Volume 33, No. 3 May/June 2012 ISSN 0143-2044
	Cryopreservation of human skeletal muscle impairs mitochondrial function Steen Larsen, Cindy Wright-Paradis, Erich Gnaiger, Jřrn W. Helge and Robert Boushel	169-175
	A study to evaluate the action of lipocryolysis Hernán R. Pinto, Eduardo Garcia-Cruz and Graciela E. Melamed	176-180
	Development of a vitrification-based cryopreservation protocol for the storage of saltcedar (Tamarix boveana Bunge) Miriam Cano-Castillo and José Luis Casas	181-189
	High-efficiency encapsulation-vitrification protocols for cryopreservation of embryogenic calli of the oriental medicinal plant Anemarrhena asphodeloides Bunge Hong Sen-Rong and Yin Ming-Hua	190-200
	Amidic and acetonic cryoprotectants improve cryopreservation of volvocine green algae Atsushi Nakazawa and Ichiro Nishii	201-212
	Caseinate protects stallion sperm during semen cooling and freezing M.A. Lagares, H.S. Martins, I.A. Carvalho, C.A. Oliveira Junior, M.R. Souza, C.F.A.M. Penna, B.C. Cruz, Stahlberg R. and M.R.J.M. Henry	213-218
	Narrowing of the critical hydration window for cryopreservation of Salix caprea seeds following ageing and a reduction in vigour Elena V. Popova, Du Hyun Kim, Sim Hee Han, Hugh W. Pritchard and Jae Cheon Lee	219-230
	Measurement of membrane hydraulic conductivity of bovine carotid artery endothelial cells using a perfusion microscope Gang Zhao, Kosaku Kurata and Hiroshi Takamatsu	231-239
	Desiccation sensitivity and cryopreservation of excised embryonic axes of Citrus suhuiensis cv. limau madu, citrumelo [Citrus paradisi Macf. × Poncirus trifoliata (L.) Raf.] and Fortunella polyandra Omar M. Al Zoubi and Normah M. N	240-250
	Editorial Announcements and Upcoming Meetings	251

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CryoLetters 33 (3), 169-175 (2012)
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CRYOPRESERVATION OF HUMAN SKELETAL MUSCLE IMPAIRS MITOCHONDRIAL FUNCTION

Steen Larsen^1*, Cindy Wright-Paradis², Erich Gnaiger³,
Jřrn W. Helge¹ and Robert Boushel^{1, 2}

¹Xlab, Center for Healthy Aging, Department of Biomedical Sciences, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
²Department of Exercise Science, Concordia University, Montreal, Quebec, Canada
³Department of Visceral, Transplant and Thoracic Surgery, D. Swarovski Research Laboratory, Medical University of Innsbruck, Austria
*Corresponding author e-mail: stelar@sund.ku.dk

Abstract

Previous studies have investigated if cryopreservation is a viable approach for functional mitochondrial analysis. Different tissues have been studied, and conflicting results have been published. The aim of the present study was to investigate if mitochondria in human skeletal muscle maintain functionality after long term cryopreservation (1 year).

Skeletal muscle samples were preserved in dimethyl sulfoxide (Me₂SO) for later analysis. Human skeletal muscle fibres were thawed and permeabilised with saponin, and mitochondrial respiration was measured by high-resolution respirometry.

The capacity of oxidative phosphorylation (OXPHOS) was significantly (P<0.05) reduced in cryopreserved human skeletal muscle samples. Cryopreservation impaired respiration with substrates linked to Complex I more than for Complex II (P<0.05). Addition of cytochrome c revealed an increase in respiration indicating cytochrome c loss from the mitochondria. The results from this study demonstrate that normal mitochondrial functionality is not maintained in cryopreserved human skeletal muscle samples.

Keywords: Cryopreservation, high-resolution respirometry, human skeletal muscle, mitochondrial function, permeabilised fibres

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CryoLetters 33 (3), 176-180 (2012)
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A STUDY TO EVALUATE THE ACTION OF LIPOCRYOLYSIS

Hernán R. Pinto*¹, Eduardo Garcia-Cruz ² and Graciela E. Melamed ³

¹Age Back Barcelona, Aesthetic Medicine Department, Spain.
²Sexual Medicine and Men's Health, Hospital Clinic Barcelona, Spain.
³MEG Group Clinic Buenos Aires, Argentina.
*Corresponding author email: portatil@ageback.es

Abstract

Since ancient times human beings have been conceiving methods that could help reduce the accumulation of undesired fat tissue in their own bodies. Lipocryolysis has already been recognized as an therapy for localized fat reduction by means of a combination of regulated and controlled vacuum and heat extraction therapy. This study was designed to quantify the claimed reduction of local adiposities. For this purpose, 16 treatments were analysed. The data suggested that lipocryolysis is effective for localized reduction of adiposities and that the reduction obtained are measurable.

Keywords: Lipocryolysis, localized adiposities, heat extraction, triglycerides.

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CryoLetters 33 (3), 181-189 (2012)
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DEVELOPMENT OF A VITRIFICATION-BASED CRYOPRESERVATION PROTOCOL FOR THE STORAGE OF SALTCEDAR (Tamarix boveana BUNGE)

Miriam Cano-Castillo and José Luis Casas*

Plant Biotechnology Laboratory. Institute of Biodiversity (CIBIO). University of Alicante. Crta. San Vicente del Raspeig s/n. E-03690 San Vicente del Raspeig, Alicante, Spain.
*Corresponding author  email: jl.casas@ua.es

Abstract

We cryopreserved in vitro shoot tips of saltcedar (Tamarix boveana Bunge) using the vitrification technique. The success of the cryopreservation protocol was strongly affected by preculture, loading duration, dehydration duration in plant vitrification solution 2 (PVS2), and medium composition during post-warming regrowth. The highest explant regrowth (50%) occurred when the following conditions were employed: preculture in 0.4 M glycerol; treatment with a loading solution (LS) consisting of 2 M glycerol + 0.4 M sucrose in culture medium for 40 min at room temperature; and dehydration in PVS2 at 0°C for 45 min before rapid immersion in liquid nitrogen (LN). Rewarming was performed in a water-bath at 40°C for 2 min. Explants were then immersed in unloading solution for 10 min before plating on recovery medium supplemented with 0.01 mg l^-1 thidiazuron (TDZ). TDZ was progressively eliminated from the medium over a period of 6 weeks. Plantlets were transferred to a double-layer medium to enhance rooting. This protocol was successfully applied to three individuals of T. boveana harvested from the wild.

Keywords: cryopreservation, double-phase system, preculture, PVS2, Tamarix, thidiazuron, vitrification

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CryoLetters 33 (3), 190-200 (2012)
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HIGH-EFFICIENCY ENCAPSULATION-VITRIFICATION PROTOCOLS FOR CRYOPRESERVATION OF EMBRYOGENIC CALLI OF THE ORIENTAL MEDICINAL PLANT Anemarrhena asphodeloides BUNGE

Hong Sen-Rong and Yin Ming-Hua*

College of Life Sciences, Shangrao Normal University, 334001 Shangrao, People's Republic of China.
*Corresponding author email: yinminghua04@163.com

Abstract

Embryogenic calli from in vitro grown tillers of Anemarrhena asphodeloides Bunge were successfully cryopreserved by the encapsulation–vitrification technique. Excised embryogenic calli were precultured for 4 days in liquid MS medium supplemented with 2 mg L^-1 kinetin (KIN), 0.1 mg L^-1 α-naphthalene acetic acid (NAA) and 0.75 M sucrose, then encapsulated in calcium alginate beads and loaded with a mixture of 2 M glycerol + 0.4 M sucrose for 60 min at 25±1°C. Calli were then dehydrated with the PVS2 solution for 80 min at 0°C. After changing the solution with fresh PVS2, calli were directly immersed in liquid nitrogen (LN). After rapid rewarming in a water-bath at 35°C for 5 min, calli were washed three times with liquid MS medium supplemented with 2 mg L^-1 KIN, 0.1 mg L^-1 NAA and 1.2 M sucrose, then transferred on solid MS medium supplemented with 2 mg L^-1 KIN, 0.1 mg L^-1 NAA, 3% (w/v) sucrose and 0.75% (w/v) agar. Cryopreserved cultures were kept in the dark for 5 days prior to exposure to a 14 h light/10 h dark photoperiod with a light intensity of 36 μmol m^-2 s^-1 provided by white cool fluorescent tubes at 25 ±1°C. Survival of cryopreserved embryogenic calli reached 80%, including after storage for c. 1 year. No significant difference was observed in the morphological development of plants coming from control and cryopreserved embryogenic calli. This encapsulation-vitrification method appears promising for the cryopreservation of A. asphodeloides Bunge germplasm.

Keywords: Anemarrhena asphodeloides Bunge, cryopreservation, encapsulation-vitrification, embryogenic calli.

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CryoLetters 33 (3), 201-212 (2012)
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AMIDIC AND ACETONIC CRYOPROTECTANTS IMPROVE CRYOPRESERVATION OF VOLVOCINE GREEN ALGAE

Atsushi Nakazawa¹ and Ichiro Nishii²*

¹Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan.
²Department of Biological Sciences, Nara Women's University, Nara 630-8506, Japan.
*Corresponding author  email: ichiron@cc.nara-wu.ac.jp

Abstract

A number of volvocalean green algae species were subjected to a two-step cryopreservation protocol with various cryoprotectants. Potential cryoprotectants were methanol (MeOH), N,N-dimethylformamide (DMF), N,N-dimethylacetamide, N-methylformamide, and hydroxyacetone (HA). We confirmed prior reports that MeOH was effective for cryopreserving Chlamydomonas, but did not work well for larger volvocaleans such as Volvox. In contrast, DMF and HA were effective for both unicellular and multicellular representatives. When we used a cold-inducible transposon to probe Southern blots of Volvox DNA samples taken before and after storage for one month in LN, we could detect no differences, indicating that the genome had remained relatively stable and that the transposon had not been induced by the cryopreservation procedure. We believe these methods will facilitate long-term storage of several volvocine algal species, including Volvox strains harboring transposon-induced mutations of developmental interest.

Keywords: cryopreservation, dimethylformamide, hydroxyacetone, Volvocaceae, Volvox, Chlamydomonas

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CryoLetters 33 (3), 13-218 (2012)
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ASEINATE PROTECTS STALLION SPERM DURING SEMEN COOLING AND FREEZING

M.A. Lagares^1*, H.S. Martins¹, I.A. Carvalho¹, C.A. Oliveira Junior¹,
M.R. Souza², C.F.A.M. Penna², B.C. Cruz¹, Stahlberg R².
and M.R.J.M. Henry¹

¹Departamento de Clínica e Cirurgia Veterinária, Escola de Veterinária, Universidade Federal de Minas Gerais, MG, ²Departamento de Tecnologia e Inspeçăo de Produtos de Origem Animal, Escola de Veterinária, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, CEP:31270-901, MG,
²Curso de Medicina Veterinária, PUC Minas Betim, R. do Rosário 1081, Bairro Angola, Betim, CEP: 32604-115, MG, Brasil
*Corresponding author  e-mail: mlagares@ufmg.br

Abstract

Extenders with a defined composition containing only components with clearly protective effects on sperm during storage would be an advantage. The aims of the present work were to assess whether caseinate, improves cooled and frozen equine semen quality. Semen from six stallions were suspended with four different cooling extenders C1) Kenney extender; C2) 0.6% caseinate; C3) 2.7% caseinate ; and C4) C1 + 2.1% caseinate, and frozen extenders: F1) INRA 82 extender; F2) 1.35% caseinate; and F3) 2.7% caseinate. Although there was no significant difference between the motility rate among the cooled (C1:45.0, C2:36.7, C3:38.3 and C4:48.3) and frozen extenders (F1:16.9, F2:21.1 and F3:18.6), significant higher values of sperm velocity variables were observed with the 1.35% caseinate extender compared to the control (VSL: 40.8x18.9 and VAP: 46.8 x 25.0 µm/s), respectively. Caseinate seemed to be responsible for sperm protection during preservation and showed to be as efficient as milk.

Keywords: casein-equine-spermatozoa-semen-cryopreservation- extender

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CryoLetters 33 (3), 219-230 (2012)
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NARROWING OF THE CRITICAL HYDRATION WINDOW FOR CRYOPRESERVATION OF Salix caprea SEEDS FOLLOWING AGEING AND A REDUCTION  IN VIGOUR

Elena V. Popova¹, Du Hyun Kim¹, Sim Hee Han¹, Hugh W. Pritchard² and Jae Cheon Lee¹

¹Division of Forest Genetic Resources, Korea Forest Research Institute, 44-3 Omokdong, Suwon 441-350, Korea.
²Seed Conservation Department, Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, U.K.

Abstract

We investigated the effects of desiccation, rehydration and cryopreservation on the viability of seeds of a wild mountain species and seven clones of Salix caprea L. Seeds responded differently to all treatments depending on clone, seed initial moisture content (MC) and seed vigour. Fresh seeds of two randomly selected clones tolerated desiccation to MC 8.5-9.6% FW (0.09-0.11 gH₂O g^-1DW) without any noticeable loss in viability and were successfully cryopreserved at MCs ranging from 8.5 to 23.4% (0.09-0.30 gH₂O g^-1DW). Storage at 5C for approximately 10 weeks significantly reduced the viability of seed lots of a wild species and of three S. caprea clones, whilst viability of seeds of four other clones remained unaffected. Since all clones tested were genetically derived from one tree, this variation is unlikely to be of maternal origin. Most probably paternal x environmental factors have influenced seed behavior during desiccation and storage. As viability decreased due to partial ageing, seeds became more susceptible to desiccation stress. When seeds of three clones were cryopreserved, the hydration window for survival was wider for highly vigorous seeds (c. 0.05-0.28 gH₂O g^-1DW) than for seeds with intermediate vigour (c. 0.10-0.24 gH₂O g^-1DW) and low vigour (c. 0.20-0.37 gH₂O g^-1DW). Rehydration to MC above 0.15 gH₂O g^-1DW improved germination of low vigour seeds, both in controls and after cryopreservation. In contrast, cryopreservation of high vigour seeds rehydrated to MCs above 0.11 gH₂O g^-1DW resulted in a sharp decrease in normal seedling production. Whilst no effect of cryogenic temperature on germination and normal seedling production was observed when seeds of seven clones were cryopreserved within their hydration windows, the results indicate the need to account for seed lot vigour when designing cryopreservation protocols.

Keywords: Salix caprea, cryopreservation, desiccation, moisture content, short-lived seeds, seed vigour

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CryoLetters 33 (3), 231-239 (2012)
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MEASUREMENT OF MEMBRANE HYDRAULIC CONDUCTIVITY OF BOVINE CAROTID ARTERY ENDOTHELIAL CELLS USING A PERFUSION MICROSCOPE

Gang Zhao¹*, Kosaku Kurata² and Hiroshi Takamatsu²

¹Department of Electronic Science and Technology, University of Science and Technology of China, Hefei 230027, Anhui, China; ²Department of Mechanical Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
*Corresponding author  email: zhaog@ustc.edu.cn

Abstract

The osmotic properties of bovine carotid artery endothelial cells (BCAECs) associated with cryobiology were investigated using a perfusion microscope. These properties include the hydraulic conductivity (L_p) and its activation energy (E_Lp). The response of isolated cells was observed when the extracellular concentration increased from 0.15 M to 0.5 M NaCl at three different temperatures. The transient volumes of the cell were calculated from the measurements of the projected areas with an assumption of a spherical cell. The hydraulic conductivity (L_p) and the osmotically inactive volume (V_b) of BCAECs were simultaneously determined using nonlinear regression to fit the change of cell volume estimated by water transport equations to measured cell volumes. The L_p values were 0.26 ± 0.08, 0.12 ± 0.02, and 0.06 ± 0.02 ྒྷm/atm/min (mean ± SD) at 23, 11 and 4^oC, respectively, yielding the activation energy of L_p of 47.6 kJ/mol according to the Arrhenius relationship.

Keywords: endothelial cells, hydraulic conductivity, activation energy, perfusion microscope

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CryoLetters 33 (3), 240-250 (2012)
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DESICCATION SENSITIVITY AND CRYOPRESERVATION OF EXCISED EMBRYONIC AXES OF Citrus suhuiensis cv. LIMAU MADU, CITRUMELO [Citrus paradisi Macf. × Poncirus trifoliata (L.) Raf.] AND Fortunella polyandra

Omar M. Al Zoubi¹ and Normah M. N^1,2*

¹School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.
²Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.
*Corresponding author  e-mail: normah@ukm.my

Abstract

Excised embryonic axes from seeds of three taxa, namely, Citrus suhuiensis cv. limau madu, Citrumelo (Citrus paradisi x Poncirus trifoliate) and Fortunella polyandra, were desiccated in a laminar airflow, over silica gel, and ultra-rapidly. Desiccation sensitivity (WC₅₀) was estimated for each taxon using the quantal response model. High desiccation tolerance (WC₅₀ = 0.11 g H₂O g^-1dw) was observed for limau madu embryonic axes desiccated in a laminar airflow and ultra-rapidly (WC₅₀ =0.10 g H₂O g^-1dw). Desiccation tolerance was substantially lower (WC₅₀ = 0.19 g H₂O g^-1dw) for silica gel dehydration. Similarly, high desiccation tolerance (WC₅₀ = 0.15 g H₂O g^-1dw) was associated with F. polyandra embryonic axes when desiccated in a laminar airflow, while a lower desiccation tolerance (WC₅₀ = 0.17 g H₂O g^-1dw) was observed with silica gel dehydration. Ultra-rapid desiccation led to the highest desiccation tolerance (WC₅₀ = 0.14 g H₂O g^-1dw). The dehydration rate, however, had no influence on desiccation tolerance (WC₅₀ ~ 0.14 g H₂O g^-1dw) for Citrumelo embryonic axes. After each desiccation period, embryonic axes were directly immersed in liquid nitrogen (LN) followed by rapid rewarming. Normal seedling recovery of 80 to 83% for excised embryonic axes of limau madu was observed for laminar airflow and ultra-rapid dehydration, but for silica gel dehydration, 57% recovery was obtained. Similarly, for Citrumelo, high recoveries of 100% and 97% were obtained from axes desiccated in a laminar airflow and using ultra-rapid dehydration, respectively, whereas a lower value was associated with silica gel dehydration (80%). For F. polyandra, 50% recovery was obtained both for laminar airflow and ultra-rapid dehydration, while much lower recovery (43%) was associated with silica gel dehydration. Regardless of the drying method employed, axis survival percentages following exposure to LN were commensurate with the desiccation sensitivity pattern.

Keywords: Citrus suhuiensis cv. limau madu, Citrumelo (Poncirus trifoliata x Citrus paradisi), Fortunella polyandra, cryopreservation, dehydration rate, desiccation sensitivity, embryonic axis.

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