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Abstracts: CryoLetters 28 (6), 2007

CryoLetters 28 (6), 403-408 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

INFLUENCE OF EXPOSURE TO VITRIFICATION SOLUTIONS ON 2-CELL MOUSE EMBRYOS:
I. INTRACELLULAR POTASSIUM AND SODIUM CONTENT§

Alexander G. Pogorelov1,2, Igor I. Katkov3, and Valentina N. Pogorelova1

1Institute of Theoretical and Experimental Biophysics, RAS, 142290 Puschino, Moscow Region, Russia.
2State University at Pushchino, Department of Biophysics and Biomedicine, 142290 Puschino, Moscow Region, Russia.
3UCSD Department of Pediatrics and The Burnham Institute for Medical Research, San Diego, California 92037, USA.
*E-mail:
pogorelov@iteb.ru

§Results of this work were in part presented at the 43rd Annual Meeting of the Society for Cryobiology, Hamburg, Germany, July 24-27, 2006

Abstract

Intracellular concentrations of potassium and sodium in two-cell mouse embryos in G1/S phase after exposure to vitrification solutions containing vitrificant agents (VFAs) ethane-1,2-diol (ethylene glycol, EG), propane-1,2-diol (propylene glycol, PG), or dimethyl sulfoxide (DMSO), and sucrose (S) after incubation in Dulbecco`s solution were measured by electron probe microanalysis (EPMA) as described earlier (CryoLetters, 2006, 27: 87-98). The 4-step protocol was as followed: 10%-VFA for 10min => 30%-VFA + 0.7 M S for 1.5 min ==> 0.5 M S for 10 min ==> 10 min pure Dulbecco's. The cytoplasmic concentration of potassium and sodium in immediately flashed out from the oviduct embryos was in range of 120 + 2 mM, with good concordance with the previous data (CryoLetters, 2006, 27:87-98). Exposure in Dulbecco's for 30 min did not alter elemental composition, neither did exposure in PG or DMSO for 1.5 min. In contrast, exposure for 1.5 min in EG dropped the level of potassium to 96 ± 2 mM while elevating level of cytoplasmic sodium to 136 ± 3 mM. Further exposure to 30%-EG for 3 min led to a two-fold decrease of both elements (60 ± 3 mM and 66 ± 2 mM for K and Na respectively).

Keywords: intracellular potassium and sodium; early mouse embryo; ethylene glycol; propylene glycol; dimethyl sulfoxide; electron probe microanalysis (EPMA).

 

 

CryoLetters 28 (6), 409-427 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

INFLUENCE OF EXPOSURE TO VITRIFICATION SOLUTIONS ON 2-CELL MOUSE EMBRYOS:
II. OSMOTIC EFFECTS OR CHEMICAL TOXICITY?§

Igor I. Katkov1* and Alexander G. Pogorelov2,

1UCSD Department of Pediatrics and The Burnham Institute for Medical Research, San Diego, California 92037, USA.
2Institute of Theoretical and Experimental Biophysics and State University at Pushchino, Department of Biophysics and Biomedicine, 142290 Puschino, Moscow Region, Russia.
*E-mail:
prodvincell@hotmail.com

§Results of this work were in part presented at the 43rd Annual Meeting of the Society for Cryobiology, Hamburg, Germany, July 24-27, 2006

Abstract

In the companion paper (CryoLetters, 2007, 28:403-408), we reported effects of exposure of two-cell mouse embryos to vitrification solutions containing different vitrificants (EG, PG and DMSO) on the intracellular potassium and sodium content. We also compared exposure of 30% v/v ethylene glycol for 1.5 min to the similar experiments with 3-min exposure reported previously (CryoLetters, 2006, 27:87-98). In all experiments, four step protocols (2 steps of addition and 2 steps of washing) were used. Here we present mathematical modeling of the cell osmotic response using the relativistic permeability (RP) approach, which allows calculation of the osmotic curves without using simulation software but by direct calculations of the cell volume, intracellular concentration, and amount of the permeable vitrificants (Cryobiology, 2006, 53:402-3). Magnitude of the maximum cell volume excursion and other important osmotic characteristics were calculated for each step of the protocol, and the results of the mathematical modeling were superimposed onto the experimental data reported and discussed in the companion paper. The osmotic damage vs. specific chemical toxicity of the vitrificants as the major cause of the elemental disturbance of intracellular potassium and sodium content are discussed.

Keywords: mouse embryo; vitrification; ethylene glycol; propylene glycol; dimethyl sulfoxide; osmotic modeling; chemical toxicity.

 

 

CryoLetters 28 (6), 429-444 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

EFFECT OF INTRA/EXTRALIPOSOMAL DISTRIBUTION OF SODIUM CHLORIDE ON THE STABILITY OF LARGE UNILAMELLAR VESICLES

Lee Fong Siowa, Thomas Radesb and Miang Hoong Lim a*

aDepartment of Food Science,
bSchool of Pharmacy, University of Otago, P. O. Box 56, Dunedin 9054, New Zealand.
*Corresponding author –
miang.lim@stonebow.otago.ac.nz

                                                                                                                        

Abstract

Three groups of 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC) large unilamellar vesicle (LUV) dispersions were studied: LUV (A) dispersions with only extraliposomal sodium chloride (NaCl), LUV (B) dispersions with intra- and extraliposomal NaCl, and LUV (C) dispersions with only intraliposomal NaCl. The NaCl concentrations ranged from 0 to 150 mM. An abrupt increase in leakage was observed around –10C for all the three groups of LUV, which coincided with the temperature of extraliposomal ice formation. Within the three groups, leakage of LUV (C) was significantly higher than the other groups. Extraliposomal ice formation and the resulting freeze-concentration of LUV may be the major cause of the leakage. Intraliposomal ice formation observed at –43C seemed to stop leakage of LUV when LUV were frozen below –43C. An exotherm of eutectic crystallization of NaCl was occasionally observed at –37C, with a higher probability of formation at 150 mM extraliposomal NaCl than at 50 mM. The eutectic crystals were thought to cause additional leakage from the LUV (B).

Keywords: large unilamellar vesicles, eutectic crystallization, intraliposomal freezing, sodium chloride, extraliposomal, liposomes

 

 

CryoLetters 28 (6), 445-460 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

 

OPTIMIZATION OF CRYOPRESERVATION OF STEM CELLS CULTURED AS NEUROSPHERES: COMPARISON BETWEEN VITRIFICATION, SLOW-COOLING AND RAPID COOLING "FREEZING" PROTOCOLS

Francis Chee Kuan Tan,1,2 Kong Heng Lee,3 Sok Siam Gouk,3 Raquel Magalhăes,3 Anuradha Poonepalli,4 Manoor Prakash Hande,4 Gavin S. Dawe1,2,* and Lilia L. Kuleshova3,*

1Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
2Neurobiology and Ageing, Centre for Life Sciences, National University of Singapore, Singapore. 
3Low Temperature Preservation Unit, National University Medical Institutes, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. 4Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
*Correspondence: e-mail:
nmikl@nus.edu.sg and gavindawe@nus.edu.sg

Abstract

We compared cryopreservation of mammalian neural stem cells (NSCs) cultured as neurospheres by slow-cooling (1 °C/min) in 10% (v/v) DMSO and cryopreservation by immersion into liquid nitrogen in ethylene glycol (EG)-sucrose solutions that support vitrification (40% (v/v) EG, 0.6 M sucrose) or that do not (37% (v/v) EG, 0.6 M sucrose and 30% (v/v) EG, 0.6 M sucrose); the concentration of penetrating cryoprotectant in the last two solutions was lowered with the intention to reduce their toxicity towards NSCs. To protect against contamination a "straw-in-straw" technique was employed. Vitrification offered the best combination of preservation of structural integrity of neurospheres, cell viability (>96%), multipotency and karyotype. Rapid cooling in 37% (v/v) EG, 0.6 M sucrose afforded good viability but did not preserve structural integrity. Rapid cooling in 30% (v/v) EG, 0.6 M sucrose additionally reduced cell viability to 77%. Slow-cooling reduced cell viability to 65% and damaged the neurospheres. This study suggests that, in contrast to "freezing", vitrification has immense potential for the cryopreservation of stem cells cultured as neurospheres or in other structured cultures.

Keywords: stem cells, vitrification, "freezing", cryopreservation, neurospheres

 

 

CryoLetters 28 (6), 461-470 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

 

CRYOPRESERVATION OF IN VITRO SHOOT TIPS OF DIOSCOREA DELTOIDEA WALL., AN ENDANGERED MEDICINAL PLANT: EFFECT OF CRYOGENIC PROCEDURE AND STORAGE DURATION

B.B. Mandal* and Sonali Dixit-Sharma1

Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi-110012, India.
1Avestha Gengraine Technologies Pvt. Ltd., Whitefield Road, Bangalore 560066, India (current address).
*e-mail:
mandalbinay@yahoo.co.in

Abstract

   In vitro shoot tips of Dioscorea deltoidea Wall., an endangered medicinal plant, were successfully cryopreserved using the vitrification and the encapsulation-dehydration techniques with subsequent high frequency plant regeneration. Using vitrification, post-liquid nitrogen (LN) shoot regeneration up to 83% was recorded when excised shoot tips were pretreated overnight on MS medium containing 0.3 M sucrose followed by loading with MS containing 2 M glycerol plus 0.4 M sucrose for 20 min at 25°C, dehydration with PVS2 for 90 min at 0°C and quenching in LN. After 1 h of storage in LN, the shoot tips were rewarmed in a water-bath at 40°C, unloaded with 1.2 M sucrose solution for 20 min and cultured on recovery growth medium. While using encapsulation-dehydration, the highest regeneration frequency recorded was 76% when sucrose-pretreated shoot tips were encapsulated with 3% calcium alginate, precultured in 0.75 M sucrose for 3 days, dehydrated to 25% moisture content (FW basis) under the laminar air flow, stored in LN for 1h and rewarmed at 40oC. The cryopreserved shoot tips maintained their viability and an unaltered level of regeneration capability after up to one year of storage in LN.

Keywords: Dioscorea deltoidea, cryopreservation, encapsulation-dehydration, vitrification.

 

 

CryoLetters 28 (6), 471-482 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

 

CRYOPRESERVATION OF GARLIC GERMPLASM COLLECTIONS USING THE DROPLET-VITRIFICATION TECHNIQUE

Haeng-Hoon Kim1*, Joung-Kwan Lee2, Hae-Sung Hwang1 and Florent Engelmann3, 4

1National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Korea. *Correspondence: hkim@rda.go.kr
2Danyang Garlic Experiment Station, Danyang 441-744, Korea.
3Institut de recherche pour le développement (IRD), UMR DIA-PC, BP 64501, 34394 Montpellier cedex 5, France.
4Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.

Abstract

The droplet-vitrification protocol was applied to unripe inflorescences of plants of two Korean garlic collections, Danyang and Mokpo, to establish a cryopreserved germplasm collection. Garlic unripe inflorescences of the 59 accessions harvested at Danyang showed a mean survival of 83.3% and regeneration of 73.5% after cryopreservation. Unripe inflorescences of accessions cryopreserved at sub-optimal developmental stages displayed lower survival and/or regeneration. Of these 59 accessions, 53 were cryopreserved and stored for long-term conservation. In the Mokpo collection, unripe inflorescences of 149 accessions were cryopreserved, displaying a mean survival of 79.9% and regeneration of 78.2%. Of these 149 accessions, 116 were cryopreserved and stored for the long-term. A total of 252 accessions of five clonal Allium species, including garlic, were cryopreserved using unripe inflorescences, cloves or bulbils, with a mean survival of 80.9% survival and regeneration of 77.0%, from which 221 accessions were stored in liquid nitrogen for long-term conservation. The real-time quantitative, reverse transcription (RT)-PCR assay of several garlic viruses showed that virus concentration was much lower in plantlets originating from cryopreserved material, compared to plantlets originating from preculture control and dehydration control samples. These results demonstrate that large-scale implementation of cryopreservation of Allium germplasm is feasible and that it can result in the regeneration of virus-free or little infected material. These findings will strongly facilitate the conservation and international exchange of Allium germplasm.

Keywords: Allium sativum L., droplet-vitrification, bulbil primordial, PVS3.

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