title: What is the best cryopreservation protocol for human testicular tissue banking?
abstract: OBJECTIVE
  Is there a better alternative to the conventional cryopreservation protocols for human testicular tissue banking?
  
  
  CONCLUSIONS
  Uncontrolled slow freezing (USF) using 1.5 M dimethylsulphoxide (DMSO) and 0.15 M sucrose as cryoprotectants appears to be a user-friendly and efficient method for the cryopreservation of human testicular tissue.
  
  
  BACKGROUND
  Currently, time-consuming controlled slow freezing (CSF) protocols that need expensive equipment are commonly used for human testicular tissue banking. USF and vitrification are cryopreservation techniques that were successfully applied in several animal models but need further exploration with human tissue.
  
  
  METHODS
  Fragments (n = 160) of testicular tissue from 14 patients undergoing vasectomy reversal were assigned to a fresh control group or one of the following cryopreservation procedures: CSF using DMSO at a concentration of 0.7 or 1.5 M in the presence (+S) or absence of sucrose (-S), USF using either 0.7 or 1.5 M DMSO combined with sucrose, solid-surface vitrification (SSV) or direct cover vitrification (DCV).
  
  
  METHODS
  Light microscopic evaluations were performed to study apoptosis, germ cell proliferation ability, spermatogonial survival, coherence of the seminiferous epithelium and integrity of the interstitial compartment after cryopreservation. Ultrastructural alterations were studied by scoring cryodamage to four relevant testicular cell types.
  
  
  RESULTS
  The USF 1.5 M DMSO + S protocol proved not solely to prevent cell death and to preserve seminiferous epithelial coherence, interstitial compartment integrity, SG and their potential to divide but also protected the testicular cell ultrastructure. A significant reduction in the number of SG per tubule from 21.4 ± 5.6 in control tissue to 4.9 ± 2.1, 8.2 ± 5.4, 11.6 ± 5.1, 8.8 ± 3.9, 12.6 ± 4.4 and 11.7 ± 5.7 was observed after cryopreservation combined with at least one other form of cryoinjury when using CSF 0.7 M DMSO -S, CSF 0.7 M DMSO + S, CSF 1.5 M DMSO + S, USF 0.7 M DMSO + S, SSV and direct cover vitrification (DCV), respectively (P < 0.001).
  
  
  CONCLUSIONS
  Supplementary research is required to investigate the effect on tissue functionality and to confirm this study's findings using prepubertal tissue.
  
  
  CONCLUSIONS
  An optimal cryopreservation protocol enhances the chances for successful fertility restoration. USF, being an easy and cost-effective alternative to CSF, would be preferable for laboratories in developing countries or whenever tissue is to be procured from a diseased child at a site distant from the banking facility.
authors.affiliation: Biology of Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel, Laarbeeklaan 103, Brussels 1090, Belgium. yoni.baert@vub.ac.be
authors.forename: Y
authors.initials: Y
authors.lastname: Baert
authors.forename: D
authors.initials: D
authors.lastname: Van Saen
authors.forename: P
authors.initials: P
authors.lastname: Haentjens
authors.forename: P
authors.initials: P
authors.lastname: In't Veld
authors.forename: H
authors.initials: H
authors.lastname: Tournaye
authors.forename: E
authors.initials: E
authors.lastname: Goossens
mesh_terms: D017209:Apoptosis; D049109:Cell Proliferation; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D006801:Humans; D008297:Male; D012670:Seminiferous Epithelium / Q000166:cytology / Q000648:ultrastructure; D013737:Testis / Q000166:cytology* / Q000648:ultrastructure
chemical_list: D003451:Cryoprotective Agents
keywords: cryopreservation; electron microscopy; germ cells; immunohistochemistry; male infertility