Abstracts: CryoLetters 31 (2), 2010

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



Volume 31, No. 2 March/April 2010

ISSN 0143-2044



Increased efficiency using the encapsulation-dehydration cryopreservation technique for Arabidopsis thaliana
Remi Bonnart and Gayle M. Volk




Cryoprotective effect of low-molecular-weight hyaluronan on human dermal fibroblast monolayers
Masanobu Ujihira, Akira Iwama, Makie Aoki, Kanako Aoki, Sayaka Omaki, Erika Goto and Kiyoshi Mabuchi




Cryopreservation of the human ovarian tissue induces the expression of fas system in morphologically normal primordial follicles
Zhun Xiao, Yan Wang, Lei Li and Shang–Wei Li




Theoretical prediction of the effect of heat transfer parameters on cooling rates of liquid-filled plastic straws used for cryopreservation of spermatozoa
M. Sansinena, M. V. Santos, N. Zaritzky , R. Baeza and J. Chirife




Establishment and cryopreservation of a fibroblast cell line derived from bengal tiger (panthera tigris tigris)
W.J. Guan, C.Q. Liu, C.Y. Li, D. Liu , W.X. Zhang, Y.H. Ma




Cryopreservation of orthodox seeds of alnus glutinosa
Paweł Chmielarz




Loading trehalose into red blood cells by electroporation and its application in freeze-drying
Xinli Zhou, Ji Yuan, Jianfeng Liu and Baolin Liu




Cryopreservation of organotypical cultures based on 3d scaffolds
Rupf T., Ebert S., Lorenz K., Salvetter J. and Bader A.




SLTB Annual Scientific Meeting, AGM and Symposium
"Application of Cryobiology from Human Tissue Engineering to Plant Genebank Integration" September 7-9, 2009, Leibniz Universitaet Hannover and the IPK Gatersleben, Germany




In Memoriam: Karl Erik Zachariassen, 1942-2009





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CryoLetters 31 (2), 95-100 (2010)
© CryoLetters,


Remi Bonnart and Gayle M. Volk*

USDA-ARS National Center for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, CO 80521, USA
* Corresponding author e-mail:


Arabidopsis thaliana shoot tips were successfully cryopreserved using encapsulation-dehydration cryopreservation methods. Between one and seven shoot tips were encapsulated within 4 mm calcium-alginate beads. Beads were formed in the presence of 2 M glycerol + 0.4 M sucrose. The time required to make 10 beads, each containing five shoot tips (4 min), was less than the time required to make 50 beads containing one shoot tip (12 min). Shoot tip regrowth after cryoexposure was between 60 and 68%, with one to seven shoot tips per bead. Using five Arabidopsis shoot tips per bead, alginate beads were formed either in the presence of 2 M glycerol + 0.4 M sucrose or 0.5 M sucrose. Beads formed in the presence of glycerol were immediately air-dried to moisture contents between 0.21 to 0.26 g H2O/g FW (0.27 to 0.38 g H2O/g DW). Alginate beads formed in 0.5 M sucrose were incubated in solutions of 0.5, 0.75, and 1 M sucrose for one day each prior to air-dehydration, achieving moisture contents of 0.19 to 0.21 g H2O/g FW (0.23 to 0.27 g H2O/g DW). Shoot tip regrowth after cryoexposure was between 42 and 65%, with no significant differences among treatments. We successfully reduced the amount of time needed for shoot tip processing for Arabidopsis by encapsulating five shoot tips per alginate bead and by using a glycerol-encapsulation method, without lowering shoot tip regrowth levels after cryopreservation.

Keywords: alginate, Arabidopsis thaliana, dehydration, encapsulation, liquid nitrogen



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CryoLetters 31 (2), 101-111 (2010)
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cryoprotective effect of low-molecular-weight hyaluronan on HUMAN dermal fibroblast monolayers

Masanobu Ujihira1,2*, Akira Iwama1, Makie Aoki2, Kanako Aoki2,
Sayaka Omaki2, Erika Goto2 and Kiyoshi Mabuchi1,2

1Graduate School of Medical Sciences and 2School of Allied Health Sciences, Kitasato University, 1-15-1 Kitazato, Sagamihara-shi, Kanagawa 228-8555, Japan
*Corresponding author  email:


The purpose of this study was to assess the availability of low-molecular-weight (low-MW) hyaluronan (HA) as a cryoprotectant for cellular cryopreservation. To clarify whether low-MW HA is cryoprotective, we evaluated the effect of HA concentration (0-5% w/w) in a cryoprotectant solution on cell membrane integrity after freeze-thaw. A test sample was created using human dermal fibroblast monolayers incubated in a culture dish for 24 h (37°C, 5% CO2). Sodium hyaluronate (MW 3x104 - 5x104) dissolved in medium served as the cryoprotectant solution. Samples were immersed in the solution for 2 h at 0-4°C. They were frozen at a cooling rate of 3°C/min from 4 to –80°C, cooled further to below –185°C, and then thawed. Cell membrane integrity after thawing was evaluated using a trypan blue exclusion assay. The sample and freezing procedures were repeated in subsequent experiments, while the conditions of the solution immersion with respect to the sample varied. Next, to clarify whether the cryoprotective action of HA is intra- or extracellular, we performed three experiments. The first studied the dependence of membrane integrity after freeze-thaw on preliminary incubation time (0.75-24 h at 37°C) with a sample immersed in the solution (5% w/w HA). In the second, membrane integrity of thawed samples that were initially frozen in a medium instead of solution, by removing extracellular HA following a preliminary 6-h incubation period, were evaluated. Thirdly, we investigated cellular uptake of fluorescein isothiocyanate-labeled HA (MW 105, 1% w/w) after a preliminary 6-h incubation period under fluorescent microscopy (without freeze-thaw). The results show that HA had a cryoprotective effect, and that this cryoprotective action was intracellular. Therefore, low-MW HA proves to be a promising cellular cryoprotectant.

Keywords: cell membrane integrity, cryopreservation, cryoprotectant, cryoprotective effect, human dermal fibroblast monolayers, low-molecular-weight hyaluronan, preliminary incubation time



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CryoLetters 31 (2), 112-119 (2010)
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Zhun Xiao, Yan Wang, Lei Li and Shang–Wei Li*

Reproductive Medical Center of West China 2nd University Hospital, Sichuan University, ChengDu 610041, China
*Corresponding author e-mail:


The aim of this study was to investigate whether the Fas system was involved in cryopreservation process of the human ovarian tissue. Human ovarian cortical tissues were cryopreserved using slow freezing method. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to investigate apoptosis of primordial follicle. Fas, Fas ligand and active caspase-3 expression were detected by means of immunohistochemistry. The results showed that immunostaining for Fas, Fas ligand and active caspase-3 were not detected in morphologically normal primordial follicles in fresh ovarian tissue. After cryopreservation, Fas, Fas ligand and active caspase-3 immunostaining were present in morphologically normal primordial follicles. The study showed that cryopreservation of the human ovarian tissue induced the expression of Fas system in morphologically normal primordial follicles.

Keywords: cryopreservation, human, ovarian tissue, Fas system, primordial follicles



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CryoLetters 31 (2), 120-129 (2010)
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M. Sansinena1*, M. V.  Santos2,  N. Zaritzky 2, R. Baeza1 and J. Chirife1

1Facultad de Ciencias Agrarias, Pontificia Universidad Católica Argentina, Cap. Gral. Ramón Freire 183, (1426) Buenos Aires, Argentina.
2Facultad de Ingeniería, Universidad Nacional de La Plata and CIDCA ( CONICET –UNLP), Argentina
*Corresponding author  email:


Heat transfer plays a key role in cryopreservation of liquid semen in plastic straws. The effect of several parameters on the cooling rate of a liquid-filled polypropylene straw when plunged into liquid nitrogen was investigated using a theoretical model. The geometry of the straw containing the liquid was assimilated as two concentric finite cylinders of different materials: the fluid and the straw; the unsteady-state heat conduction equation for concentric cylinders was numerically solved. Parameters studied include external (convection) heat transfer coefficient (h), the thermal properties of straw manufacturing material and wall thickness. It was concluded that the single most important parameter affecting the cooling rate of a liquid column contained in a straw is the external heat transfer coefficient in LN2. Consequently, in order to attain maximum cooling rates, conditions have to be designed to obtain the highest possible heat transfer coefficient when the plastic straw is plunged in liquid nitrogen.

Keywords: cryopreservation, spermatozoa, heat conduction, thermal diffusivity, straw



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CryoLetters 31 (2), 130-138 (2010)
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W.J. Guan1*, C.Q. Liu1, 2*, C.Y. Li3, D. Liu 4, W.X. Zhang1, Y.H. Ma1**

1Institute of Beijing Animal Science and veterinary, CAAS, Beijing, 100094, China
2Bioscience Department, Bengbu Medical College, Bengbu, 233000, China
3College of Wildlife Resources, Northeast Forestry University, Harbin, 150040, China
4The Northeast Tiger Wooden Land of Heilongjiang; Harbin 150028; China
*These authors contributed equally to this work.
**Corresponding author: e-mail:;


The Bengal tiger ear marginal tissue fibroblasts cell line (BTF22), containing 157 tubes of frozen cells, was successfully established by using primary explants technique and cell cryoconservation technology. Biological analysis showed that the population doubling time (PDT) for revival cells was approximately 28 h. Measurement of LDH and MDH isoenzymes showed no cross-contamination among the cells. Karyotyping showed that the frequency of cells with chromosome number 2n=38 was 90.6–92.2%. Tests for bacteria, fungi, viruses and mycoplasma were negative. Plasmids encoding the fluorescent proteins pEGFP-N3, pEGFP-C1, pECFP-N1, pECFP-mito, pDsRed1-N1, and pEYFP-N1 were transfected into cells to study exogenous gene expression in the cells. The plasmid transfection efficiency was between 4.4% and 31.9%. Every index of the BTF22 cell line meets all the standard quality controls of American type Culture Collection (ATCC). Not only has the germline of this important Bengal tiger species been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somacloning research. Moreover, the establishment of this technical platform would provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.

Keywords: Bengal tiger; Fibroblast Cell line; Establishment; Characterization; Cryopreservation



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CryoLetters 31 (2), 139-146 (2010)
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Paweł  Chmielarz

Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik, Poland
Corresponding author e-mail:


We evaluated the sensitivity of orthodox seeds of black alder (Alnus glutinosa (L.) Gaertn.) to extreme desiccation and/or to the ultra-low temperature of liquid nitrogen (LN; -196°C). The critical water content (WC) of desiccated seeds and the high-moisture freezing limit of seeds desiccated or imbibed to various WCs and frozen for 24 h or up to two years in LN was determined. Germination tests revealed that desiccation to 0.03 g H2O.g–1 dry mass was not detrimental to these seeds. Seeds tolerated LN freezing when the WC was 0.03-0.18g H2O g–1 dm (nuts). Seeds desiccated to this WC and stored in LN for two years showed similar germination as seeds stored at -3°C for two years. Therefore, long-term cryopreservation of A. glutinosa seeds in genebanks is feasible.

Keywords: desiccation, germination, liquid nitrogen, seed storage, water content, black alder



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CryoLetters 31 (2), 147-156 (2010)
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Loading Trehalose into Red Blood Cells by Electroporation and its application in Freeze-drying

Xinli Zhou, Ji Yuan, Jianfeng Liu and Baolin Liu

Institute of Biomedical Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China.
Corresponding author e-mail:


Freeze-drying is one potentially ideal method for long-term storage of red blood cells (RBCs). Intracellular trehalose is believed to be an effective protectant to stabilize cells during freezing and drying. In this study, we adopted electroporation to load trehalose into human RBCs first. The effects of electroporation parameters (extracellular trehalose concentration, field strength, pulse length and frequency) on loading efficiency were studied. The results show that RBCs can be loaded with 63.7 mM trehalose at 800 mM extracellular trehalose concentration, 1.5 kV/cm,field strength, 1 ms pulse length, 4 pulses in one minute. Then, RBCs loaded with different amount of trehalose by electroporation were freeze-dried and rehydrated. Recovery rates of trehalose-loaded and freeze-dried RBCs increased with intracellular trehalose concentration. The recovery rate of RBCs loaded with 63.7 mM trehalose reached 70.9 %. In conclusion, electroporation is an effective method for loading of nonpermeating trehalose into RBCs and therefore benefit freeze-drying of RBCs.

Keywords: Electroporation; trehalose; freeze-drying; red blood cell



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CryoLetters 31 (2), 157-168 (2010)
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Cryopreservation of organotypical cultures based on 3D scaffolds

Rupf T., Ebert S., Lorenz K., Salvetter J. and Bader A.

University of Leipzig, Center for Biotechnology and Biomedicine Cell Techniques and Applied Stem Cell Biology, Germany.
*Corresponding author  email:


An integral component of the manufacture of a skin substitute is the cryopreservation of the complete skin construct. Under this demand, investigations were carried out in the present work in the case of cryopreservation of human fibroblasts and keratinocytes composed to organotypical skin substitutes (OTS). Two scaffolds made up of gelatine and collagen/elastin were seeded with human fibroblasts via centrifugation method. Subsequent human keratinocytes were applied on the preceded scaffolds and cultivated under air-exposed conditions. For the investigation of the cryopreservation, OTS were frozen after 10 days cultivation via computer-controlled CryoMed included defined freezing conditions. After 24 hours storage in fluid nitrogen the OTSs were thawed and recultivated under airlift conditions. After that metabolic activity and immunfluorescent staining was analyzed in comparison with conventionally produced OTSs on basis of collagengel and/or OTSs based on scaffolds without cryopreservation. It could be assessed that cryopreservation has no negative influence on vitality and differentiation capacity of the cultivated constructs. The determination of OTS vitality after 14 days airlift culture delivered persistent higher metabolic activities of the scaffold based constructs in comparison with the corresponding controls. This could be confirmed by investigation of OTSs with and without cryopreservation. All expression patterns of differentiation marker could be detected after cryopreservation and subsequent recultivation. The results from cryopreservation of OTSs introduced here prove the possibility of temporally independent tailor-made applications by means of a complete skin substitute for example in the area pharmascreening.

Keywords: cryopreservation, skin equivalent, 3D scaffold, tissue engineering, organotypical cultures

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