrium desiccation with saturated salt solutions. Further investigation is required to elucidate the mechanisms that contribute to variable cryopreservation tolerance.

Keywords: intermediate seeds, desiccation tolerance, seed storage, equilibrium desiccation, saturated salt solutions, tropical plant germplasm conservation.




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CryoLetters 35 (5), 427-437 (2014)
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Fatih Ögretmen1* and Burak E. İnanan2

1Muğla Sıtkı Koçman University, Faculty of Fisheries Department of Aquaculture, 48000 Kötekli, Muğla, Turkey
2Muğla Sıtkı Koçman University, Faculty of Science, Department of Biology, Muğla, Turkey tel:+90 252 2113179   fax: +90 252 2113179.
*Corresponding author:


BACKGROUND: The cryopreservation procedures that allow preserving sperm cells have been applied for sperm of many species. A sugar like glucose, fructose and sucrose were frequently used in cryomedia but up to the present pine honey was not used for cryopreservation of sperm cells. OBJECTIVE: The objective of present study is to investigate the effect of pine honey in various concentrations of 100, 200, 300, 400 and 500 mg ml-1 solutions on cryopreservation and fertilization ability of spermatozoa of common carp (Cyprinus carpio).MATERIALS AND METHODS: Totally 12.5% (v/v) Me2SO as a cryoprotectant and 10% (v/v) egg yolk added all extenders. Pine honey also compared with sugars as glucose, fructose (monosaccharide) and sucrose (disaccharide). Collected semen samples were diluted at the ratio of 1:9 with the extenders. After dilutions, the sperm motility was assessed for each group and then the diluted semen samples were cryopreserved. RESULTS: The extenders containing 300 mg ml-1 pine honey group showed both highest post thaw motility 75.3±5.1%, motility duration (s) 47.3±2.5% and hatching ratio 42.6±4.2% than other cryopreserved groups (P<0.05). CONCLUSION: Using the pine honey in cryomedia is effective for cryopreservation especially about hatching success of egg fertilized by frozen-thawed sperm of common carp.

Keywords: Pine honey, sperm cryopreservation, C. carpio spermatozoa




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CryoLetters 35 (5), 438-448 (2014)
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Xueying Lu 1,2, Jieqiong Li 1, Xiaoning Liu 1, Xiaobo Li 2 and Ji Ma 1

1 Key Laboratory of Xinjiang Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqiô¬China.
2 Key Laboratory of Chemistry of Plant Resources in Arid Regions, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, China.
*Corresponding author email:


BACKGROUND: Chitinase is responsible for chitin metabolism in a wide range of organisms. However, current knowledge on insect chitinase and their possible functions in relation to low temperature stress is very limited. OBJECTIVE: Six chitinase genes from cold treated desert beetle Microdera punctipennis obtained by RNA-seq technology were characterized, and their expression patterns in different tissues and in response to cold were investigated. METHODS: Multiple sequence alignment was carried out using ClustalW1.81 and Phylogenetic trees were generated by MEGA5. The expression patterns were studied by quantitative real-time PCR. RESULTS: These genes were belong to three different chitinase groups. Almost all of them were highly expressed in midgut, and some are expressed in fat body or hindgut. Subzero-4şC had stronger effect than 4şC in inducing chitinase expression. CONCLUSION: The tissue specific and cold inducible expressions suggest that the chitinases may have diverse functions and play roles in insect cold adaptation.

Keywords: Microdera punctipennis, chitinase genes, cold stress, tissue-specific gene expression.

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