Abstracts: CryoLetters 34 (5), 2013

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



Volume 34, No. 5 September/October 2013

ISSN 0143-2044



Effects of non-toxic cryoprotective agents on the viability of cord blood derived MNCs
Akalabya Bissoyi and K. Pramanik




Is sucrose required in open pulled straw (ops) vitrification of mouse embryos?
A.N. Al Yacoub, M. Gauly and W. Holtz




Vitrification of human umbilical cord Wharton's jelly-derived mesenchymal stem cells
Ezzatabadipour Massood, Kaviani Maryam,
Salehinejad Parvin, Mohammadi Mojgan
and Nematollahi-Mahani Seyed Noureddin




Cryopreservation of Neottopteris nidus prothallus and green globular bodies by droplet-vitrification
Tengmin Li, Li Xu, Zhiying Li and Bart Panis




Medium- and long-term storage of the Pycnanthemum (mountain mint) germplasm collection
Maria M. Jenderek, Gregory E. Holman, Jeanine DeNoma
and Barbara M. Reed




Establishment and cryopreservation of a skin fibroblast cell line derived from Yunnan semi-fine wool sheep in the presence of synthetic ice blocker
Shuai Shuai Wu, Dong Jiang Li, Chun Rong Lv,
Hong Yuan Yang, Lan Zhu, Wei Juan Li, Guo Bo Quan
and Qiong Hua Hong




Development of cryopreservation for Loxocarya cinerea - an endemic Australian plant species important for post-mining restoration
Anja Kaczmarczyk, Bryn Funnekotter, Shane R. Turner,
Eric Bunn, Gary Bryant, Taavi E. Hunt and Ricardo L. Mancera




In vitro culture thawed human ovarian tissue: NIV versus slow freezing method
Zhun Xiao, Yan Wang, Ling-ling Li and Shang–Wei Li




The relation between cryoprotective and physico-chemical properties of oxyethylated methyl cellosolve-based media
Nikolenko A.V., Chekanova V.V., Schetinsky M.I.
and Vyazovska O.V.




Human spermatozoa cryopreservation: comparison of three different freezing protocols
Claudia Omes, Anna Lisa Marchetti, Maria Luisa Masanti,
Roberto Bassani, Carmine Tinelli, Rossella Chiara Sanarica,
Arsenio Spinillo and Rossella Elena Nappi




Influence of pectins on NADPH oxidase and phagocytic activity of neutrophils during cryopreservation
O.O. Zaitseva, T.V. Polezhaeva, A.N. Khudyakov,
O.N. Solomina, D.S. Laptev, E.P. Svedentsov,
S.V. Utemov and A.A. Kostyaev






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CryoLetters 34 (5), 453-465 (2013)
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Akalabya Bissoyi and K. Pramanik*

Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, India.
*Corresponding author  email:


The present work investigates the effects of a variety of natural cryoprotectants in combination on post-thaw viability and apoptosis of cryopreserved mononuclear cells (MNCs) derived from umbilical cord blood.  The extracellular cryoprotectants (10mM) namely trehalose, hydroxyl ethyl starch, polyvinyl pyrrolidine and intracellular CPAs (5mM) like erythritol, taurine and ectoine were used to prepare different combinations of freezing medium following L9 (34) Taguchi orthogonal array. Catalase, coenzyme Q10 and n-acetyl cystine (100 μg/m) were added as antioxidants. Among various combinations, freezing medium consisting of hydroxyl ethyl starch, ectoin and co-enzyme Q10 with 10% FBS is found to be most effective combination achieving maximum cell viability of 93%, 5.6% early apoptotic, 0.7% late apoptotic and 0.1% necrotic cells. SEM and phase contrast microscopy confirmed the normal cell morphology of the post-thaw cultured cells with retaining their membrane integrity. The survival rate of MNCs is higher than the rate achieved using conventional Me2SO.

Keywords: Cryopreservation, cryoprotective agents, MNCs, Umbilical Cord Blood, apoptosis, necrosis, antioxidant




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CryoLetters 34 (5), 466-470 (2013)
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A.N. Al Yacoub, M. Gauly and W. Holtz*

Department of Animal Science, Georg-August-University Goettingen, Albrecht-Thaer-Weg 3, 37075 Goettingen, Germany
*Corresonding author email:


The study addresses the relevance of sucrose with OPS vitrification of murine blastocysts. In a 3x3 factorial experiment, blastocysts were subjected to vitrification solution (20% Me2SO and 20% EG) containing 0.0, 0.4 or 0.8M sucrose and warming solution (HEPES buffered TCM199) containing 0.00, 0.25 or 0.50M sucrose. After 48h of in vitro culture, with 0.4 M sucrose in vitrification solution, 84-87% of embryos had reached the expanded blastocyst stage, as compared to 76-82% with 0.0M and 40-54% (P<0.01) with 0.8M sucrose. Hatching rates confirmed the tendency. The sucrose content of the warming solution had no significant effect on expansion or hatching rates (P>0.05). It may be concluded that, whereas, vitrification solution should contain a moderate concentration of sucrose, in dilution medium sucrose is dispensable. This implies that embryos may be transferred directly after warming, which, if applicable to farm animals, would greatly facilitate vitrification in practice.

Keywords: Cryopreservation; Open Pulled Straw; Vitrification; Sucrose; Embryo; Mouse




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CryoLetters 34 (5), 471-480 (2013)
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vitrification of human umbilical cord Wharton's jelly-derived mesenchymal stem cells

Ezzatabadipour Massood1,2, Kaviani Maryam2,*, Salehinejad Parvin5, Mohammadi Mojgan1,6, and Nematollahi-Mahani Seyed Noureddin 3,4

1Physiology Research Centre, Kerman University of Medical Sciences, Kerman, Iran.
2Department of Anatomy, Medical School, Kerman University of Medical Sciences, Kerman, Iran.
3Kerman Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran.
4Afzal Research Institute (NGO), Kerman, Iran.
5Department of Midwifery, Kerman University of Medical Sciences, Kerman, Iran.
6Department of Microbiology, Virology and Immunology, Medical School, Kerman University of Medical Sciences, Kerman, Iran.
*Corresponding author: Tel: +983413221666, email:


The aim of this study was to evaluate whether vitrification is an effective cryopreservation method for storage of human umbilical cord mesenchymal cells (HUCMs). HUCMs were vitrified using a two step method. The viability of vitrified HUCMs (v-HUCM) and non-vitrified HUCMs (n-HUCM) was determined by Trypan Blue staining. The expression of several markers was evaluated using flow cytometry. The osteogenic, adipogenic and chondrogenic differentiation potential of v-HUCMs and n-HUCMs was determined. The post-warming viability of HUCMs was 95.54 ± 2.30. The expression of surface antigens (strong positive for CD44, CD90 and CD105; negative for CD34 and CD45) was similar in the above mentioned groups. The v-HUCM cells retained ability to differentiate into osteoblasts, adipocytes and chondrocytes under appropriate culture conditions. The analysis of these results showed that vitrification is a reliable and effective method for cryopreservation of human umbilical cord mesenchymal cells.

Keywords: vitrification, human umbilical cord, mesenchymal cells, viability, stem cell markers, differentiation.




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CryoLetters 34 (5), 481-489 (2013)
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Tengmin Li1†, Li Xu1†, Zhiying Li1* and Bart Panis2

1Tropical Crops Genetic Resources Institute, Academy of Tropical Agricultural Sciences, Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China, 571737, Hainan, China.
Li Xu and Tengmi Li contributed equally to this work.
2Laboratory of Tropical Crop Improvement, Division of Crop Biotechnics, Department of Biosystems, K. U. Leuven, B3001 Leuven, Belgium.
*Corresponding author email:


Neottopteris nidus prothalli and green globular bodies (GGBs) were successfully cryopreserved by droplet-vitrification. Prothalli were subjected to different treatments. The following parameters were studied: the age of in vitro mother plants from which prothalli were originated (30 to 90-day old), length of exposure to loading solution (LS) (20 to 40 min) and length of exposure to the plant vitrification solution (PVS2) (10 to 55 min). N. nidus GGBs and GGBs in suspension were subjected to PVS2 for 20, 30 and 40 min before liquid nitrogen exposure. The highest prothalli regrowth (92%) occurred when they were exposed for 40 min to LS, followed by 20 min to PVS2 and when they originated from non-preconditioned 45-day old mother plants. The highest GGB (100%) and GGB suspension regrowth (100%) after cryopreservation occurred when they were exposed to PVS2 for either 20 min or 40 min.

Keywords: cryopreservation, droplet-vitrification, green globular bodies, Neottopteris nidus, prothallus




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CryoLetters 34 (5), 490-496 (2013)
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Maria M. Jenderek1*, Gregory E. Holman1, Jeanine DeNoma2 and Barbara M. Reed2

1USDA-ARS National Center for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, CO 80521
2USDA-ARS National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, OR 97333-2521
*Corresponding author  email:


The United States of America collection of mountain mint (Pycnanthemum Michx.) is held at the USDA-ARS National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon as seed, potted plants and tissue cultures and a long-term storage collection is preserved at the USDA-ARS National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, Colorado. The clonal collection is comprised of 34 accessions as potted plants that are duplicated with 31 accessions stored as in vitro cultures at 4°C in tissue culture bags for medium-term storage at NCGR and as cryopreserved shoot tips in liquid nitrogen at NCGRP for long-term storage. This study reports on these two models of preservation of mountain mint at the U.S. National Plant Germplasm System. In vitro plants required 2 to 7 months for propagation on MS medium without growth regulators before storage at 4°C. Plants remained in storage with good vigour in bags on ˝x nitrogen MS medium without growth regulators for a mean of 2.08 y. An encapsulation-dehydration protocol was successful for cryopreservation of shoot tips from cold acclimated in vitro plants. Post-cryo viability, indicated by shoot tips with developed leaves and roots, ranged from 60 to 100 % for 27 accessions and 40 to 50 % for the other four. The encapsulation-dehydration cryopreservation method proved suitable for long-term preservation of the 31 Pycnanthemum accessions. These alternative storage forms allow for active use of the collection as well as base storage for clonally propagated accessions.

Keywords: cold storage, cryopreservation, encapsulation-dehydration, genetic resources, micropropagation




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CryoLetters 34 (5), 497-507 (2013)
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Shuai Shuai Wu,  Dong Jiang Li, Chun Rong Lv, Hong Yuan Yang, Lan Zhu, Wei Juan Li, Guo Bo Quan and Qiong Hua Hong*

Yunnan Animal Science and Veterinary Institute, Qinglongshan, Jindian, the Panlong County, Kunming 650224, Yunnan province, China
*Corresponding Author e-mail:

Statement of funding: This study was funded by the National Wool Caprine Industrial Technology System (Grant No. CARS-40) and the National Infrastructure of Animal Genetic Resources (NIAGR).


In this study, the fibroblasts cell line derived from ear marginal tissue of Yunnan semi-fine wool sheep was successfully established using the primary explants technique and cryopreservation technology. Additionally, the protective effect of synthetic ice blocker (SIB) including 1, 3-cyclohexanediol (1, 3-CHD) and 1, 4-cyclohexanediol (1, 4-CHD) on frozen fibroblast cells was also assessed and compared. Propidium iodide (PI) was used to stain the dead cells following cryopreservation and thawing. The results showed that compared with Medium 199 (M199) and Dulbecco's modified Eagle's medium : Nutrient Mixture F-12 (1:1) Mixture (DMEM/F12), Dulbecco's modified Eagle's medium (DMEM) may be more suitable for the primary culture of fibroblast cells of Yunnan semi-fine wool sheep. The growth curve of cells is a typical "S" type. After subculture for four days, the cells entered the plateau phase and began to degenerate. Biological analysis showed that the population doubling time (PDT) for subculturing fibroblast cells was approximately 26h. The Karyotyping data indicated that the percentage of fibroblast cells with normal chromosome number 2n=54 was over 90% following subculture for 10 passages. Moreover, the tests for bacteria, fungi, viruses and mycoplasma were negative. After serial subculture for 5 generations, the fibroblast cells were cryopreserved in the presence or absence of 1, 3-CHD or 1, 4-CHD. The data indicated that with increase of the synthetic ice blocker concentrations, the viability of frozen-thawed fibroblast cells was firstly increased and then decreased. When the concentration of 1, 3-CHD or 1, 4-CHD was 50mM, the viable percentage of frozen-thawed fibroblast cells was 91.93%±2.24% and 94.13%±0.55% respectively and significantly higher than that of the cells frozen in the absence of synthetic ice blockers (88.10%±1.49%, P<0.05). In conclusion, the skin fibroblast cell line of Yunnan semi-fine wool sheep was firstly established in this study. Additionally, the presence of synthetic ice blocker can increase the viability of frozen-thawed sheep fibroblast cell line.

Keywords: Yunnan semi-fine wool sheep; fibroblast cell line; cryopreservation; synthetic ice blocker




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CryoLetters 34 (5), 508-519 (2013)
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Anja Kaczmarczyk1, 2, Bryn Funnekotter1,2, Shane R. Turner 2,3, Eric Bunn 2,3, Gary Bryant4, Taavi E. Hunt4 and Ricardo L. Mancera1*

1School of Biomedical Sciences, CHIRI Biosciences Research Precinct, Faculty of Health Sciences, Curtin University, GPO Box U1987, Perth WA 6845, Australia.
2Botanic Gardens and Parks Authority, Fraser Avenue, West Perth WA, 6005, Australia.
3School of Plant Biology, Faculty of Natural and Agricultural Sciences, University of Western Australia, Crawley WA, 6009, Australia.
4Applied Physics, School of Applied Sciences, RMIT University, GPO Box 2476, Melbourne, VIC 3001, Australia.
*Corresponding author email:


We report the development of a cryopreservation protocol for the endemic Western Australian plant species Loxocarya cinerea (Restionaceae). Shoot tips from two genotypes, SXH404 and SXH804, were cryopreserved using the droplet-vitrification technique. Control explants, which were cryoprotected, but not cooled, showed regeneration for both genotypes (SXH404, 22.1 ± 5.9%; SXH804, 67.7 ± 9.6%). Extension of incubation in PVS2 from 30 to 60 min did not lead to survival after cryopreservation. Thermal analysis using differential scanning calorimetry confirmed the beneficial effect of a loading phase but also revealed no or very little ice formation after cryoprotection of shoot tips in other treatments. Regeneration following cryopreservation was obtained for genotype SXH804 (4.3 ± 2.1%) but not for SXH404. Regenerated explants of L. cinerea SXH804 were morphologically identical to tissue-cultured plants. As an alternative to shoot tips, callus tissues of clone SXH404 were successfully cryopreserved (>66.7% post LN survival) using the same protocol.

Keywords: cryopreservation, differential scanning calorimetry, droplet vitrification, Loxocarya cinerea, tissue culture




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CryoLetters 34 (5), 520-526 (2013)
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Zhun Xiao1, Yan Wang1, Ling-ling Li 2and Shang–Wei Li1*

1Reproductive Medical Center of West China 2nd University Hospital, Sichuan University, Chengdu 610041, China.
2Department of Obstetrics and Gynecology of Sichuan Provincial Hospital for Women and Children, Chengdu 610031, China.
*Correspondence author e-mail:


The aim of this study was to determine if the needle immersed vitrification method (NIV) can improve the growth potential of thawed ovarian tissue in vitro culture. Human ovarian cortical tissues were cryopreserved using NIV and slow freezing method. After 14 days of culture, the preservation outcomes of NIV and slow freezing groups were analyzed histologically using light microscope and apoptosis was assessed by TUNEL assay. The result showed that the percentage of morphologically abnormal primordial follicles was lower in NIV group than in slow freezing group (P<0.05). The incidence of TUNEL-positive primordial follicles was lower in NIV group than in slow freezing group (P<0.05). The study showed that cryopreservation of human ovarian tissue with NIV was effective in improving the growth potential of frozen-thawed ovarian tissue in vitro culture.

Keywords: cryopreservation, human, ovarian tissue, vitrification, primordial follicle




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CryoLetters 34 (5), 527-534 (2013)
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Nikolenko A.V., Chekanova V.V., Schetinsky M.I. and Vyazovska O.V.*

Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine, Kharkov, Ukraine.
*Corresponding author: email:


Oxyethyl derivatives of polyol class members such as glycerol and ethylene glycol with various polymerization degrees possess low toxicity and cryoprotective properties via an exocellular mechanism. To develop the cryoprotective media based on non-penetrating cryoprotectants, we studied cryoprotective and physico-chemical properties of oxyethylated methyl cellosolve (OEMC)-based media. Media containing 20% and 30% OEMC were prepared with 50 mmol/l and 150 mmol/l NaCl. In some media, NaCl content was reduced, and sucrose, mannitol or glucose was added to attain the isotonic media. Cryopreservation of red blood cells depends on the media composition and physico-chemical properties. The increases in dynamic viscosity due to higher OEMC concentration and in ionic strength resulted from higher NaCl concentration lead to greater erythrocytes dehydration prior to freezing, and lower red blood cell preservation upon freezing. Salt substitution with carbohydrates in 20% OEMC media reduces ionic strength and increases OEMC distribution coefficient. The correlation of red blood cell protection upon freezing is discussed in terms of the media physico-chemical properties (ionic strength, viscosity and surface tension), cryoprotectant distribution coefficient, and cell dehydration.

Keywords: cryopreservation, oxyethylated methyl cellosolve, red blood cells




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CryoLetters 34 (5), 535-543 (2013)
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Human spermatozoa cryopreservation: comparison of three different FREEZING protocols

Claudia Omes1*, Anna Lisa Marchetti2, Maria Luisa Masanti3, Roberto Bassani1, Carmine Tinelli4, Rossella Chiara Sanarica1, Arsenio Spinillo1,3,5 and Rossella Elena Nappi1,3,5

1Research Center for Reproductive Medicine, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
2Scientific Direction, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
3Department of Clinical, Surgical, Diagnostic and Pediatric Sciences, University of Pavia
4Clinical Epidemiology and Biometric Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
5Maternal Infant Department, Obstetrics and Gynecology Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
*Corresponce author e-mail:


Human spermatozoa cryopreservation has significantly improved over the last few decades, but the actual protocols are neither optimal nor standardized between different laboratories in spite of the importance of preserving male fertility or treating severely infertile males.
In the present study we aimed to determine the best in-house method of rapid freezing in terms of sperm motility and vitality by comparing three different rapid methods of human spermatozoa cryopreservation.
Our data showed that M1 (triphasic cooling) is the method associated with a significantly lower deterioration of semen quality in comparison with mono or biphasic cooling. Differences observed among the three protocols were supported by statistical analysis.
These data reinforce previous evidences about the influence of sperm quality on IVF outcome and suggest the importance of improving sperm cryopreservation techniques especially when semen is seriously compromised at baseline.

Keywords: freezing protocol, human spermatozoa cryopreservation, fertility preservation, sperm viability, sperm motility.




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CryoLetters 34 (5), 544-548 (2013)
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O.O. Zaitseva1*, T.V. Polezhaeva1, A.N. Khudyakov1*, O.N. Solomina1, D.S. Laptev1, E.P. Svedentsov, S.V. Utemov2 and A.A. Kostyaev2

1Laboratory of Cryophysiology of Blood (T.V. Polezhaeva) of the Physiology Institute at the Komi Scientific Center affiliated to the Ural Branch of the Russian Academy of Sciences, Komi Republic, Syktyvkar, Russian Federation.
2Kirov Research Institute of Haematology and Blood Transfusion, Kirov, Russian Federation
*Corresponding authors:;


NADPH oxidase activation was studied by detecting the reactive oxygen species (ROS) and the dynamics of neutrophil phagocytic activity in blood before freezing and one day after exposure to -20°C in the presence of pectic polysaccharides. Pectins (excluding lemnan) were found to increase the phagocytic activity of neutrophils, promote the formation of ROS in these cells and enhance NADPH oxidase activity in the following order: bergenan, comaruman, lemnan, apiuman, and potamogetonan. Cryoprotective action of lemnan and comaruman was proposed to be related to their unique chemical structures.

Keywords: NADPH oxidase, phagocytosis, neutrophils, pectins, cryopreservation.

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