Abstracts: CryoLetters 37 (4), 2016

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



Volume 37, No. 4 July/August 2016

ISSN 0143-2044



Seed desiccation tolerance and cryopreservation of Philippine Calamansi [x Citrofortunella microcarpa (BUNGE) wijnands]
Olivia P. Damasco and Luzminda C Refuerzo




Cryopreservation of adventitious roots of Cleome rosea Vahl (CLEOMACEAE) using a vitrification technique and assessment of genetic stability
Lívia da Silva Cordeiro, Claudia Simões-Gurgel
and Norma Albarello




Viability, in vitro differentiation and molecular characterization of Equine adipose tissue-derived mesenchymal stem cells cryopreserved in serum and serum-free medium
Barbara Merlo, Stefania Pirondi, Eleonora Iacono,
Barbara Rossi, Francesca Ricci and Gaetano Mari




ROS-induced oxidative stress in nobile-type Dendrobium protocorm-like bodies (PLBS) during vitrification
Mengxue Jia, Wei Di, Yan Liu, Yin Shi and Yingran Xie




Characteristics and fertility of Sumatran Tiger spermatozoa cryopreserved with different sugars
Ni Wayan Kurniani Karja, Mokhamad Fahrudin,
Mohamad Agus Setiadi, Ligaya ITA Tumbelaka,
Retno Sudarwati, Yohana Tri Hastuti, Bongot Huaso Mulia,
Ardyta Widianti, Keni Sultan, Tsukasa Terazono, Zhao Namula,
Masayasu Taniguchi, Fuminori Tanihara, Tatsuya Takemoto,
Kazuhiro Kikuchi, Yoko Sato and Takeshige Otoi




The effects of a simple method for cryopreservation and thawing procedures on cord blood derived DC-based esophageal carcinoma vaccine : Running title: Cryopreservation Of fused carcinoma vaccine
Jing Yu, Lihua Xie Suzuan Chen, Juan Zhang, Guanghua Guo
and Binming Chen




Post-harvest embryo development in Ginseng seeds increases desiccation sensitivity and narrows the hydration window for cryopreservation
Eun-Ju Han, Elena Popova, Gyu-Taek Cho, Sang-Un Park,
Sheong-Chun Lee, Hugh W. Pritchard and Haeng-Hoon Kim




Effect of NAC on Mouse GV oocyte survival and subsequent embryonic development following vitrfication
Shun-Li Yue, Yu-Ting Zhang, Shun-Wei Wang, Meng Sun,
Yue-Chao Xing, Jing Wen and Jia-Bo Zhou






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CryoLetters 37 (4), 219-230 (2016)
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Olivia P. Damasco* and Luzminda C Refuerzo

National Plant Genetic Resources Laboratory-Institute of Plant Breeding-Crop Science Cluster, College of Agriculture, University of the Philippines Los Banos, College, 4031 Laguna, Philippines
*Corresponding author email:


BACKGROUND: The traditional on farm conservation of Calamansi [x Citrofortunella microcarpa (Bunge) Wijnands], an important indigenous Citrus species in the Philippines, is now being threatened by shifting agricultural crop production, climate change, and increasing biotic and abiotic stresses. OBJECTIVE: The study aimed to characterize the desiccation and cryopreservation tolerance of seeds as the basis for complementary long term ex situ conservation.  MATERIALS AND METHODS: Intact seeds were desiccated in an airtight container filled with activated silica gel for 0-96 h. Seeds placed in cryotubes were subjected to rapid freezing in liquid nitrogen, rapid thawing in a water bath at 50C for 3 min, and cultured on MS basal medium for seedling recovery and growth. Recovered seedlings were potted out in plastic bags filled with coir dust: garden soil mixture (1:1 v/v) and maintained in the nursery.  RESULTS: Significant reduction in percentage germination was obtained at in a moisture content (MC) window between 24.3% and 4.2% and complete loss of viability at below 3.2%. The number of germinated embryos per seed was significantly reduced following desiccation from a mean of 4.2 embryos per seed for the untreated control to 1.2 to 1.02 embryos per seed at 33.3-4.2% MC, respectively. Recovery and germination of seeds after cryopreservation were obtained in a MC window between 24.3% and 4.2% with the maximum seed germination (27%) obtained at 13.4%. Germination abnormalities such as incomplete germination, greening and or enlargement of cotyledon without shoot emergence were observed in both desiccated and cryopreserved seeds. Variations in response to seed desiccation and cryopreservation were observed among Calamansi accessions tested. Maximum seedling recovery after liquid nitrogen storage varied between 12.5% and 61.5%. Recovered seedlings from desiccation and cryopreservation treatments survived ex vitro establishment and showed normal growth and similar morphology with the non-treated control seedlings. CONCLUSION: The partial tolerance of Calamansi seeds to desiccation and subsequent recovery after cryopreservation provides the basis for long term ex situ preservation of this valuable germplasm, although further optimization is needed.

Keywords: seed desiccation, cryopreservation, Calamansi, Citrofortunella microcarpa




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CryoLetters 37 (4), 231-242 (2016)
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Lívia da Silva Cordeiro*, Claudia Simões-Gurgel and Norma Albarello

Núcleo de Biotecnologia Vegetal (NBV), Instituto de Biologia Roberto Alcantara Gomes (IBRAG), Universidade do Estado do Rio de Janeiro (UERJ), Rua São Francisco Xavier, 524, PHLC, sala 509, CEP 20550-013, Rio de Janeiro, RJ, Brazil
*Corresponding author e-mail:


BACKGROUND: Cleome rosea, a Brazilian native species, has medicinal potential. Previously a cryopreservation protocol for in vitro roots using the vitrification solution PVS2 has been developed. However, the genetic stability of the cryopreserved material is yet to be assessed. OBJECTIVES: To evaluate the effects of loading and vitrification solutions (PVS2 and PVS3) on post-cryopreservation recovery of C. rosea roots, and to assess their genetic stability using Random Amplified Polymorphic DNA (RAPD) markers. MATERIALS AND METHODS: Root segments were pretreated with increasing concentrations of sucrose (0.2 to 0.4 M), followed by osmoprotection with loading solution and treatment with one of the vitrification solutions tested.  RESULTS: The highest recoveries using PVS2 and PVS3 were obtained when root segments were exposed to these solutions for 15 min, reaching 77% and 100% respectively. The RAPD band profiles were monomorphic with most of the primers used. This molecular analysis revealed high genetic similarity (similarity coefficients among 0.98 and 1.00) between the cryopreserved roots and their mother plants. CONCLUSION: Roots from in vitro-propagated plants of C. rosea, were successfully cryopreserved using the vitrification technique. No major variations were observed on the genetic stability of cryopreserved roots, validating the use of this protocol as an efficient long-term conservation option for this species.

Keywords: in vitro roots, long-term conservation, medicinal plant, PVS2, PVS3, RAPD.




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CryoLetters 37 (4), 243-252 (2016)
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Barbara Merlo1*, Stefania Pirondi2, Eleonora Iacono1, Barbara Rossi1,
Francesca Ricci3 and Gaetano Mari1

1Department of Veterinary Medical Sciences, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia (BO), Italy;
2 IRET Foundation, Via Tolara di Sopra 41/E, Ozzano Emilia (BO), Italy;
3Service of Transfusion Medicine, S.Orsola-Malpighi Hospital, Via Massarenti 9, 40126 Bologna, Italy.
*Corresponding author email:


BACKGROUND: The effect of freezing-thawing equine adipose tissue-derived mesenchymal stem cells (eATMSCs) have been poorly investigated.  OBJECTIVE: This study is to test the influence of cryopreservation solution and temperature when adding the cryoprotectant for freezing eATMSCs, and to investigate the effects of cryopreservation on their stemness features.  MATERIALS AND METHODS: Four freezing protocols were evaluated. Viability and proliferation ability of cryopreserved cells were investigated by MTT assay. Fresh and frozen thawed eATMSCs were compared for morphology, phenotypic characteristics (flow cytometry), and differentiation potential. RESULTS: A higher value of viable cells for samples frozen in FBS and a positive effect of CPA equilibration at low temperature in samples frozen in medium were observed. Morphology was similar for fresh and cryopreserved cells, such as CD expression and differentiation potential. CONCLUSION: eATMSCs can be safely stored for clinical use. FBS is superior to medium for freezing, but CPA equilibration at low temperature is beneficial when freezing in serum- free medium.

Keywords: Equine, adipose tissue, mesenchymal stem cells, cryopreservation.




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CryoLetters 37 (4), 253-263 (2016)
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Mengxue Jia#, Wei Di#, Yan Liu*, Yin Shi and Yingran Xie

Beijing Laboratory of Urban and Rural Ecological Environment, Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, National Engineering Research Center for Floriculture, College of Landscape Architecture, Beijing Forestry University, Beijing 100083, People’s Republic of China
*Corresponding author email:;
#contributed equally to this work


BACKGROUND: Oxidative stress involved in cryopreservation protocols may be responsible for the poor survival of tissues after cryopreservation. OBJECTIVE: In the current study, we aimed to clarify the role of oxidative stress and its relationship with survival rate during cryopreservation of PLBs from nobile-type Dendrobium. MATERIALS AND METHODS: ROS, antioxidants and oxidative products and the survival rate in PLBs from Dendrobium Hamanal Lake Dream were determined during vitrification. RESULTS: Relative survival of PLBs decreased significantly after preculture and rewarming (P<0.01). Generation of ·O2 and protein carbonyl (PCO) increased significantly after preculture. Dramatic increases in ·O2, H2O2 and MDA, and significant decreases in AsA content, activities of SOD and CAT were observed after rewarming. CONCLUSION: ROS-induced oxidative stress was associated with the poor survival of PLBs following vitrification. ·O2 was the predominant ROS resulting in the decreased survival after preculture, while H2O2 together with ·O2 appear to be responsible for the survival decrease after rewarming.

Keywords: reactive oxygen species, oxidative damage, cryopreservation,·orchid




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CryoLetters 37 (4), 264-271 (2016)
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Ni Wayan Kurniani Karja1, Mokhamad Fahrudin1,
Mohamad Agus Setiadi1, Ligaya ITA Tumbelaka1,2,
Retno Sudarwati2, Yohana Tri Hastuti2, Bongot Huaso Mulia2,
Ardyta Widianti2, Keni Sultan2, Tsukasa Terazono3,
Zhao Namula3, Masayasu Taniguchi3, Fuminori Tanihara3,4,
Tatsuya Takemoto5, Kazuhiro Kikuchi6, Yoko Sato3*
and Takeshige Otoi3,4

1Faculty of Veterinary Science, Bogor Agricultural University, Indonesia.
2Taman Safari Indonesia, Bogor, Indonesia.
3The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan.
4Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan.
5Fujii Memorial Institute of Medical Sciences, Tokushima University, Tokushima, Japan.
6Department of Animal Science, National Institute of Agrobiological Sciences, Ibaraki, Japan
*Corresponding author email:


BACKGROUND: Cryopreservation of semen is one of the most important methods for the preservation of endangered tigers. OBJECTIVE: This study evaluated the effects of sugar supplementation on the cryosurvival of spermatozoa from Sumatran tigers (Panthera tigris sumatrae). MATERIALS AND METHODS: The post-thaw characteristics and fertility of spermatozoa cryopreserved with different sugars (glucose, lactose, and trehalose) were evaluated using heterologous in-vitro fertilisation with cat oocytes. RESULTS: All parameters of post-thaw spermatozoa significantly decreased as compared with those of fresh spermatozoa. The index of sperm motility for semen cryopreserved with lactose was significantly higher than that for semen cryopreserved with trehalose. The percentage of total fertilisation for tiger spermatozoa cryopreserved with trehalose was significantly lower than that for control cat spermatozoa. CONCLUSION: Our findings indicated that supplementation with lactose or glycerol as the main sugar in the egg yolk extender resulted in a better motility and fertility potential for post-thawed spermatozoa.

Keywords: artificial insemination, endangered species, extender, semen freezing




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CryoLetters 37 (4), 272-283 (2016)
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Jing Yu, Lihua Xie Suzuan Chen, Juan Zhang, Guanghua Guo
and Binming Chen*

Department of Gastroenterology, The First Affiliated Hospital of Shantou University Medical College, N0.57 Changping Road, Shantou 515041, China.
*Corresponding author email:
Tel: +86 0754-88258290   Fax: +86 0754-88905000


BACKGROUND: Producing sufficient numbers of DCs at one time point and subsequently cryopreserving the generated DCs in ready-for-use aliquots for clinical application is useful in cancer treatment.  OBJECTIVE: To study the effects of a simplified cryopreservation method and thawing procedures acting on the biological characteristics and specific cytotoxic activity of cord blood derived DC-based esophageal carcinoma vaccine. MATERIALS AND METHODS: CD34+ hematopoietic stem cells were isolated from cord blood using CD34+ Progenitor Cell Isolation Kit by magnetic cell sorting system (MACS). The CD34+ cells were expanded with cytokines as DCs, and fused with EC109 cells by PEG-3600. The fused cells were transferred to a freezing tube without rate-controlled freezing and stored at -80 ℃ for three weeks. During cryopreservation, 2.5% DMSO, 2.5% glucose and 10% FCS at final concentration was used as stock solution. After thawing, cells were assayed for Typan blue viability, morphology, immunophenotypes and T-cell stimulatory capacity, and specific CTL activity.  RESULTS: Cryopreservation does not cause significant changes in the phenotypes expression or morphology of the fused cells, and the viability were well preserved (Typan blue viability was 77.2±1.8%). After being stimulated by DC-based esophageal carcinoma vaccine either before or after cryopreservation, the numbers of CD3+T/CD4+T and CD3+T/CD8+T lymphocytes increased obviously, especially for CD3+T/CD4+T, and the ratio of CD4/CD8 changed from 0.85 to 1.29 and 1.25 respectively. Specific CTL activity were well preserved (compare to the fresh fused vaccine, P>0.05).  CONCLUSION: A simple -80 ℃ freezing and storage method is practical for cord blood derived DC-based esophageal carcinoma vaccine. It will greatly facilitate the clinical use of DC-based vaccine for immunotherapy.

Keywords: Dendritic cell; Fused vaccine; Cryopreservation; Biological characteristics; CTL activity.




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CryoLetters 37 (4), 284-294 (2016)
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post-harvest Embryo development in ginseng seedS Increases desiccation sensitivity AND NARROWS THE HYDRATION WINDOW FOR cryopreservation

Eun-Ju Han1, Elena Popova2, Gyu-Taek Cho1, Sang-Un Park3,
Sheong-Chun Lee4, Hugh W. Pritchard5 and Haeng-Hoon Kim4*

1 National Agrobiodiversity Center, RDA-NAAS, Suwon 441-707, Korea.
2 Gosling Research Institute for Plant Preservation, Department of Plant Agriculture, Univ. Guelph, Ontario
N1G 2W1, Canada.
3 Division of Plant Science and Resources, Chungnam National University, Daejeon 305-764, Korea
4 Dept. of Well-being Resources, Sunchon National University, Suncheon 540-742, Korea.
5 Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK.
*Corresponding author email:


BACKGROUND: Despite its self-pollinating characteristics, Korean ginseng germplasm is mainly maintained in clonal gene banks as there is no defined approach to the long-term conservation of its seed, including the most appropriate stage of embryo development for storage. OBJECTIVE: The aim of this study was to reveal the effect of embryo development on desiccation tolerance and cryopreservation success in ginseng seeds. MATERIALS AND METHODS: Seeds of Korean ginseng (Panax ginseng C.A. Meyer) at three post-harvest stages (immediately after harvesting and following treatments to enable internal growth of the embryo) were desiccated and cryopreserved. RESULTS: The hydration window for the >80% dehiscence and germination of cryopreserved ginseng seeds varied with embryo developmental stage: 3-9% moisture content (MC) for both unpulped and undehisced seeds when the embryo was 0.1 the length of the endosperm, 7-10% MC for dehisced seeds (0.5 embryo:endosperm) and 9-11% MC for seeds with fully developed embryos (0.9 embryo:endosperm). Whilst dried (4-8% moisture content) and undehisced seeds within fruits (unpulped seeds) lost more than half their viability during 1 year’s storage at room temperature, cryopreservation enabled germination levels of c. 90%. Overall, 432 accessions of Korean ginseng landraces have been cryopreserved using undehisced seeds with or without fruits. CONCLUSION: Post-harvest treatment of Korean ginseng seeds to enable embryo development decreases tolerance of very low MCs, and thus narrows the hydration window for cryopreservation. Fresh-harvested and unpulped seeds that have been dried to c. 5 % MC are recommended for long-term cryogenic storage. 

Keywords: Panax ginseng, embryo:endosperm ratio, cryobanking, seed storage.




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CryoLetters 37 (4), 295-302 (2016)
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Shun-Li Yue, Yu-Ting Zhang, Shun-Wei Wang, Meng Sun,
Yue-Chao Xing, Jing Wen and Jia-Bo Zhou*

College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang 150030, PR China
*Corresponding author email:


BACKGROUND: Oocytes that survive cryopreservation may accumulate ROS which are known to bring harmful effects on embryonic development. NAC is an antioxidant which can be a supplement to reduce oxidative stress. However, whether NAC can improve the developmental competence of vitrified GV-oocytes remains unclear. OBJECTIVE: The study was to investigate the effect of NAC on subsequent embryonic developmental competence of mice vitrified GV-oocytes. MATERIALS AND METHODS: This study compared the effects of different concentration of NAC on the cleavage and blastocyst rates of mice vitrified GV-oocytes. Then the effects of NAC on mitochondria distribution, ROS level and embryonic development of vitrified oocytes were tested.  RESULTS: ROS activity of vitrified oocytes was significantly annihilated and mitochondrial distribution pattern was improved by 1.5 mM NAC (P<0.05). NAC supplementation throughout vitrification/warming and IVM media significantly improved the developmental competence of vitrified oocytes. CONCLUSION: Supplementation of NAC could partially overcome the damages by vitrification and improve the development ability of mice vitrified GV-oocytes.

Keywords: N-acetyl-L-cysteine; Oocyte; Vitrification; ROS

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