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Volume 32, No. 5 September/October 2011
ISSN 0143-2044
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Cryopreservation
of winter-dormant apple buds: II - tissue water status after desiccation at -4°c and before further cooling
C. Vogiatzi, B. W.W Grout, A.Wetten and
B. T. Toldam-Andersen
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367-376
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In vitro conservation of Passiflora suberosa L.: slow growth and cryopreservation
R.O. Garcia, G. Pacheco, M.G. Vianna and E. Mansur
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377-388
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RGDS-Fuctionalized
alginates improve the survival rate of encapsulated embryonic stem cells during cryopreservation
S Sambu, X Xu, HA Schiffter, ZF Cui and H Ye
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389-401
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Cryopreservation
of adult bovine testicular tissue for spermatogonia enrichment
Jianying Wu, Tiejun Hu, Bin Guo, Zhanpeng Yue,
Zhengtao Yang, and Xueming Zhang
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402-409
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Effects of
cryoprotectant agents and equilibration methods on developmental competence of porcine oocytes
Masayasu Taniguchi, Rie Arikawa,
Yukine Kaedei, Fuminori Tanihara, Zhao Namula, Vien Luu Viet, Yoko Sato, and
Takeshige Otoi
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410-414
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Mediated
trehalose un-loading for reduced erythrocyte osmotic fragility and phosphatidylserine translocation
Andrew L. Lynch and Nigel K.H. Slater
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415-424
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Effect
of various freezing solutions on cryopreservation of mesenchymal stem cells from different animal species
Yang Liu, Xia Xu, Xuehu Ma, Jing Liu and Zhanfeng Cui
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425-435
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Cryoprotective effect of an insect antifreeze protein MpAFP698
and its mutants from the desert beetle Microdera punctipennis
Min Jiang, Ji. Ma and Liming Qiu
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436-446
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CryoLetters 32 (5), 367-376 (2011)
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CRYOPRESERVATION OF WINTER-DORMANT APPLE BUDS:
II - TISSUE WATER STATUS AFTER DESICCATION AT -4°C AND BEFORE FURTHER COOLING
C. Vogiatzi1, B. W.W Grout1*, A.Wetten2 and B. T. Toldam-Andersen1
1Department of Agriculture and Ecology, University of Copenhagen, Højbakkegård Allé 13, DK-2630 Taastrup, Denmark
2 School of Biological Sciences, University of Reading, Harborne Building 132, RG6
6AS, United Kingdom
*Corresponding author email: bwg@life.ku.dk
Abstract
The established protocol for the cryopreservation of winter-dormant Malus buds
requires that stem explants, containing a single, dormant bud are desiccated at -4°C, for up to 14 days, to reduce their water content to 25-30% of fresh weight. Using three
apple cultivars, with known differences in response to cryopreservation, the pattern of evaporative water loss has been characterised, including early freezing events in the
bud and cortical tissues that allow further desiccation by water migration to extracellular ice. There were no significant differences between cultivars in this respect
or in the proportions of tissue water lost during the desiccation process. Differential Scanning Calorimetry (to -90°C) of intact buds indicated that bud tissues of the cultivar
with the poorest response to cryopreservation had the highest residual water content at the end of the desiccation process and froze at the highest temperature
Keywords: Malus, cryopreservation, dormant bud, dehydration
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CryoLetters 32 (5), 377-388 (2011)
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IN VITRO CONSERVATION OF PASSIFLORA SUBEROSA L.: SLOW GROWTH AND CRYOPRESERVATION
R.O. Garcia 1, 2, G. Pacheco1, M.G. Vianna1 and E. Mansur1*
1Instituto de Biologia Roberto Alcantara Gomes, Núcleo de Biotecnologia Vegetal,
Universidade do Estado do Rio de Janeiro, Rua São Francisco Xavier 524 PHLC sala 505, Maracanã, Rio de Janeiro 20550-013, Brazil.
2Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde
Pós-Graduação em Biotecnologia Vegetal, Av. Carlos Chagas Filho, 373, Bloco K, 2° andar, sala 032 - Ilha do Fundão, Cidade Universitária - Rio de Janeiro 21941-590, Brazil.
*Corresponding author email: elisabeth.mansur@gmail.com
Abstract
Passiflora suberosa is a tropical species used as an ornamental, in popular medicine
and in improvement programs. The goal of this study was the development of in vitro conservation strategies for this species, including medium-term storage through slow
growth, and long-term storage through cryopreservation using vitrification-based techniques. Plants were maintained under slow growth conditions on ½ MSM or ¼
MSM medium for 12 months without decrease in regrowth ability. The efficiency of vitrification and encapsulation-vitrification protocols was compared in order to
determine the optimal conditions for successful cryopreservation. Several parameters were evaluated, including pregrowth on medium with high sucrose concentrations, type
of vitrification solution (PVS2 and PVS3), exposure time to vitrification solutions, and recovery conditions. The highest recovery was obtained with the
encapsulation-vitrification protocol after a pretreatment with 0.3 M sucrose and post-cryopreservation incubation in the dark for 30 days on MSM medium supplemented with 0.44 μM BA.
Keywords: passion fruit, medium-term conservation, long-term conservation, vitrification, encapsulation-vitrification.
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CryoLetters 32 (5), 389-401 (2011)
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RGDS-FUCTIONALIZED ALGINATES IMPROVE THE SURVIVAL RATE OF ENCAPSULATED EMBRYONIC STEM CELLS DURING CRYOPRESERVATION
S Sambu, X Xu, HA Schiffter, ZF Cui and H Ye*
Institute of Biomedical Engineering, Department of Engineering Science, Old Road
Campus Research Building, University of Oxford, Oxford, OX3 7DQTel: +44 1865 617689, Fax: +44 01865 617701
*Corresponding author email: Hua.ye@eng.ox.ac.uk
Abstract
Cryopreservation of stem cells, especially embryonic stem cells, is problematic
because of low post-thaw cell survival rates and spontaneous differentiation following recovery. In this investigation, mouse embryonic stem cells (mESCs) were
encapsulated in arginine-glycine-aspartic acid-serine (RGDS)-coupled calcium alginates (1.2 wt%), allowed to attach to the substratum and then cryopreserved in
10% vol. dimethyl sulfoxide (DMSO) solution at a slow cooling rate of 1˚C/min. RGDS coupling to alginate was confirmed by Transmission Fourier Transform Infra-Red
spectroscopy (T-FTIR) and quantified by using ninhydrin-Ultraviolet/Visible light (ninhydrin-UV/VIS) assay. Flow cytometry data showed that mESCs cryopreserved in
RGDS-alginate beads had a higher expression of stem cell markers compared with cells cryopreserved in suspension or cells cryopreserved in unmodified alginates. Cell
viability after thawing was assessed using trypan blue exclusion assay and monitored using Alamar blue assay for 6 hours. It was shown that post-thaw cell survival rate was
significantly higher for cells encapsulated in RGDS-modified alginate (93±2%, mean and standard error) than those in suspension (52±2%) or in unmodified alginates
(62±3%). These results showed that cells encapsulated and attached to a substratum have better survival rate and stem cell marker expression 24 hours after
cryopreservation than those in suspension. Encapsulation in RGDS-alginate was optimized for peptide concentration, cryoprotective agent loading time and cooling rate.
The best result was obtained when using 12.5 mg peptide /g alginate, 30 minutes loading time and 1C/min cooling rate.
Keywords: RGDS, cryopreservation, alginate, mESCs, encapsulation
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CryoLetters 32 (5), 402-409 (2011)
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CRYOPRESERVATION OF ADULT BOVINE TESTICULAR TISSUE FOR SPERMATOGONIA ENRICHMENT
Jianying Wu2, 1, Tiejun Hu3, Bin Guo1, Zhanpeng Yue1, Zhengtao Yang1, and
Xueming Zhang1*
1Jilin Province Key Laboratory of Animal Embryo Engineering, and the Center for
Animal Embryo Engineering of Jilin Province, College of Animal Sciences and Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun 130062, Jilin Province, China.
2Department of Laboratory Medicine, the 90th Hospital of Jinan, Jinan 250031,
Shandong Province, China.
3Jilin Province Changchun Haoyue Islamic Meat Co.LTD., Changchun 130062, Jilin Province, China.
*Corresponding authors email: zhangxuem@jlu.edu.cn
ABSTRACT
To develop a procedure for cryopreservation of adult bovine testis tissue, the effects of
dimethyl sulphoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), and their concentrations (v/v), as well as different thawing temperatures, on the cell viability of
bovine testis tissue after freezing/thawing were examined. The highest testicular cell viabilities came from the media containing DMSO (85.3 ± 1.2%), PG (82 ± 1.0%) and
EG (83.4 ± 1.0%) at 10% concentration respectively. Using 10% DMSO gave significantly higher spermatogonia percentage (61.1 ± 1.2%, P < 0.001) than
processing with 10% PG (54.3 ± 0.6%) or 10% EG (55 ± 1.8%) after differential plating. Thawing in water bath of 37 or 97-100°C also provided significantly higher
viabilities (85.1 ± 1.0, 85 ± 1.0%, P < 0.01, respectively) and spermatogonia percentages (56.6 ± 2.0, 56.6 ± 2.6%, P < 0.01, respectively) than that thawing at 4°C
(23.4 ± 0.8% for total viability, 8.97 ± 1.0% for spermatogonia percentage). Collectively, 10% DMSO and thawing in 37-100°C water baths were appropriate for the
cryopreservation of bovine testicular tissue and subsequent spermatogonia enrichment.
Keywords: bovine; cellular viability; cryopresevation; cryoprotectant; testis tissue
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CryoLetters 32 (5), 410-414 (2011)
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EFFECTS OF CRYOPROTECTANT AGENTS AND EQUILIBRATION METHODS ON DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES
Masayasu Taniguchi, Rie Arikawa, Yukine Kaedei, Fuminori Tanihara, Zhao Namula, Vien Luu Viet, Yoko Sato, and Takeshige Otoi*
Laboratory of Animal Reproduction, Department of Veterinary Medicine, Faculty of
Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan.
*Corresponding author email: otoi@yamaguchi-u.ac.jp
Abstract
Chemical toxicity of cryoprotectants to in vitro developmental competence of porcine
oocytes was examined. In vitro-matured oocytes were exposed to 40% ethylene glycol (EG), glycerol (GLY), or 1,2-propanediol (PD), fertilized with spermatozoa, and cultured
for 8 d. Compared to treatment with other cryoprotectants, exposure to EG resulted in the development of significantly more blastocysts, but the rate was significantly lower
than that of non-exposed control oocytes. In vitro-matured oocytes were also equilibrated in 40% EG by 3 multi-step methods, after which their developmental
competence was evaluated. The rate of blastocyst development was higher in the 4-step method than in the 2- and 3-step methods of equilibrium. These results indicate
that cryoprotectants and equilibration methods affect the developmental competence of porcine oocytes and that EG may be a superior cryoprotectant for vitrification of these cells.
Keywords: Cryoprotectant, equilibration, porcine, oocyte, toxicity, vitrification
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CryoLetters 32 (5), 415-424 (2011)
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MEDIATED TREHALOSE UN-LOADING FOR REDUCED ERYTHROCYTE OSMOTIC FRAGILITY AND PHOSPHATIDYLSERINE TRANSLOCATION
Andrew L. Lynch1* and Nigel K.H. Slater1
1Department of Chemical Engineering and Biotechnology, University of Cambridge,
Pembroke Street, Cambridge, CB2 3RA, United Kingdom
*Corresponding author email: all39@cam.ac.uk
Abstract
Recently, high concentrations of intracellular trehalose (>200mM) were employed to
enhance the cryoprotection and desiccation protection of human erythrocytes. However, significant challenges must be overcome if this advancement is to be
translated into clinical practice. It is here demonstrated that 247 ± 5 mM intracellular trehalose caused the lysis of 60 ± 2% of erythrocytes upon resuspension in PBS of
physiological osmolality (300 mOsm) and caused surviving cells to swell up to 140 ± 2% of isotonic cell volume. Trehalose loaded cells also exhibited 24 ± 1% incidence of
phosphatidylserine translocation upon resuspension in 300 mOsm PBS, likely due to loading induced cell swelling. Un-loading of trehalose from erythrocytes using the
membrane-permeabilizing biopolymer PP-50 was investigated as a technique to mitigate these damaging effects. After erythrocyte un-loading from 247 ± 5 mM to 39 ±
2 mM intracellular trehalose, cell lysis at 300 mOsm PBS was reduced from 60 ± 2% to 17 ± 3%. Un-loading also reduced cellular incidence of PS translocation in resuspended cells from 24 ± 1% to 13 ± 1%.
Keywords: erythrocyte, trehalose, biopolymer, phosphatidylserine, osmotic fragility
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CryoLetters 32 (5), 425-435 (2011)
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EFFECT OF VARIOUS FREEZING SOLUTIONS ON CRYOPRESERVATION OF MESENCHYMAL STEM CELLS FROM DIFFERENT ANIMAL SPECIES
Yang Liu1,2,3, Xia Xu1,4, Xuehu Ma3, Jing Liu2 and Zhanfeng Cui1,*
1Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford, UK.
2Sino-UK Regenerative Medicine Center, The 1st Affiliated Hospital of Dalian Medical
University, Dalian 116011, China
3Dalian R& D Center for Stem Cell and Tissue Engineering, School of Chemical Engineering, Dalian University of Technology, Dalian 116024, China
4National Key Laboratory of Biochemical Engineering, Institute of Process
Engineering, Chinese Academy of Sciences, Beijing, 100190, China.
*Corresponding author Tel: 44-1865-283273, email: zhanfeng.cui@eng.ox.ac.uk
Abstract
The objective of this study is to compare the effects of different well defined freezing
solutions with a reduced concentration of dimethylsulfoxide (DMSO) combined with polyethylene glycol (PEG) and/or trehalose on cryopreservation of mesenchymal stem
cells (MSCs) from mice, rats and calves. Post-thaw cell viability, proliferation capacity and differentiation potential of MSCs from different species were assessed after
cryopreservation with the conventional slow freezing method. Although the post-thaw viabilities and metabolic activities varied among the different species, satisfactory
results were obtained with 5% (v/v) DMSO, 2% (w/v) PEG, 3% (w/v) trehalose and 2% (w/v) bovine serum albumin (BSA) as the freezing solution. Our results showed that
mouse MSCs were more robust to cryopreservation compared with rat and bovine MSCs.
Keywords: cryopreservation, mesenchymal stem cells, well defined freezing solutions
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CryoLetters 32 (5), 436-446 (2011)
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CRYOPROTECTIVE EFFECT OF AN INSECT ANTIFREEZE PROTEIN MPAFP698 AND ITS MUTANTS FROM THE DESERT BEETLE MICRODERA PUNCTIPENNIS
Min Jiang, Ji. Ma* and Liming Qiu
Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of
Life Science and Technology, Xinjiang University, Urumqi, China
*Corresponding author email: majibrge@yahoo.cn
Abstract
An insect antifreeze protein, MpAFP698, from the desert beetle Microdera punctipennis, shares 77% similarity with TmTHP (YL-3) from Tenebrio molitor. The
predicted tertiary structure for MpAFP698 was modeled as a regular -helix with TCT motifs arrayed to form the putative ice-binding surface. This model was validated via
site-directed mutagenesis. The results suggest that the desert beetle in central Asia has evolved antifreeze proteins similar to those from North America continent. In an
additional study, the recombinant MpAFP698 and its mutants were expressed in Escherichia coli strain BL21 (DE3). The thermal hysteresis activity of MpAFP698 was
1.73°C at 1.0 mg/ml. MpAFP698 was also tested by in vitro antifreeze activity assay to evaluate its cryoprotective effect at -20°C. MpAFP698 displays high cryoprotective
effect on bacteria cells at freezing temperatures.
Keywords: antifreeze protein, Microdera punctipennis, site-directed mutagenesis, thermal hysteresis
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