Abstracts: CryoLetters 34 (3), 2013

CryoLetters is a bimonthly, international journal for low temperature science and technology



Volume 34, No. 3 May/June 2013

ISSN 0143-2044



The effects of 1, 4-cyclohexanediol on frozen ram spermatozoa
Guo Bo Quan, Shuai Shuai Wu, Zhi Gang Lan,
Hong Yuan Yang, Qing Yong Shao and Qiong Hua Hong




Effects of different cryoprotectant combinations on primordial follicle survivability and apoptosis incidence after vitrification of whole rat ovary
Rouhollah Fathi, Mojtaba Rezazadeh Valojerdi
and Mojdeh Salehnia




Immediate induction of heat shock proteins is not protective against cryopreservation in normal human fibroblasts
Sung-Jun Park, Hye-Ryung Choi, Kyoung-Mi Nam,
Jung-Im Na, Chang-Hun Huh and Kyoung-Chan Park




Confocal raman microscopy as a non-invasive tool to investigate the phase composition of frozen complex cryopreservation media
Asger Kreiner-Møller, Frank Stracke and Heiko Zimmermann




Heat transfer coefficient of cryotop during freezing
Weijie-Li, Xinli-Zhou, Haisong-Wang, Baolin-Liu
and Jianjun-Dai




Thermocouple design for measuring temperatures of small insects
A.A. Hanson and R.C. Venette




A refinement to the liquidus-tracking method for vitreous preservation of articular cartilage
Xiaoyi Yu, Guangming Chen and Shaozhi Zhang




Translating cryobiology principles into trans-disciplinary storage guidelines for biorepositories and biobanks : A concept paper
Erica E Benson, Fotini Betsou, Barry J Fuller, Keith Harding
and Olga Kofanova






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CryoLetters 34 (3), 217-227 (2013)
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Guo Bo Quan, Shuai Shuai Wu, Zhi Gang Lan, Hong Yuan Yang, Qing Yong Shao and Qiong Hua Hong*

Yunnan Animal Science and Veterinary Institute, Qinglongshan, Jindian, the Panlong County, Kunming 650224, Yunnan province, China
*Corresponding author: Mrs Qiong Hua Hong, M.D. e-mail:


In order to improve the quality of frozen spermatozoa of Yunnan semi-fine wool sheep, 1, 4-cyclohexanediol (1, 4-CHD) as a synthetic ice blocker was used for cryopreservation of ram spermatozoa in this study. Briefly, following collection by electric stimulation, equilibration at 5°C following dilution with the freezing extender, and pre-freezing in liquid nitrogen vapor, the ram spermatozoa were preserved in liquid nitrogen for one month. In addition, the effects of osmolarity of the diluting extenders used for evaluation of frozen spermatozoa quality were also assessed. The results indicated addition of 1, 4-CHD could not increase the motility of ram spermatozoa after cryopreservation and thawing. With the elevation of the concentrations of 1, 4-CHD, the motility and moving velocity of frozen ram spermatozoa showed a steady decrease. Additionally, the presence of 1, 4-CHD can not increase the percentage of frozen spermatozoa with intact acrosome and membrane. When the isotonic binding buffer was used to dilute the thawed spermatozoa, the percentage of cells labeled with propidium iodide (PI) after cryopreservation in the presence of 1, 4-CHD was significantly higher than that of spermatozoa frozen in the absence of 1, 4-CHD (P<0.05). However, the percentage of frozen-thawed spermatozoa with exposed PS in the presence of 1, 4-CHD was significantly less than that of spermatozoa frozen in the absence of 1, 4-CHD (P<0.01). When the basic extenders with an osmolarity of 404mOsm, 528mOsm, 648mOsm, or 853mOsm were used to dilute the frozen-thawed spermatozoa respectively, there is no significant difference between the four groups with respect to the moving velocity and membrane integrity (P>0.05). In conclusion, the presence of 1, 4-CHD can not improve the motility, moving velocity, acrosome staus, and membrane integrity of frozen ram spermatozoa. However, 1, 4-CHD may inhibit apoptosis caused by freezing and thawing.

Keywords: ram; spermatozoa; cryopreservation; synthetic ice blocker; 1, 4-cyclohexanediol, freezing




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CryoLetters 34 (3), 228-238 (2013)
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Rouhollah Fathi1, Mojtaba Rezazadeh Valojerdi1*, 2, Mojdeh Salehnia1

1Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-111, Tehran, Iran
2Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
*Corresponding author e-mail: or


This study was aimed at obtaining best vitrification conditions for preservation of primordial follicles after vitrification of whole ovarian tissue of rats. Ovaries of prepubertal ~5-week old female Wistar rats were divided randomly into 7 groups: Control (non-vitrified), VI (EG+DMSO), VII (EG+PROH), VIII (DMSO+PROH), VIV (EG+DMSO+Sucrose), VV (EG+PROH+Sucrose) and VVI (DMSO+PROH+Sucrose). Control and vitrified-warmed samples were sectioned serially and stained either with H&E or anti & pro active caspase-3 kit. The number of intact follicles in different stages of development was lower and the number of atretic and apoptotic follicles was higher in vitrification groups than those of the control group. Cryoprotectant combinations in VIV group showed better follicular preservation especially for primordial follicle. VIII, VIV and VV were best cryoprotectant mixtures, after the control group, according to the number of atretic follicles but the incidence of apoptotic primordial follicles was lowest in VIII, VIV and VVI. Incidence of apoptosis and the number of atretic follicles were lowest in VIII and VIV groups, and there was better primordial follicle preservation and survivability in VIV group. Thus, the combination of EG + DMSO with sucrose appears to be better suited for vitrification of whole ovarian tissue of rats.

Keywords: rat ovary, primordial follicles, vitrification, different cryoprotectants, caspase 3




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CryoLetters 34 (3), 239-247 (2013)
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Sung-Jun Parka, Hye-Ryung Choia, Kyoung-Mi Nam, Jung-Im Na, Chang-Hun Huh and Kyoung-Chan Park*

Department of Dermatology, Seoul National University Bundang Hospital, Gumi-ro 166 Bundang-gu, Seongnam-si, Gyeonggi-do, 463-707, Republic of Korea,
aThese authors contributed equally to this work.
*Corresponding author  e-mail:


Heat shock proteins (HSPs) were first identified as proteins whose synthesis was enhanced by stresses, such as increased temperature. HSPs can protect cells from various cytotoxic factors by stabilizing proteins. Thus, it could be hypothesized that heat induced HSPs can provide protective effects against cryopreservation-induced cell death. The aim of this study was to determine whether induction of HSPs can increase the cell viability of normal human fibroblasts after cryopreservation. Cytotoxic effects of heat treatment were tested and the induction of HSPs was assessed by examining time-dependent HSP expression. A cell counting method using fluorescence microscopy was used to determine the viability of cells. In addition, the effects of geranylgeranylacetone were evaluated in terms of HSP expression and cytoskeleton changes. The results of this study showed that immediate induction of HSPs does not protect normal human fibroblasts against cryopreservation-induced cell death possibly by inducing cytoskeleton changes.

Keywords: Heat shock proteins, cryopreservation, actin, normal human fibroblasts




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CryoLetters 34 (3), 248-254 (2013)
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Confocal Raman microscopy as a non-invasive tool to investigate the phase composition of frozen complex cryopreservation media

Asger Kreiner-Møller, Frank Stracke and Heiko Zimmermann*

Fraunhofer Institute for Biomedical Engineering, Ensheimer Strasse 48, 66386 St. Ingbert, Germany.
*Corresponding author e-mail:


Various cryoprotective agents (CPA) are added to cell media in order to avoid cell injury during cryo preservation. The resulting complex environment of the preserved cell, consisting of crystalline and liquid phases can however not be investigated non-invasively by established methods in cryobiology. This study shows how scanning confocal Raman microscopy can non-invasively extract information on chemical composition, phase domain and distribution at cryogenic temperatures. The formation of the salt hydrate, hydrohalite NaCl∙H2O, in solutions comprised of phosphate buffered saline (PBS) and dimethyl sulphoxide (DMSO) is studied in particular. Scanning confocal Raman microscopy can be used to unambiguously identify hydrohalite in a medium containing DMSO and saline. The confocal Raman microscopy imaging along with differential scanning calorimetric measurements further show that the hydrohalite is formed without eutectic formation. This method also allows for discrimination between closely packed hydrohalite crystals that are oriented differently.

Keywords: Scanning Confocal Raman Microscopy, Hydrohalite, DMSO, Eutectic formation, Cryopreservation, Phase Texture




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CryoLetters 34 (3), 255-260 (2013)
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Heat Transfer Coefficient of Cryotop during Freezing

Weijie-Li1, Xinli-Zhou1, Haisong-Wang1, Baolin-Liu1 and Jianjun-Dai

1Institute of Biomedical Technology, University of Shanghai for Science and Technology, Shanghai, 200093, China. E-mail:
2Animal and Veterinary Research Institute, SAAS, Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding, Shanghai 201106, China


Cryotop is an efficient vitrification method for cryopreservation of oocytes. It has been widely used owing to its simple operation and high freezing rate. Recently, the heat transfer performance of cryotop was studied by numerical simulation in several studies. However, the range of heat transfer coefficient in the simulation is uncertain. In this study, the heat transfer coefficient for cryotop during freezing process was analyzed. The cooling rates of 40% ethylene glycol (EG) droplet in cryotop during freezing were measured by ultra-fast measurement system and calculated by numerical simulation at different value of heat transfer coefficient. Compared with the results obtained by two methods, the range of the heat transfer coefficient necessary for the numerical simulation of cryotop was determined, which is about 9000 W/(m2·K)<h<10000 W/(m2·K).

Keywords:  Cryotop, heat transfer coefficient, numerical simulation, vitrification.




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CryoLetters 34 (3), 261-267 (2013)
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Thermocouple design for measuring  temperatures of small  insects

A.A. Hanson1 and R.C. Venette2*

1Department of Entomology, University of Minnesota, St. Paul, MN 55108, USA.
2USDA, Forest Service Northern Research Station, St. Paul, MN 55108, USA.
*Corresponding author e-mail:


Contact thermocouples often are used to measure surface body temperature changes of insects during cold exposure. However, small temperature changes of minute insects can be difficult to detect, particularly during the measurement of supercooling points. We developed two thermocouple designs, which use 0.51 mm diameter or 0.127 mm diameter copper-constantan wires, to improve our ability to resolve insect exotherms. We tested the designs with adults from three parasitoid species: Tetrastichus planipennisi, Spathius agrili, and S. floridanus. These species are <3 mm long and <0.1 mg. Mean exotherms were greater for fine-gauge thermocouples than thick-gauge thermocouples for the smallest species tested, T. planipennisi. This difference was not apparent for larger species S. agrili and S. floridanus. Thermocouple design did not affect the mean supercooling point for any of the species. The “cradle” thermocouple design developed with the fine gauge wire was reusable and allowed for easy insect recovery after cold exposure.

Keywords: supercooling point, exotherm, parasitoid




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CryoLetters 34 (3), 267-276 (2013)
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A refinement to the liquidus-tracking method for vitreous preservation of articular cartilage

Xiaoyi Yu, Guangming Chen and Shaozhi Zhang*

Institute of Refrigeration and Cryogenics, Zhejiang University, Hangzhou 310027, China
*Corresponding author e-mail:


The recent liquidus-tracking method developed by Pegg et al. (2006a), as an alternative pathway to vitrification, achieved reasonable survival of post-thawing chondrocytes in situ. One of the main drawbacks of this method is the long duration of the cryoprotectant addition/removal process. This study was conducted to investigate the possibility of reducing the time by rationalizing the final dimethyl sulfoxide (Me2SO) concentration loaded in tissue before being plunged into liquid nitrogen. Using the differential scanning calorimetric technique, the critical cooling and warming rates for solutions of Me2SO in CPTes2 (a potassium-rich medium, modified slightly from Taylor’s original formulation by Pegg et al.) were obtained. The critical cooling and warming rates for 47.5%(w/w) solution are < 2.5°C/min and < 10° C/min, respectively, which could be readily realized for 4 ml solution samples held in polypropylene cryovials as demonstrated by experiments. For articular cartilage, 47.5%(w/w) may be recommended as the final concentration of Me2SO loaded in the tissue, which will lead to a time cut of about one-third compared with the original protocol of Pegg et al. (2006a).

Keywords: Critical cooling rate, Critical warming rate, Dimethyl sulfoxide, Liquidus tracking, Articular cartilage, Vitrification




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CryoLetters 34 (3), 277-312 (2013)
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Erica E Benson1*, Fotini Betsou2, Barry J Fuller3, Keith Harding1
and Olga Kofanova2

1Damar Research Scientists, Drum Road, Cuparmuir, Fife, Scotland, UK
2Integrated Biobank of Luxembourg, 6, rue Ernest Barblé, L-1210 Luxembourg
3University Department of Surgery, Royal Free and University College Medical School, UCL, London UK
*Corresponding author  e-mail:


Low temperatures are used routinely to preserve diverse biospecimens, genetic resources and non-viable or viable biosamples for medical and clinical research in hospital-based biobanks and non-medical biorepositories, such as genebanks and culture, scientific, museum, and environmental collections. However, the basic knowledge underpinning preservation can sometimes be overlooked by practitioners who are unfamiliar with fundamental cryobiological principles which are more usually described in research literature rather than in quality and risk management documents. Whilst procedures vary, low temperature storage is a common requirement and reaching consensus as to how best it is applied could facilitate the entire biopreservation sector. This may be achieved by encouraging an understanding of cryoprotection theory and emphasizing the criticality of thermal events (glass transitions, ice nucleation, thawing) for sample integrity, functionality and stability. The objective of this paper is to inspire diverse biopreservation sectors to communicate more clearly about low temperature storage and, raise awareness of the importance of cryobiology principles to field newcomers and biopreservation practitioners, by considering how the principles may be translated into evidence-based guidelines for biobank and biorepository operations.

Keywords: biobank, biorepository, biopreservation, cryobiology, storage guidelines

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