title: Comparison of Cryotip vs. Cryotop for mouse and human blastomere vitrification.
abstract: OBJECTIVE
  Compare the efficiency of two vitrification methods for single-blastomere cryopreservation with mouse or human embryos.
  
  
  METHODS
  Experimental prospective controlled study.
  
  
  METHODS
  Research center.
  
  
  METHODS
  Human Blastomeres were obtained after biopsy.
  
  
  METHODS
  Mouse blastomeres were obtained after diluting the zona pellucida of embryos with Tyrode acid and manual isolation. Individual human blastomeres were biopsied from embryos following established clinical protocols. The modified open Cryotop and classical closed Cryotip vitrification systems were assayed. After thawing, some mouse blastomeres were fixed and analyzed for apoptotic markers annexin V and caspase 3 with the use of immunofluorescence and confocal microscopy. Ultrastructural morphology was examined using electron microscopy. The human blastomere division rate was assessed 24 hours after thawing.
  
  
  METHODS
  Blastomere survival and division rates after thawing, apoptotic markers, and electron microscopy; adhesion and outgrowth rates of human blastomeres.
  
  
  RESULTS
  After thawing, survival rates in mouse blastomeres using Cryotop vs. Cryotip were 38.46% vs. 85.41%, respectively. As expected, thawed morphologically alive blastomeres were classified negative for annexin V and caspase 3, whereas degenerated blastomeres were positive for both. Further, nuclear chromatin was compacted. Survival rates of human blastomeres vitrified with Cryotop were 22.78% vs. 53.77% with Cryotip. Division capabilities were 16.6% and 47.16%, respectively, in Cryotop and Cryotip.
  
  
  CONCLUSIONS
  The closed system is more efficient in preserving mouse and human blastomeres in terms of acceptable survival and division rates, and it also complies with European Union directives.
authors.affiliation: Valencia Stem Cell Bank, Centro de Investigación Príncipe Felipe, Valencia, Spain.
authors.forename: Diana
authors.initials: D
authors.lastname: Valbuena
authors.forename: Maria Eugenia
authors.initials: ME
authors.lastname: Póo
authors.forename: Cristobal
authors.initials: C
authors.lastname: Aguilar-Gallardo
authors.forename: Sebastian
authors.initials: S
authors.lastname: Martinez
authors.forename: Ana Cristina
authors.initials: AC
authors.lastname: Cobo
authors.forename: Antonio
authors.initials: A
authors.lastname: Pellicer
authors.forename: Carlos
authors.initials: C
authors.lastname: Simón
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D001706:Biopsy; D001757:Blastomeres / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D053148:Caspase 3 / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D015925:Cryopreservation / Q000379:methods*; D046149:Embryo Culture Techniques / Q000379:methods*; D005260:Female; D005780:Gelatin; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains; D008854:Microscopy, Electron; D011446:Prospective Studies; D058989:Vitrification; D015044:Zona Pellucida / Q000502:physiology
chemical_list: D017304:Annexin A5; D005780:Gelatin; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3