title: Character, distribution and biological implications of ice crystallization in cryopreserved rabbit ovarian tissue revealed by cryo-scanning electron microscopy.
abstract: 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 degrees C/min or 3.0 degrees C/min after nucleating ice at -7 degrees 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 degrees C and the crystals, often >30 microm 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.
authors.affiliation: Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, 1305 York Avenue, New York, NY 10021, USA. rgg2004@med.cornell.edu
authors.forename: Roger G
authors.initials: RG
authors.lastname: Gosden
authors.forename: Hang
authors.initials: H
authors.lastname: Yin
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Bodine
authors.forename: G John
authors.initials: GJ
authors.lastname: Morris
mesh_terms: D000818:Animals; D020285:Cryoelectron Microscopy / Q000379:methods; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D005615:Freezing; D007053:Ice; D008855:Microscopy, Electron, Scanning / Q000379:methods; D009926:Organ Preservation / Q000379:methods*; D010053:Ovary / Q000648:ultrastructure*; D011817:Rabbits
chemical_list: D007053:Ice