Skip Navigation



Hum. Reprod. Advance Access published online on November 21, 2009
Human Reproduction, doi:10.1093/humrep/dep395
This Article
Right arrow Full Text
Right arrow Full Text (PDF )
Right arrow All Versions of this Article:
25/2/470    most recent
dep395v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Gosden, R. G.
Right arrow Articles by Morris, G. J.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gosden, R. G.
Right arrow Articles by Morris, G. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Character, distribution and biological implications of ice crystallization in cryopreserved rabbit ovarian tissue revealed by cryo-scanning electron microscopy

Roger G. Gosden1,3, Hang Yin1, Richard J. Bodine1 and G. John Morris2

1 Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, 1305 York Avenue, New York, NY 10021, USA 2 Asymptote Ltd, St Johns Innovation Centre, Cowley Road, Cambridge CB4 0WS, UK

3 Correspondence address. Tel: +1-212-746-1287; Fax: +1-212-746-2101; E-mail: rgg2004{at}med.cornell.edu

BACKGROUND: Ovarian tissue banking is an emerging strategy for fertility preservation which has led to several viable pregnancies after transplantation. However, the standard method of slow cooling was never rigorously optimized for human tissue nor has the extent and location of ice crystals in tissue been investigated. To address this, we used cryo-scanning electron microscopy (cryo-SEM) to study ice formation in cryopreserved ovarian tissue.

METHODS: Rabbit ovarian tissue slices were equilibrated in 1,2-propanediol-sucrose solution and cooled at either 0.3°C/min or 3.0°C/min after nucleating ice at –7°C, or snap-frozen by plunging in liquid nitrogen. Frozen tissues were fractured, etched and coated with gold or prepared by freeze substitution and sectioning for cryo-SEM.

RESULTS: The size, location and orientation of extracellular ice crystals were revealed as pits and channels that had grown radially between freeze-concentrated cellular materials. They represented 60% of the total volume in slowly cooled samples that were nucleated at –7°C and the crystals, often >30 µm in length, displaced cells without piercing them. Samples cooled more rapidly were much less dehydrated, accounting for the presence of small ice crystals inside cells and possibly in organelles.

CONCLUSIONS: Cryo-SEM revealed the internal structure of ovarian tissue in the frozen state was dominated by elongated ice crystals between islands of freeze-concentrated cellular matrix. Despite the grossly distorted anatomy, the greater degree of dehydration and absence of intracellular ice confirmed the superiority of a very slow rate of cooling for optimal cell viability. These ultrastructural methods will be useful for validating and improving new protocols for tissue cryopreservation.

Key words: cryopreservation/electron microscopy/freezing/ovary/rabbit

Submitted on July 23, 2009; resubmitted on October 13, 2009; accepted on October 15, 2009.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.