Volume 34, No. 4 July/August 2013 ISSN 0143-2044
	Effects of freezing rates and cryoprotectant on thermal expansion of articular cartilage during freezing process Yi Xu, Hui-Jun Sun, Ya Lv, Jin-Cheng Zou, Bao-Lin Liu and Tse-Chao Hua	313-323
	Computerized training of cryosurgery – a system approach Robert Keelan, Soji Yamakawa, Kenji Shimada and Yoed Rabin	324-337
	Shoot-tip cryopreservation by droplet vitrification of Byrsonima intermedia A. Juss.: a woody tropical and medicinal plant species from Brazilian Cerrado Luciano Coutinho Silva, Renato Paiva, Rony Swennen, Edwige Andrèand Bart Panis	338-348
	The effect of temperature elevation on cryopreserved mesenchymal stem cells M. Norkus, L. Kilmartin, D. Fay, M. J. Murphy, G. ÓLaighin and F. Barry	349-359
	A device to record ultra-rapid cooling profiles Leonardo Juan de Paz, Daniel Adolfo Graf, Angel Luis Scandizzi, Edgardo Elvio Guibert and Joaquin Valentin Rodriguez	360-368
	Development of three vitrification-based cryopreservation procedures for shoot tips of China's potato Biao Wang, Ren-rui Wang, Jing-wei Li, Yan-li Ma, Wan-min Sheng, Min-fu Li and Qiao-chun Wang	369-380
	Enhanced metabolic function of human hepatocytes cryopreserved with low concentration Me2SO and polyol additives at –80°C Bo Yang, Baolin Liu, Xinli Zhou, Li Shen and Danhua Huang	381-387
	Cryopreservation of nematode Caenorhabditis elegans in the adult stage Masakazu Hayashi, Hisako Amino, Kiyoshi Kita and Norio Murase	388-395
	Cryoprotection of fibroblasts by carboxylated poly-l-Lysine upon repeated freeze/thaw cycles Oh Seong Jin, Jong Ho Lee, Yong Cheol Shin, Eun Ji Lee, Jun Jae Lee, Kazuaki Matsumura, Suong-Hyu Hyon and Dong-Wook Han	396-403
	Parameter conversion from Kedem-Katchalsky model to two-parameter model revisited Jian Ren and Gang Zhao	404-412
	Biochemical characterization of Ecuadorian wild Solanum lycopersicum mill.plants produced from non-cryopreserved and cryopreserved seeds Byron Zevallos, Inaudis Cejas, René Carlos Rodríguez, Lourdes Yabor, Carlos Aragón, Justo González, Florent Engelmann, Marcos Edel Martínez and José Carlos Lorenzo	413-421
	Effect of pasteurized egg and Rosmarinus officinalis supplementation on quality of cryopreserved ram semen Francisco Mascaró, Lydia Gil, Clara Malo, Noelia González, Felisa Martínez and Ignacio de Blas	422-431
	Biopreservation of hepatocytes: current concepts on hypothermic preservation, cryopreservation, and vitrification Barry J. Fuller, Alexander Y. Petrenko, Joaquín V. Rodriguez, Alexander Y. Somov, Cecilia L. Balaban and Edgardo E. Guibert	432-452

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CryoLetters 34 (4), 313-323 (2013)
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EFFECTS OF FREEZING RATES AND CRYOPROTECTANT ON THERMAL EXPANSION OF ARTICULAR CARTILAGE DURING FREEZING PROCESS

Yi Xu^*, Hui-Jun Sun, Ya Lv, Jin-Cheng Zou, Bao-Lin Liu
and Tse-Chao Hua

Institute of Cryomedicine, University of Shanghai for Science and Technology, Shanghai 200093, P. R. China
*Corresponding author  e-mail: xu_hongyi@263.net

Abstract

The intact articular cartilage has not yet been successfully preserved at low temperature most likely due to the volume expansion from water to ice during freezing. The objective of this current study focuses on examining thermal expansion behavior of articular cartilage (AC) during freezing from 0°C to -100° C. Thermo Mechanical Analysis (TMA) was used to investigate the effects of different concentrations of dimethyl sulphoxide (DMSO) (0%, 10%, 30% and 60% v/v) and different freezing rates (1℃æ/min, 3℃æ/min and 5℃æ/min). The results showed that: (1) the inhomogeneous thermal expansion (or contraction) presents due to inhomogeneous water distributions in articular cartilage during freezing, which also may be the most likely reason that the matrix has been damaged in cryopreserved intact articular cartilage; (2) at the phase transition temperature range, the maximum thermal strain change value for 5℃æ/min is approximately 1.45 times than that for 1℃æ/min, but the maximum thermal expansion coefficient of the later is about six times than that of the former; (3) the thermal expansion coefficient decreases with increasing cooling rate at the unfrozen temperature region, but some opposite results are obtained at the frozen temperature region; (4) the higher the DMSO concentration is, at the phase change temperature region, the smaller the thermal strain change as well as the maximum thermal expansion coefficient are, but DMSO concentration exhibits little effect on the thermal expansion coefficient at both unfrozen and frozen region. Once the DMSO concentration increasing enough, e.g. 60%v/v, the thermal strain decreases linearly and smoothly without any abrupt change due to little or no ice crystal forms (i.e. vitrification) in frozen articular cartilage. This study may improve our understanding of the thermal expansion (or contraction) behavior of cryopreserved articular cartilage and it may be useful for the future study on cryopreservation of intact articular cartilage.

Keywords: Articular cartilage, cryopreservation, freezing process, thermal expansion, thermal strain, Thermo Mechanical Analysis (TMA)

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CryoLetters 34 (4), 324-337 (2013)
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COMPUTERIZED TRAINING OF CRYOSURGERY – A SYSTEM APPROACH

Robert Keelan, Soji Yamakawa, Kenji Shimada and Yoed Rabin*

Department of Mechanical Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA 15213
*Corresponding author  email: rabin@cmu.edu

Abstract

The objective of the current study is to provide the foundation for a computerized training platform for cryosurgery. Consistent with clinical practice, the training process targets the correlation of the frozen region contour with the target region shape, using medical imaging and accepted criteria for clinical success. The current study focuses on system design considerations, including a bioheat transfer model, simulation techniques, optimal cryoprobe layout strategy, and a simulation core framework. Two fundamentally different approaches were considered for the development of a cryosurgery simulator, based on a finite-elements (FE) commercial code (ANSYS) and a proprietary finite-difference (FD) code. Results of this study demonstrate that the FE simulator is superior in terms of geometric modeling, while the FD simulator is superior in terms of runtime. Benchmarking results further indicate that the FD simulator is superior in terms of usage of memory resources, pre-processing, parallel processing, and post-processing. It is envisioned that future integration of a human-interface module and clinical data into the proposed computer framework will make computerized training of cryosurgery a practical reality.

Keywords: cryosurgery, simulation, training, computerized planning, system design

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CryoLetters 34 (4), 338-348 (2013)
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SHOOT-TIP CRYOPRESERVATION BY DROPLET VITRIFICATION OF BYRSONIMA INTERMEDIA A. JUSS.: A WOODY TROPICAL AND MEDICINAL PLANT SPECIES FROM BRAZILIAN CERRADO

Luciano Coutinho Silva^1*, Renato Paiva¹, Rony Swennen²,
Edwige Andrè² and Bart Panis²

¹ Universidade Federal de Lavras (UFLA), Departamento de Biologia, Setor de Fisiologia Vegetal, Campus Universitário, CP 3037, Lavras – MG, 372000-000, Brasil.
² Laboratory of Tropical Crop Improvement, Katholieke Universiteit Leuven (K.U. L.), Kasteelpark Arenberg 13, B-3001 Leuven, Belgium
* Correspondent author email: lucoutsilva@yahoo.com.br

Abstract

Cryopreservation of plant species is poorly investigated in Brazil. The aim of this study was to cryopreserve Byrsonima intermedia shoot apical meristems through droplet vitrification. A culture medium for shoot-tips growth was established using the Woody Plant Medium supplemented with 2.22 µM 6-benzylaminopurine. Excised shoot-tips were subjected to pre-culture and/or post-culture treatments on Murashige and Skoog medium with 0.3 M sucrose for 24 h prior dehydration on PVS2 at 0°C for 15, 30 or 45 minutes prior to plunging in liquid nitrogen. The effect of 15 days of shoot pre-growth on a high osmotic medium (0.3 M sucrose or 0.21 M sorbitol + 0.09 M sucrose) prior to meristem excision and cryopreservation was also investigated. Pre-culturing shoot-tips on 0.3 M sucrose for 24 h prior to cryopreservation increased the regrowth level after thawing to 90%. Shoot-tips excised from shoots pre-grown on MS + 0.21 M sorbitol + 0.09 M sucrose for 15 days presented a satisfactory regrowth level (67%).

Keywords: pre-culture effect, post-culture effect, pre-growth effect, sorbitol, medicinal plant, vitrification

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CryoLetters 34 (4), 349-359 (2013)
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THE EFFECT OF TEMPERATURE ELEVATION ON CRYOPRESERVED MESENCHYMAL STEM CELLS

M. Norkus^1,2,*, L. Kilmartin^1,2, D. Fay³, M. J. Murphy⁴, G. ÓLaighin^1,2
and F. Barry⁴

¹School of Engineering and Informatics, National University of Ireland Galway, University Road, Galway, Ireland.
²Bioelectronics Research Cluster, National Centre for Biomedical Engineering Science, National University of Ireland Galway, University Road, Galway, Ireland.
³Computer Laboratory, Cambridge University, 15 JJ Thompson Ave, Cambridge, United Kingdom.
⁴Regenerative Medicine Institute, National Centre for Biomedical Engineering Science, National University of Ireland Galway, University Road, Galway, Ireland.
*Corresponding author e-mail: m.norkus1@nuigalway.ie

Abstract

Background: Cryopreservation is of particular importance in stem cell research and regenerative medicine as it permits long term stabilisation of biological cells. Cells retain their regenerative capacity after years of storage at cryogenic temperatures. However, elevation of temperature may occur due to variety of reasons, for example in the event of equipment malfunction or during delays in transportation. To date, a limited amount research has been carried out to examine the effects of temperature elevation on stem cell survival during cryopreservation. Methods: Mesenchymal Stem Cells (MSCs) obtained from 8-12 week Sprague Dawley male rats were cryopreserved according to the standard procedures. Under experimental conditions, cryopreserved specimens were exposed to elevated temperatures ranging from -20°C to 37°C and cellular membrane integrity assessed via trypan-blue exclusion at various time points. Results: An approximating model of multiple regression was fitted to the experimental data and optimisation of model parameters was carried out. This model provides an approximation of cell viability in response to elevated temperature conditions. Discussion: The results demonstrate that elevation of temperature has a dramatic effect, even over short periods of time, on the viability of cryopreserved specimens. The model presented here could be used to predict the damage suffered by a specimen due to exposure to elevated temperature over a defined period of time.

Keywords: Cell viability, cryopreservation, mesenchymal stem cells, temperature elevation

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CryoLetters 34 (4), 360-368 (2013)
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A DEVICE TO RECORD ULTRA-RAPID COOLING PROFILES

Leonardo Juan de Paz^1,2, Daniel Adolfo Graf^1,2,
Angel Luis Scandizzi¹, Edgardo Elvio Guibert¹
and Joaquin Valentin Rodriguez¹*

¹Centro Binacional (Argentina -Italia) de Investigaciones en Criobiología Clínica y Aplicada (CAIC), Universidad Nacional de Rosario, Avda. Arijón 28 bis, S2011BXN Rosario, Argentina.
²Servicio de Electrónica y Óptica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531-S2002LRK Rosario, Argentina.
*Corresponding author  email: jrodrig@fbioyf.unr.edu.ar

Abstract

This work deals with the construction and performance of a measuring system capable of estimating temperature at sufficiently high speed (up to 1000 samples/sec). Due to its simple design and the utilization of standard materials, it could serve to recording the cooling profile of ultra-rapid procedures. An immersion device was also developed with the purpose of normalize the penetration speed of the sample in the LN₂. The device allows also the comparative analysis of different cooling profiles. The system consists of an immersion device of the sample in the cooling agent, a temperature measurement system developed by Kleihans F and a laptop computer. To test the system, we recorded the cooling profiles of 10 L of distilled water and 6 M glycerol solution, obtaining a cooling rate of 8732 °C.min^-1 and 4441 °C.min^-1 respectively. Also we determine a cooling rate of 204.012 °C.min^-1 during the immersion of the thermocouple assembly in LN₂. Although, the same device, with small technical modifications related to the handling of the sample, could be used to evaluate the recovery from LN₂ temperature to room temperature (re-warming).

Keywords: ultra-rapid cooling, vitrification, cooling rate, thermal history

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CryoLetters 34 (4), 369-380 (2013)
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DEVELOPMENT OF THREE VITRIFICATION-BASED CRYOPRESERVATION PROCEDURES FOR SHOOT TIPS OF CHINA'S POTATO

Biao Wang¹, Ren-rui Wang^{1, 2}, Jing-wei Li¹, Yan-li Ma¹,
Wan-min Sheng³, Min-fu Li² and Qiao-chun Wang¹*

^1.State Key Laboratory of Crop Stress Biology for Arid Region, College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, PR China.
^2.Institute of Animal & Plant Quarantine, Chinese Academy of Inspection and Quarantine (CAIQ), Chaoyang District 10029, Beijing 100029, PR China.
^3.Keshan State Potato Germplasm Centre, Heilongjiang Academy of Agricultural Sciences, Keshan 161606, Heilongjiang, PR China.
*Corresponding author  email: qiaochunwang@nwsuaf.edu.cn

Abstract

China is now the largest potato producing country worldwide. The establishment of efficient conservation techniques for potato germplasm is a prerequisite for breeding of elite cultivars. Potato viral diseases have been a great threat for sustainable potato production in China. Use of virus-free seed tubers is an effective and practical means to control viral diseases. In the present study, three vitrification-based cryopreservation techniques, i.e. droplet-vitrification, encapsulation-vitrification and vitrification were successfully developed for the first time for China's potato. Cultivar 'Zihuabai' was used to optimize key parameters involved in the three vitrification-based procedures. With the optimized parameters, shoot regrowth percentages of 71%, 76% and 43% were obtained for droplet-vitrification, encapsulation-vitrification and vitrification, respectively. The three protocols developed were further tested with eight China's major cultivars, with average shoot regrowth of 61%, 38% and 28% for droplet-vitrification, encapsulation-vitrification and vitrification, respectively. Successful development of the three cryopreservation procedures using a single cultivar will facilitate a number of comparative studies such as cryo-injury, regrowth pattern, genetic stability and efficiency of virus elimination. Testing these three cryogenic procedures for potato major cultivars representing a wide range of genetic background, will help the establishment of potato cryobanking in China and the production of virus-free plants.

Keywords: Long-term conservation, droplet-vitrification, encapsulation-vitrification, Solanum tuberosum, survival, regrowth

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CryoLetters 34 (4), 381-387 (2013)
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ENHANCED METABOLIC FUNCTION OF HUMAN HEPATOCYTES CRYOPRESERVED WITH LOW CONCENTRATION ME₂SO AND POLYOL ADDITIVES AT –80 °C

Bo Yang, Baolin Liu, Xinli Zhou, Li Shen and Danhua Huang

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

Abstract

The metabolic function of cryopreserved cells, in addition to cell viability after thawing, is an important parameter in any successful cryopreservation protocol. Dimethyl sulfoxide (Me₂SO) is known to affect the differentiation of recovered cells. In this study, we report that sugars and sugar alcohols increases cell recovery, and also improves the metabolic function of human hepatocytes that are cryopreserved using low concentration Me₂SO (5%). Three sugars (glucose, sucrose, and trehalose) and three sugar alcohols (xylitol, maltol, and sorbitol) have been tested. Cell viability after thaw and 24-h post-thaw attachment rate of cryopreserved human hepatocytes were assessed. Post-thaw metabolic activities (albumin, glucose, urea content) were measured, and cell proliferation was observed with inverted microscope. Cell viability, post-thaw attachment rate and metabolic activity of cryopreserved hepatocytes are enhanced by the addition of 0.4M sorbitol into 5% Me₂SO solution. The study concludes that 5% Me₂SO + 0.4M sorbitol can replace the 10% Me₂SO method for cryopreservation of human hepatocytes at -80C freezer. The new solution may reduce the side effects on the patients and improve the safety of using cryopreserved hepatocytes.

Keywords: dimethyl sulfoxide, cryoprotectant, human hepatocytes, sorbital.

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CryoLetters 34 (4), 388-395 (2013)
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CRYOPRESERVATION OF NEMATODE CAENORHABDITIS ELEGANS IN THE ADULT STAGE

Masakazu Hayashi¹*, Hisako Amino², Kiyoshi Kita²
and Norio Murase¹

¹School of Science and Engineering, Tokyo Denki University, Hatoyama, Hiki-gun, Saitama350-0394, Japan.
²Graduate School of Medicine, the University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
*Corresponding author  email: masakazuhayashii@gmail.com

Abstract

Cryopreservation of nematode Caenorhabditis elegans in the adult stage is of importance as the nematode is a powerful research model organism. In this study, we applied the protocol previously established for cryopreservation of the L4 nematode to the adult one, and achieved a survival rate of 84%. When ice seeding was induced with bacteria P. syringae directly added to the nematode suspension instead of using a pre-cooled steel sticking needle, comparable survival rate was obtained after thawing. Moreover, a simple freezing device composed of a polystyrene foam box surrounded by a Dewar vessel put in a deep freezer was developed for a practical use. This simple method obtained a survival rate of 69 ± 4% for the adult nematode after thawing.

Keywords: Caenorhabditis elegans, dimethyl sulfoxide, ice seeding, Pseudomonas syringae

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CryoLetters 34 (4), 396-403 (2013)
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CRYOPROTECTION OF FIBROBLASTS BY CARBOXYLATED POLY-L-LYSINE UPON REPEATED FREEZE/THAW CYCLES

Oh Seong Jin ¹, Jong Ho Lee ¹, Yong Cheol Shin ¹, Eun Ji Lee ¹,
Jun Jae Lee ¹, Kazuaki Matsumura ², Suong-Hyu Hyon ^3*
and Dong-Wook Han ^1*

¹ Department of Applied Nanoscience and BK21 Nano Fusion Technology Division, Pusan National University, Busan, Korea.
² School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa, Japan.
³ Department of Biobased Materials Science, Kyoto Institute of Technology, Kyoto, Japan
* Corresponding authors emails: ma37ha73@kcn.jp and nanohan@pusan.ac.kr

Abstract

The cryoprotection of carboxylated ε-poly-L-lysine (COOH-PLL) was investigated on fibroblasts [L-929 cells and human dermal fibroblasts (HDFs)] during multiple freeze/thaw cycles. COOH-PLL was not toxic to two fibroblast cell types even at 25% (w/v) concentration, whereas dimethylsulphoxide (DMSO) was highly toxic over 3.13% (v/v). When L-929 cells were subjected to 5 freeze/thaw cycles, the media containing 7.5% (w/v) COOH-PLL maintained cell morphology and significantly suppressed growth inhibition as well as cell detachment (P < 0.05). The result was comparable to the media containing 10% (v/v) DMSO. For HDFs, COOH-PLL could effectively retain cell viability and proliferation against 3 freeze/thaw cycles. Cell viability of HDFs was decreased after 5 freeze/thaw cycles, but COOH-PLL exerted better cryoprotection. The cell type might account for the difference in the observations. The data demonstrated that COOH-PLL is a good cryoprotectant for mammalian cells against repeated freeze/thaw cycles, and may be used for cell preservation in fields of cell transplantation, tissue engineering and regenerative medicine.

Keywords: Carboxylated ε-poly-L-lysine, cryopreservation, dimethyl sulphoxide, fibroblast cells, freezing/thawing.

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CryoLetters 34 (4), 404-412 (2013)
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PARAMETER CONVERSION FROM KEDEM-KATCHALSKY MODEL TO TWO-PARAMETER MODEL REVISITED

Jian Ren and Gang Zhao^*

Department of Electronic Science and Technology, University of Science and Technology of China, Hefei, Anhui, P.R. China.
^*Corresponding author email: ZhaoG@ustc.edu.cn

Abstract

Successful cryopreservation needs to avoid osmotic injury during the addition and removal of cryoprotective agents. The Kedem-Katchalsky model (KK) and more recently the two-parameter model (2P) are applied to study the volumetric responses of cells for process optimization. The difference between two models is the presence of the reflection coefficient () in the KK model. Earlier studies found that the inclusion of is unnecessary and in some cases may lead to conceptual errors when the model is applied cell membranes. Since the 2P model is accurate and simple to use, Chuenkhum et al. had derived the numerical values of 2P parameters for many cell types from KK parameters. We noted, however, that the calculation of Chuenkhum et al. needs further verification because they did not take into consideration of the original experimental conditions in their simulations. The present paper presents the corrected dataset for many cell types.

Keywords: cryopreservation, membrane hydraulic conductivity, Kedem-Katchalsky model, two-parameter model.

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CryoLetters 34 (4), 413-421 (2013)
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BIOCHEMICAL CHARACTERIZATION OF ECUADORIAN WILD SOLANUM LYCOPERSICUM MILL. PLANTS PRODUCED FROM NON-CRYOPRESERVED AND CRYOPRESERVED SEEDS

Byron Zevallos¹, Inaudis Cejas², René Carlos Rodríguez³,
Lourdes Yabor³, Carlos Aragón³, Justo González³,
Florent Engelmann⁴, Marcos Edel Martínez³
and José Carlos Lorenzo³

¹Escuela Superior Politécnica Agropecuaria de Manabí Manuel Félix López (ESPAM), Campus Politécnico El Limón, Carrera de Ingeniería Agrícola, Calceta, Manabí, Ecuador,
²Faculty of Agronomy, Universidad de Ciego de Ávila, Ciego de Ávila 69450, Cuba.
³Laboratory for Plant Breeding, Centro de Bioplantas, Universidad de Ciego de Ávila, Ciego de Ávila 69450, Cuba.  URL: www.bioplantas.cu.
⁴IRD, UMR DIADE, 911 Avenue Agropolis, BP 64501, 34394 Montpellier Cedex, 5, France.
*Corresponding author  e-mail: bzevallos@espam.edu.ec. 

Abstract

This paper presents some of the effects of cryopreservation of wild Solanum lycopersicum Mill. seeds on the early stages of germination post liquid nitrogen exposure.  Percentage of germination, conversion into plantlets and plant fresh mass were evaluated after cryostorage. Levels of chlorophyll pigments (a, b, total), malondialdehyde, other aldehydes, phenolics (cell wall-linked, free, and total) and proteins were determined. Peroxidase and superoxide dismutase activities were recorded. Liquid nitrogen exposure increased the percentage of seed germination at 5 days but at 7 days, the conversion into plantlets and the plant fresh mass were not statistically different between non-cryopreserved and cryopreserved samples. Several significant effects of cryopreservation were recorded at the biochemical level at 7 days of germination under controlled conditions. Highly significant effects due to liquid nitrogen exposure were observed in leaves: increased levels of peroxidase enzymatic and specific activities and cell wall-linked phenolics. Very remarkable effects were also recorded in roots: decreased contents of chlorophylls and cell wall-linked phenolics.

Keywords: tomato; cryostorage; biochemical changes; phenotypic variation.

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CryoLetters 34 (4), 422-431 (2013)
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EFFECT OF PASTEURIZED EGG AND ROSMARINUS OFFICINALIS SUPPLEMENTATION ON QUALITY OF CRYOPRESERVED RAM SEMEN

Francisco Mascaró¹, Lydia Gil¹, Clara Malo¹*, Noelia González ¹,
Felisa Martínez¹ and Ignacio de Blas²

Department of Animal Pathology, Obstetrics and Reproduction Area¹ and Epidemiology Unit², Faculty of Veterinary Medicine, Universidad de Zaragoza, C/ Miguel Servet, 177 CP: 50013 Zaragoza (Spain)
Corresponding author email: claramalo@hotmail.com

Abstract

The aim was to assess the in vitro effect of pasteurized egg (PE) and rosemary (Rosmarinus officinalis) on frozen-thawed ram semen. Ejaculates from three mature rams of the Rasa Aragonesa breed were cryopreserved using a 2-step dilution method (Fraction 1: F1; Fraction 2: F2). In Experiment 1, semen was frozen in egg yolk (EY) or PE extenders. After thawing, similar results were obtained in terms of total and progressive motility, viability, hypo-osmotic swelling test (HOST) and acrosome integrity after 2 h incubation. In Experiment 2, addition of rosemary to F1, F2 or both fractions to EY extenders was evaluated. Rosemary in F1 decreased progressive motility (p=0.013) after 2 h incubation. Finally, PE can be used as a substitute for EY to reduce hygienic risks in extenders and is easier to standardize. Supplementation of EY extender with rosemary in F1 reduced progressive motility. Rosemary supplementation in F2 does not affect semen quality.

Keywords: ram semen; cryopreservation; pasteurized egg; rosemary; ROS

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CryoLetters 34 (4), 432-452 (2013)
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BIOPRESERVATION OF HEPATOCYTES: CURRENT CONCEPTS ON HYPOTHERMIC PRESERVATION, CRYOPRESERVATION, AND VITRIFICATION

Barry J. Fuller^1*, Alexander Y. Petrenko², Joaquín V. Rodriguez³,
Alexander Y. Somov², Cecilia L. Balaban³ and Edgardo E. Guibert³.

^1*Cell, Tissue and Organ Preservation Unit, Department of Surgery & Liver Transplant Unit, UCL Medical School, Royal Free Hospital Campus, London, UK.
²Department of Cryobiochemistry, Institute for Problems of Cryobiology and Cryomedicine, Ukraine Academy of Sciences, Kharkov, Ukraine.
³Centro Binacional (Argentina-Italia) de Investigaciones en Criobiología Clínica y Aplicada (CAIC), Universidad Nacional de Rosario, Argentina.
*Corresponding author  e-mail: bfuller@ucl.ac.uk.

Abstract

Isolated liver cells (primarily isolated hepatocytes) have found important applications in science and medicine over the past 40 years in a wide range of areas, including physiological studies, investigations on liver metabolism, organ preservation and drug de-toxification, experimental and clinical transplantation. An integral component of many of these works is the need to store the isolated cells, either for short or long-term periods. This review covers the biopreservation of liver cells, with a focus on the history of liver cell biopreservation, the application of hypothermia for short-term storage, standard cryopreservation methods for isolated hepatocytes, the biopreservation of other types of liver cells, and recent developments such as vitrification of hepatocytes. By understanding the basis for the different approaches, it will be possible to select the best options for liver cell biopreservation in different applications, and identify ways to improve preservation protocols for the future.

Keywords: Isolated hepatocytes; isolated liver cells; biopreservation; hypothermic storage; cryopreservation; vitrification; sub-zero non-freezing storage; drying

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