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Abstracts: CryoLetters 30 (6), 2009

 

 

Volume 30, No. 6 November/December 2009

ISSN 0143-2044

 

 


MORPHO-HISTOLOGICAL STUDY OF BANANA (MUSA SPP. CV. GRANDE NAINE [AAA]) CELL SUSPENSIONS DURING CRYOPRESERVATION AND REGENERATION
Frédéric Georget, Florent Engelmann, Régis Domergue and François Côte

398-407

 

 


ADDITION OF FICOLL AND DISACCHARIDES TO VITRIFICATION SOLUTIONS IMPROVE IN VITRO VIABILITY OF VITRIFIED EQUINE EMBRYO
Lagares M.A., Castanheira P.N., Amaral D.C. G., Vasconcelos A. B., Veado, J.C.C., Arantes R. M. E. and Stahlberg R.

408-413

 

 


CAN THERMAL EXPANSION DIFFERENCES BETWEEN CRYOPRESERVED TISSUE AND CRYOPROTECTIVE AGENTS ALONE CAUSE CRACKING?
Paul S. Steif, Daniel A. Noday, and Yoed Rabin

414-421

 

 


CAN VITRIFIED ZEBRAFISH BLASTOMERES BE USED TO OBTAIN GERM-LINE CHIMAERAS?
Cardona-Costa J, Francisco-Simão M and García-Ximénez F

422-428

 

 


CRYOPRESERVATION STRATEGIES FOR Cyathea australis (R. BR.) DOMIN.
Anna Mikuła, Kamila Jata, Jan J. Rybczyński

429-439

 

 


CRYOPRESERVATION OF EMBRYOGENIC CULTURES OF DIOSCOREA BULBIFERA L. BY ENCAPSULATION- DEHYDRATION
B.B. Mandal, Sonali Dixit-Sharma and P.S. Srivastava

440-448

 

 


CRYOBANKING OF HUMAN OVARIAN TISSUE FOR ANTI-CANCER TREATMENT: COMPARISON OF VITRIFICATION AND CONVENTIONAL FREEZING
Vladimir Isachenko, Evgenia Isachenko, Juergen M. Weiss, Plamen Todorov, Rolf Kreienberg

449-454

 

 


EFFECT OF THE SIZE OF ZONA PELLUCIDA THINNING BY LASER ASSISTED HATCHING ON CLINICAL OUTCOME OF HUMAN FROZEN-THAWED EMBRYO TRANSFERS
Xiao-Jian Zhang, Ye-Zhou Yang, Qun Lv, Li-Hua Min, Xiao-Jie Li and Pin Bai

455-461

 

 


OVERCOMING RECALCITRANCE IN PORPHYRIDIUM AERUGINEUM GEITLER EMPLOYING ENCAPSULATION-DEHYDRATION CRYOPRESERVATION METHODS
R. Amaral, M.F. Santos and L.M.A. Santos

462-472

 

 

 

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CryoLetters 30 (6), 398-407 (2009)
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MORPHO-HISTOLOGICAL STUDY OF BANANA (MUSA SPP. CV. GRANDE NAINE [AAA]) CELL SUSPENSIONS DURING CRYOPRESERVATION AND REGENERATION

Frédéric Georget1*, Florent Engelmann2, 3, Régis Domergue1 and François Côte1

1CIRAD, - TA B-26 / PS4 - 34398 Montpellier cedex 5, France
2IRD, 911 avenue Agropolis, BP 64501, 34394 Montpellier cedex 5, France
3Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.
*Corresponding author email:
frederic.georget@cirad.fr

Abstract

In this work, a morpho-histological study of banana (Musa spp. cv. Grande Naine [AAA]) embryogenic cell suspensions during cryopreservation and regeneration was performed. It was demonstrated that the regeneration process of somatic embryos originating from cryopreserved cell suspensions was different from that of control cell suspensions. Somatic embryos originating from cryopreserved cell suspensions had a unicellular origin. The regeneration process was modified not only by freezing in liquid nitrogen but also by the plasmolyzing effect of the 0.5 M sucrose solution employed during pretreatment. This result explained the high number of embryonic structures formed on M3 medium, compared with the control. Proembryos blocked at the globular stage could pursue their development when they were plated on new culture medium at a lower density after 30 days of culture on M3 medium. The unicellular origin of somatic embryos produced from cryopreserved cell suspensions offers the prospect of using cryopreservation to select non-chimeral transformed plants.

Keywords: banana; embryogenic cell suspension; cryopreservation; histology; cellular selection.

 

 

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CryoLetters 30 (6), 408-413 (2009)
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ADDITION OF FICOLL AND DISACCHARIDES TO VITRIFICATION SOLUTIONS IMPROVE IN VITRO VIABILITY OF VITRIFIED EQUINE EMBRYO

Lagares M.A.*1, Castanheira P.N.1, Amaral D.C. G.1,
Vasconcelos A. B.1, Veado, J.C.C.1, Arantes R. M. E.2
 and Stahlberg R.3

1Department of Veterinary Clinic and Surgery of the Veterinary School of the Federal University of Minas Gerais,
2Department of Pathology of the Biological Science Institute of the University of Minas Gerais, Belo Horizonte, Av. Antonio Carlos 6627 CEP: 31270-901
3Veterinary School of the Pontifícia Catholic University, Betim, Minas Gerais, Brazil
Department of Veterinary Clinic and Surgery of the Veterinary School of the University of Minas Gerais, Av. Antonio Carlos 6627, CEP:31270-901, Belo Horizonte, MG, Brazil.
*Corresponding author  e-mail:
mlagares@vet.ufmg.br

Abstract

The aim of the present study was to evaluate the in vitro viability of equine embryos vitrified in three different solutions. Day 6 and 6.5 embryos were measured and morphologically evaluated. Only grade 1 or 2 morulae and early blastocysts were vitrified. Eighteen embryos were distributed in Group 1: 40% ethylene glycol in PBS, Group: 2 and 3: 40% ethylene glycol, 18% ficoll, 0.3M sucrose or 0.3M trehalose in PBS, respectively. The vitrified embryos were loaded individually into 0.25 ml straws, which were cooled and immersed in liquid nitrogen. After warming at 20°C for 20s, the embryos were expelled out into 0.5M sucrose in PBS and transferred to PBS solution. The embryonic diameter was measured again and morphology and viability were evaluated with Propidium iodide and Hoechst 33258 dyes. Embryos vitrified with sucrose (19.2%) and trehalose (26.7%) showed the highest percentage of viable cells and morphological quality.

Keywords: embryo, vitrification, trehalose, equine, sucrose, viability

 

 

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CryoLetters 30 (6), 414-421 (2009)
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CAN THERMAL EXPANSION DIFFERENCES BETWEEN CRYOPRESERVED TISSUE AND CRYOPROTECTIVE AGENTS ALONE CAUSE CRACKING?

Paul S. Steif, Daniel A. Noday, and Yoed Rabin*

Biothermal Technology Laboratory, Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213

Abstract

One of the limiting factors in large-scale cryopreservation is the formation of fractures. The prevalence of cracking in cryopreserved bulky tissues is frequently associated with temperature gradients, which lead to non-uniform thermal contraction of the tissue. With new cryoprotectants available, it may be possible to reduce temperature gradients to much lower levels, in which case other contributions to mechanical stress development and cracking will become more significant. One potential contributor to such stress is the difference in thermal expansion between tissue and the cryoprotectant. The current study addresses the role of thermal expansion mismatch by drawing upon recently obtained experimental data and engineering models for the development of thermal stresses. This question is addressed for the case of cryopreservation via vitrification (glass formation), for which crystal formation is avoided, and tissues and solutions gradually transition from fluid-like to solid-like response, as the viscosity increases with decreasing temperature.

Keywords: Cracking, Cryopreservation, Vitrification, Solid Mechanics, Modeling

 

 

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CryoLetters 30 (6), 422-428 (2009)
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CAN VITRIFIED ZEBRAFISH BLASTOMERES BE USED TO OBTAIN GERM-LINE CHIMAERAS?

Cardona-Costa J*, Francisco-Simão M and García-Ximénez F

Laboratory of Animal Reproduction and Biotechnology (LARB-UPV), Polytechnic University of Valencia, Camino de Vera 14, 46022 Valencia, Spain.
*Corresponding author email:
cardona_costa_j@hotmail.com

Abstract

Blastomere cryopreservation plays an important role in maintaining the genetic diversity for valuable fish species. Recently, an original procedure for blastomere vitrification in zebrafish (Danio rerio) was developed in our lab. In the present work, blastomeres from the wild strain embryos, previously vitrified-thawed by this procedure, were injected into embryos from the gold strain in order to assess their ability to colonise the germ-line of recipient embryos.

The blastomere survival rate at thawing (higher than 90%) as well as the whole number of recovered blastomeres per donor embryo (around 20%), were in the ranges previously reported for this vitrification technique.  Despite this, only 2 adult chimaeric specimens were finally obtained from a total of 47 injected embryos. Signals of chimaerism were not detected at any stage of development of the chimaeric embryos (somatic chimaerism) or in adulthood (somatic and germ-line chimaerism). In relation to this, difficulties during blastomere insertion are thought to be responsible for the poor results obtained, their aspects being discussed in detail in this work. More improvements to overcome such technical difficulties are needed and, until then, blastomere vitrification may only be of interest for germplasm cryobanking.

Keywords: blastomere, vitrification, germ-line chimaerism, reproductive techniques, zebrafish.

 

 

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CryoLetters 30 (6), 429-439 (2009)
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CRYOPRESERVATION STRATEGIES FOR Cyathea australis (R. BR.) DOMIN.

Anna Mikuła*, Kamila Jata, Jan J. Rybczyński

Botanical Garden – Center for Biological Diversity Conservation, Polish Academy of Sciences, ul. Prawdziwka 2, 02-973 Warsaw, Poland
*Corresponding author  e-mail:
amikula@obpan.pl

Abstract

The influence of liquid nitrogen (LN) on the germination of C. australis spores and survival of gametophytes at various stages of development was investigated. Exposure to LN did not change the viability of mature spores (80%) but stimulated the germination of immature spores from 1.9% to 41%. Disinfection before cryopreservation contributed to loss of spore survival. However, some germination capacity was regained (48%) if the sterilized spores were enclosed in alginate capsules and subsequently exposed to osmotic desiccation and 5-hour air drying. Development of gametophytes derived from frozen and non-frozen spores was similar. Preculture factors (the period, type and abscisic acid treatment) affected gametophyte viability and growth. A two week preculture on agar significantly increased survival compared to preculture in a liquid medium. Addition of abscisic acid (ABA) to solid or liquid media stimulated explant survival. Highest viability (85%) of frozen-thawed gametophytes was achieved by a 2-week preculture in agar with 0.25 M sucrose and 10 μM ABA. Gametophytes developed directly from spores grew and multiplied in vitro at a uniform rate. Young, intensively growing gametophytes and large, proliferating ones survived better (73-80%) following cryoexposure than mature, non proliferating gametophytes (50%). Less than one quarter of the explant surface was alive in 60-80% of the gametophytes that survived cryoexposure.

Keywords: ABA, encapsulation, flow cytometry, gametophyte, rough tree fern, spore

 

 

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CryoLetters 30 (6), 440-448 (2009)
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CRYOPRESERVATION OF EMBRYOGENIC CULTURES OF DIOSCOREA BULBIFERA L. BY ENCAPSULATION- DEHYDRATION

B.B. Mandal1,3*, Sonali Dixit-Sharma1,4 and P.S. Srivastava2

1Tissue Culture and Cryopreservation Unit, NBPGR, Pusa, New Delhi-110012, India
2Department of Biotechnology, Jamia Hamdard, Hamdard Nagar, New Delhi-62, India
3A-2, Parijat Apartments, Sector-4, Plot No. 28, Dwarka, New Delhi-110078, India.
4Avestha Gengraine Technologies Pvt. Ltd., Whitefield Road, Bangalore 560066, India.
*Corresponding author e-mail:
mandalbinay@yahoo.co.in

Abstract

Embryogenic cultures of Dioscorea bulbifera were cryopreserved using an encapsulation-dehydration procedure with subsequent plant regeneration. Embryogenesis was induced by culturing in vitro grown axillary bud meristems on MS medium supplemented with 2.0 mg l-1 2,4-D. After cryopreservation, recovery growth of embryogenic culture up to 53.3% was recorded when excised proliferating embryogenic cultures of 1.5-2.0 mm in diameter were: encapsulated in 3% calcium alginate containing 0.15 M sucrose followed by preculturing with 0.5 M sucrose for 3 d; dehydrated in the laminar air flow for 4 h, thereby reducing the bead moisture content to 19.4% ( fresh weight basis); plunged into liquid nitrogen; thawed at 40oC; and cultured on recovery growth medium, i.e. MS supplemented with 2.0 mg l-1 2,4-D and 0.3 mg l-1 BAP. However, preculturing for an extended period of 7 d increased the recovery growth further to 67.8%. During recovery growth the embryogenic tissue protruded out of the beads without loss of structural integrity of the cryopreserved embryos. Subculturing of these cultures on to embryo conversion medium, i.e. MS medium with 0.5 mg l-1 zeatin and 400 mg l-1 glutamine, resulted in production of plantlets through embryo conversion. The regenerated plantlets exhibited the same morphology as that of originally maintained in vitro plantlets and were established in vivo, in a net house with 80% success.

Keywords: Dioscorea bulbifera, cryopreservation, embryogenic tissue, liquid nitrogen, encapsulation- dehydration.

 

 

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CryoLetters 30 (6), 449-454 (2009)
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CRYOBANKING OF HUMAN OVARIAN TISSUE FOR ANTI-CANCER TREATMENT: COMPARISON OF VITRIFICATION AND CONVENTIONAL FREEZING

Vladimir Isachenko1*, Evgenia Isachenko1,2, Juergen  M. Weiss1, Plamen Todorov2, Rolf Kreienberg1

1Department of Obstetrics and Gynaecology, University of Ulm, Prittwitzstrasse 43, 89075 Ulm, Germany
2Institute of Biology and Immunology of Reproduction, Tzarigradsko Shose 73, 1113 Sofia, Bulgaria
*Corresponding author: e-mail:
v.isachenko@yahoo.com

Abstract

The first case of cryopreservation of human ovarian tissue with good survival of follicles after warming was described in 1996. Childbirth after cryopreservation of ovarian tissue is now a reality. Cryopreservation of ovarian tissue can be performed using one of two methods: conventional ("slow") freezing and cryopreservation by direct plunging into liquid nitrogen (so called vitrification or "rapid" freezing). Comparative investigations of vitrification and conventional freezing performed on mammalian ovarian tissue are limited, and authors present different conclusions. The higher effectiveness of vitrification in comparison with conventional freezing for human oocytes and embryos was shown, whereas data on human ovarian tissue are limited. The aim of different studies was to compare the safety and effectiveness of conventional freezing and vitrification of human ovarian tissue. Below we shortly summarize the results of some investigations with different conclusions. The discussion on the post-warming quality of follicles as well as on the problems of microbial contamination of cells in liquid nitrogen at vitrification is presented. In our opinion, for cryopreservation of human ovarian tissue, conventional freezing is more promising than vitrification.

Keywords: human ovary, aseptic freezing, vitrification, follicles

 

 

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CryoLetters 30 (6), 455-461 (2009)
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EFFECT OF THE SIZE OF ZONA PELLUCIDA THINNING BY LASER ASSISTED HATCHING ON CLINICAL OUTCOME OF HUMAN FROZEN-THAWED EMBRYO TRANSFERS

Xiao-Jian Zhang, Ye-Zhou Yang*, Qun Lv, Li-Hua Min, Xiao-Jie Li and Pin Bai

Department of Assisted Reproductive Medical Center, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, 32 Yi Huan Lu Xi Er Duan, Chengdu, 610072, Sichuan, People's Republic of China.
* Corresponding author e-mail: yezhou.y@gmail.com

Abstract

Of 122 consecutive procedures, 31 were not assisted hatching (AH), which served as the control group, 34 were AH with 40 µm thinning of the zona, and 57 were AH with 80 µm of the zona thinning. The pregnancy and implantation rates were significantly higher in 80µm thinning group compared to control group (40.35 vs. 16.13%, P=0.03; 21.54 vs. 7.5%, P=0.007, respectively). In addition, the implantation rate was signigicantly higher in 80 µm group than in 40 μm group (21.54%vs.9.41%, P=0.024). The results indicated that the size of the zona pellucida thinning by laser may influence the pregnancy and implantation rates following frozen-thawed cleaved embryo transfer.

Keywords: frozen-thawed embryo transfers, laser-assisted hatching

 

 

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CryoLetters 30 (6), 462-472 (2009)
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OVERCOMING RECALCITRANCE IN PORPHYRIDIUM AERUGINEUM GEITLER EMPLOYING ENCAPSULATION-DEHYDRATION CRYOPRESERVATION METHODS

R. Amaral, M.F. Santos and L.M.A. Santos*

Department of Botany, University of Coimbra, 3001-455 Portugal.
*Corresponding author email:
liliamas@ci.uc.pt.

Abstract

Cultures of the recalcitrant microalga Porphyridium aerugineum were cryopreserved. A two-step, uncontrolled rapid freezing protocol, using methanol as cryoprotectant resulted in 23.8% viable cells. Cultures in the exponential growth phase, grown under low light intensity to prevent vacuole formation in cells, cryopreserved using a passive freezer, showed 22.4% viability. This value was enhanced to 31.5% when a controlled-rate freezer was employed. Optimized cultures in the exponential growth phase, cultivated in medium supplemented or not with vitamin B12, were then tested for freezing using the encapsulation-dehydration protocol. High cell loss was observed early during the sorbitol dehydration steps, but 63.6% of the remaining encapsulated cells were viable after thawing. This study confirmed the potential of encapsulation-dehydration as a method allowing to improve the low viability obtained with two-step freezing protocols. It also showed the importance of monitoring the response of algal cells to bead osmotic and evaporative dehydration pretreatments before freezing.

Keywords: Microalgae, cryopreservation, Porphyridium aerugineum, ACOI, encapsulation-dehydration

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