Volume 33, No. 2 March/April 2012 ISSN 0143-2044
	Cryopreservation of cryosensitive basidiomycete cultures by application and modification of perlite protocol Masanori Sato, Junji Sukenobe and Akira Nakagiri	86-94
	Comparison of cell membrane water permeability in monolayers and suspensions Adam Z. Higgins and Jens O.M. Karlsson	95-106
	Cryoprotectants protect medaka (Oryzias latipes) embryos from chilling injury Qing-Jing Zhang, Guang-Bin Zhou, Yan-Ping Wang, Xiang-Wei Fu and Shi-En Zhu	107-116
	A new quantitative method to measure activity of ice structuring proteins using differential scanning calorimetry Majid Hassas-Roudsari and H. Douglas Goff	117-124
	Increasing storage capability of pacu (Piaractus mesopotamicus) embryos by chilling: development of a useful methodology for hatcheries management D. C. Fornari, R. P. Ribeiro, D. P. Streit Jr, L. Vargas, L. C. Godoy, C. A. L. Oliveira, M. Digmayer, J. M. Galo and P. R. Neves	125-133
	Relationships between cold hardiness, and ice nucleating activity, glycerol and protein contents in the hemolymph of caterpillars, Aporia crataegi L. N.G. Li	134-142
	Numerical analysis to determine the performance of different oocyte vitrification devices for cryopreservation Weijie Li, Xinli Zhou, Haisong Wang and Baolin Liu	143-149
	Effects of cryopreservation with polyethylene glycol on the expression of cd11b and cd62l on the surface of polymorphonuclear leukocytes M. Feuerecker, I. Kaufmann, A. P. Salam and A. Choukèr	150-159
	Cryopreservation of winter-dormant apple: III – bud water status and survival after cooling to -30°C and during recovery from cryopreservation C. Vogiatzi, B.W.W. Grout and A. Wetten	160-168

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CryoLetters 33 (2), 86-94 (2012)
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CRYOPRESERVATION OF CRYOSENSITIVE BASIDIOMYCETE CULTURES BY APPLICATION AND MODIFICATION OF PERLITE PROTOCOL

Masanori Sato^1*, Junji Sukenobe¹, and Akira Nakagiri²

¹NITE Patent Microorganisms Depositary (NPMD) 2-5-8, Kazusakamatari, Kisarazu-shi, Chiba, 292-0818, Japan Tel. +81-438-20-5580 Fax. +81-438-5581
²NITE Biological Resource Center (NBRC) 2-5-8, Kazusakamatari, Kisarazu-shi, Chiba, 292-0818, Japan; Present address: Fungus/Mushroom Resource and Research Center (FMRC), Faculty of Agriculture, Tottori University, 4-101 Koyama-Minami, Tottori 680-8553, Japan
^*Corresponding author email: sato-masanori2@nite.go.jp

Abstract

Homolka's perlite protocol (HPP) for cryopreservation of fungal cultures was evaluated in 12 strains (7 species) of cryosensitive basidiomycete cultures maintained in NBRC culture collection by investigating viability, time to recover, and basic morphological study after freezing and storing at -80ºC for 6 months. The viability of the fungal strains was 60% in Phallus hadriani and 100% in remaining 11 strains, indicating the efficacy of HPP method for cryopreservation of some cryosensitive basidiomycetes. The HPP method was modified by changing the addition of cryoprotectant (glycerol) from prior precultivation to post precultivation, limiting the cryoprotectant exposure time to 48 hours, and increasing the glycerol concentration from 5% to 12%. The viability of P. hadriani strain increased from 60% to 100% with the modified perlite protocol after storage at -80ºC for 6 months

Keywords: cryopreservation, basidiomycete, perlite, glycerol

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CryoLetters 33 (2), 95-106 (2012)
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COMPARISON OF CELL MEMBRANE WATER PERMEABILITY IN MONOLAYERS AND SUSPENSIONS

Adam Z. Higgins¹ and Jens O.M. Karlsson^2*

¹School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, Oregon 97331, USA
²Department of Mechanical Engineering, and Cellular & Molecular Bioengineering Research Group, Villanova University, 800 Lancaster Avenue, Villanova, Pennsylvania 19085, USA
*Corresponding author email: karlsson@alum.mit.edu

Abstract

 We previously measured the membrane water permeability of monolayers and suspensions of MIN6 mouse insulinoma cells at room temperature, and found that water transport was faster in monolayers.  Here, we compare water transport kinetics in monolayers and suspensions over a range of temperatures for two different cell types, MIN6 cells and bovine pulmonary artery endothelial cells (BPAEC). At room temperature the results for BPAEC and MIN6 cells were similar, with approximately 2-fold faster water transport in monolayers than suspensions.  The activation energy for water transport (E_a) was estimated from Arrhenius plots of the water permeability data. The values of E_a for monolayers and suspensions of MIN6 cells were not significantly different. However, the activation energy was significantly lower for BPAEC monolayers (E_a = 49±2 kJ/mol) than suspensions (E_a = 70 ± 4 kJ/mol). Predictions of water transport during cryopreservation revealed substantial differences in supercooling between monolayers and suspensions. 

Keywords: beta cell, endothelial cell, hydraulic conductivity, isolation, trypsinization

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CryoLetters 33 (2), 107-116 (2012)
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CRYOPROTECTANTS PROTECT MEDAKA (Oryzias latipes) EMBRYOS FROM CHILLING INJURY

Qing-Jing Zhang^1,2, Guang-Bin Zhou³, Yan-Ping Wang¹, Xiang-Wei Fu¹ and Shi-En Zhu^1,*

¹College of Animal Science and Technology, State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing, 100193, P.R.China
²Beijing Fisheries Research Institute, Beijing 10068, P.R. China
³Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University (Chengdu Campus), Wenjiang 611130, P.R. China
^*Corresponding author email: zhushien@cau.edu.cn

Abstract

This study was conducted to investigate the effect of six cryoprotectants (dimethyl sulfoxide (DMSO), glycerol (Gly), methanol (MeOH), ethylene glycol (EG), 1,2-propylene glycol (PG) and N,N-dimethylformamide (DMF) on the survival of medaka (Oryzias lapites) embryos at low temperatures (0 and -5°C). Firstly, the embryos at 8 to 16-cell stages were exposed to different concentrations (1 to 4 mol/L) of DMSO, Gly, MeOH, EG, PG and DMF for 40min at 26°C. After removal of the cryoprotectants (CPAs), the embryo survivals were assessed by their development into live fries following 9 day of culture. The results showed that the higher concentration of the CPA, the lower survival of the embryos; and that the toxicity of the six CPAs to medaka embryos is in the order of PG < MeOH = DMSO < Gly < EG < DMF (P<0.05). Secondly, based on the results obtained above, embryos at 8 to 16-cell stages or other stages were exposed to 2 mol/L of PG, MeOH or DMSO for up to 180 min at 0°C and up to 80 min at -5°C respectively. The 8 to 16-cell embryos treated with MeOH at low temperatures showed highest survival. Thirdly, when embryos at different stages were treated with 2 mol/L of MeOH at -5°C for 60 min, 16-somite stage embryos showed highest survival, followed by 4-somite, neurula, 50% epiboly, blastula, 32-cell and 8 to 16-cell embryos. These results demonstrated that PG had the lowest toxicity to medaka embryos among the six permeable CPAs at 26°C, whereas MeOH showed highest cryoprotective efficiency under chilling conditions and chilling injury decreased gradually with the development of medaka embryos.

Keywords: medaka embryo, cryoprotectant, toxicity, chilling injury, protective efficiency

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CryoLetters 33 (2), 117-124 (2012)
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A NEW QUANTITATIVE METHOD TO MEASURE ACTIVITY OF ICE STRUCTURING PROTEINS USING DIFFERENTIAL SCANNING CALORIMETRY

Majid Hassas-Roudsari and H. Douglas Goff*

Department of Food Science, University of Guelph, Guelph, ON, NIG 2W1, Canada.
*Corresponding author: email: dgoff@uoguelph.ca

Abstract

There are very few quantitative assays to measure the activity of antifreeze proteins (AFPs, or Ice Structuring Proteins, ISPs) and these can be prone to various inaccuracies and inconsistencies. Some methods rely only on unassisted visual assessment. When microscopy is used to measure ice crystal size, it is critical that standardized procedures be adopted, especially when image analysis software is used to quantify sizes. Differential Scanning Calorimetry (DSC) has been used to measure the thermal hysteresis activity (TH) of AFPs. In this study, DSC was used isothermally to measure enthalpic changes associated with structural rearrangements as a function of time. Differences in slopes of isothermal heat flow vs. time between winter wheat ISP or AFP type I containing samples, and those without ISP or AFP type I were demonstrated. ISP or AFP type I containing samples had significantly higher slopes compared to those without ISP or AFP type I. Samples with higher concentration of ISP or AFP type I showed higher slope values during the first hour and took up to 3 hr to attain equilibrium. Differences were attributed to activity of the proteins at the ice interface. Proteinaceous activity of ISPs or AFP type I was confirmed by loss of activity after treatment with protease.

Keywords: ice structuring proteins (ISPs) activity, antifreeze proteins (AFP) Activity, differential scanning calorimetry (DSC), recrystallization inhibition (RI) activity, thermal hysteresis (TH) activity

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CryoLetters 33 (2), 125-133 (2012)
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INCREASING STORAGE CAPABILITY OF PACU (Piaractus mesopotamicus) EMBRYOS BY CHILLING: DEVELOPMENT OF A USEFUL METHODOLOGY FOR HATCHERies MANAGEMENT

D. C. Fornari¹, R. P. Ribeiro¹, D. P. Streit Jr², L. Vargas¹, L. C. Godoy^2*, C. A. L. Oliveira¹, M. Digmayer¹, J. M. Galo¹ and P. R. Neves¹

¹PeixeGen Research Group, Maringá State University, Department of Animal Science, Maringá, Brazil.
²Aquam Research Group, Federal University of Rio Grande do Sul, Department of Animal Science, Porto Alegre, Brazil.
*Corresponding author  email: godoyaqua@yahoo.com.br

Abstract

Cryopreservation of fish gametes has been studied extensively in the last few decades, but the successful cryopreservation of fish embryos remains elusive. However, recent studies using short-term chilling techniques have shown that it is possible to store embryos at low temperatures with no significant loss in viability. Information on cryopreservation of Neotropical freshwater fish embryos has so far been very limited in the literature. In the present study, chilling protocols for storage of pacu embryos at -8°C for up to 24 h were studied using different concentrations of sucrose in methanol. Embryos tolerated the subzero temperature for up to 6 h with no adverse effects (P > 0.05). After 12 h chilling, hatching rate of 64.0 ± 3.5% was recorded. Low temperature storage of pacu embryos by chilling is detailed here for the first time. Further studies are needed to extend the storage time and to improve the hatching rate.

Keywords: Cryopreservation, chilled embryos, Neotropical fish, Brazilian fish farming, breeding program

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CryoLetters 33 (2), 134-142 (2012)
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RELATIONSHIPS  BETWEEN COLD HARDINESS, AND ICE NUCLEATING ACTIVITY,  GLYCEROL  AND PROTEIN  CONTENTS  IN THE  HEMOLYMPH OF CATERPILLARS, Aporia crataegi L.

N.G. Li

Department of Ecology and Systematic of Invertabrates, Institute for Biological Problems of  Cryolithozone Siberian Branch of  Russian Academy of Sciences, Yakutsk, Lenin avenue, 41, 677980, Russia
Correspondence author email: li_natalia@mail.ru

Abstract

Insects in Siberia must tolerate some of the coldest conditions on earth.  The relationship between hemolymph ice nucleating activity, glycerol and total protein concentrations, and cold hardiness was explored in Aporia crataegi L. (Lepidoptera: Pieridae). Cold-hardened overwintering caterpillars were collected at a time of year when temperatures are regularly below -50°C, and warm–acclimated at +22ºC, to see how changes in the physical and chemical properties of the hemolymph influence their cold hardiness potential. Warm acclimation led to a decrease in glycerol and proteins content in the hemolymph, which was associated with the decrease in ice nucleating activity and dramatic loss of cold hardiness potential of the caterpillars.  It is suggested that one of the effects of cryoprotection in the freeze tolerant insects, caused by glycerol, might be associated with its ability to form larger aggregates of ice nucleating polypeptides that initiate the ice nucleation at high subzero temperatures. Such ice nucleating structures seem to ensure a high probability of ice nucleation at relatively high temperatures, which may contribute to the extraordinary cold hardiness of A. crataegi caterpillars, which may tolerate temperatures below -85°С. 

Keywords: glycerol, ice nucleating agents, cold hardiness potential, warm acclimation, freeze-tolerance

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CryoLetters 33 (2), 143-149 (2012)
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NUMERICAL  ANALYSIS  TO  DETERMINE THE  PERFORMANCE OF  DIFFERENT OOCYTE VITRIFICATION  DEVICES FOR  CRYOPRESERVATION

Weijie Li, Xinli Zhou, Haisong Wang and Baolin Liu

Institute of Biomedical Technology, University of Shanghai for Science and Technology, Shanghai, 200093, China.
Corresponding author email: zjulily@163.com

Abstract

Vitrification is widely used for cryopreservation of oocytes. The present study has built the theoretical models of four vitrification systems (micro-droplet, open pulled straws, quartz micro-capillary and cryotop), and performed numerical analysis to predict the cooling rates. The numerical analysis shows that the average cooling rate of the cryotop system was higher than those of other three systems between 298K and 100K. In addition, the effects of other process parameters on the cooling rate with the cryotop system were also investigated, including the thickness of the carrier, the volume of cryoprotectant agent, the temperature of cold source as well as the heat transfer coefficient, when plunging into liquid nitrogen.

Keywords: numerical analysis, vitrification, oocyte, cooling rate

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CryoLetters 33 (2), 150-159 (2012)
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EFFECTS OF CRYOPRESERVATION WITH POLYETHYLENE GLYCOL ON THE EXPRESSION OF CD11B AND CD62L ON THE SURFACE OF POLYMORPHONUCLEAR LEUKOCYTES

M. Feuerecker¹, I. Kaufmann¹, A. P. Salam² and A. Choukèr^1*

¹Department of Anaesthesiology, Klinikum Großhadern, University of Munich, Marchionini-strasse 15, 81377 Munich.
²Department of Infection, Guy's and St Thomas' NHS Trust, London, United Kingdom. *Corresponding author email: alexander.chouker@med.uni-muenchen.de

Abstract

In experimental and clinical studies, expression of surface adhesion molecules such as ß₂-integrine (CD11b) and L-selectin (CD62L) on polymorphonuclear leukocyte (PMNL) are investigated to assess certain crucial innate immune functions. Because the expression of CD11b and CD62L on PMNL can alter they cannot be quantified reliably when the time between blood draw and measurements is prolonged. Goals of this study were to test effects of cryopreservation on the expression of CD11b and CD62L on human PMNLs either under native conditions as well as after stimulation-dependant adhesion molecules' expression pattern.

CD11b and CD62L expression on PMNL can be cryopreserved with 10% of PEG-solution for at least one month at -60°C. This was observed in native, unstimulated as well as in stimulated cell-preparations. CD11b is very stable in contrast to CD62L expression which appears to be more susceptible to alteration due to freezing-thawing. However, the relative stimulus-dependant changes of activation can still be reflected.

Keywords: adhesion molecules, cryopreservation, flow cytometry, polyethylene glycol

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CryoLetters 33 (2), 160-168 (2012)
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CRYOPRESERVATION OF WINTER-DORMANT APPLE: III – BUD WATER STATUS AND SURVIVAL AFTER COOLING TO -30°C AND DURING RECOVERY FROM CRYOPRESERVATION

C. Vogiatzi¹, B.W.W. Grout^1* and A. Wetten²

¹Department of Agriculture and Ecology, University of Copenhagen, Højbakkegård Allé 13, DK-2630 Taastrup, Denmark
²School of Biological Sciences, University of Reading, Harborne Building, RG6 6AS, United Kingdom
^*Corresponding author email: bwg@life.ku.dk

Abstract

In a continuing study to improve the efficiency of dormant bud cryopreservation for tissues hardened in maritime climates, the water status of dormant buds was monitored between -4°C and recovery from liquid nitrogen (LN). Measurement of water content, simple thermal analysis and differential scanning calorimetry were employed. Buds did not lose water during cooling to, or holding at -30°C indicating that cryodehydration and/or other adaptive responses contributed during this essential step. A bud exotherm that was an artefact of warming was detected due to necessary handling at -4°C before cooling to -30°C. There were no significant differences between cultivars with respect to water status at -30°C or immediately upon rewarming from LN despite significant differences in post-LN survival. Buds rehydrated in 5 days, but up to 14 days may be needed for recovery for some cultivars. In some instances buds could be grafted without rehydration, taking up water across the early graft union.

Keywords: Malus, dormant bud, water status, cryopreservation

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