title: The effect of long-term storage on the biological and histological properties of cryopreserved amniotic membrane.
abstract: OBJECTIVE
  Cryopreserved amniotic membrane (AM) is widely used in ophthalmology because of its anti-angiogenic, anti-inflammatory, and wound healing promoting capabilities. A common method to conserve the tissue is the storage in cryo-medium containing 50% glycerol at -80°C. The aim of this study was to examine the influence of storage time on the sterility as well as the histological and biological properties of cryopreserved AM.
  
  
  METHODS
  Amniotic membrane from different donors was stored in cell culture media containing 50% glycerol for different time periods, on average 4 months (group 1), 15 months (group 2), and 24 months (group 3), at -80°C. Samples of the tissue and cryo-medium were examined for bacterial and fungal contamination. Tissue samples were incubated in 0.5 ml/cm(2) serum-free medium at 37°C. The medium was changed after 1, 2, and 3 days. The proteins released by AM were TCA-precipitated and the presence of the proteins TIMP-1 and IL-1ra was analyzed using Western blotting and semi quantified by means of image analysis. Integrity of the amniotic epithelium and the basement membrane components collagen IV, collagen VII, laminin, laminin 5, and fibronectin were examined by haematoxylin eosin stain and immunohistochemistry in cryosections of AM.
  
  
  RESULTS
  None of the examined samples showed bacterial or fungal contamination. The soluble proteins TIMP-1 and IL-1ra were found in all samples of medium incubated for all time periods. The examined proteins were detectable after one-day incubation but the staining signal diminished significantly in the second and third wash after 48 hr and 72 hr. Differences in the intensity of the Western blot signal between the three particular groups were statistically not significant. The epithelia of all samples were intact. The basement membranes of all samples showed a similar distribution of collagen IV, collagen VII, laminin, laminin 5, and fibronectin.
  
  
  CONCLUSIONS
  Long-term storage of amniotic membrane in cell culture media with 50% glycerol does not significantly impair sterility, histology, or biological properties of AM.
authors.affiliation: Department of Ophthalmology, University of Duisburg-Essen, Essen, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Thomasen
authors.forename: M
authors.initials: M
authors.lastname: Pauklin
authors.forename: B
authors.initials: B
authors.lastname: Noelle
authors.forename: G
authors.initials: G
authors.lastname: Geerling
authors.forename: J
authors.initials: J
authors.lastname: Vetter
authors.forename: P
authors.initials: P
authors.lastname: Steven
authors.forename: K-P
authors.initials: KP
authors.lastname: Steuhl
authors.forename: D
authors.initials: D
authors.lastname: Meller
mesh_terms: D000650:Amnion / Q000166:cytology* / Q000378:metabolism / Q000382:microbiology; D001419:Bacteria / Q000302:isolation & purification; D001684:Biological Dressings; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D015925:Cryopreservation / Q000379:methods*; D003470:Culture Media; D016207:Cytokines / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005658:Fungi / Q000302:isolation & purification; D006801:Humans; D009926:Organ Preservation / Q000379:methods*; D019188:Organ Preservation Solutions; D013997:Time Factors; D014015:Tissue Banks
chemical_list: D015415:Biomarkers; D003470:Culture Media; D016207:Cytokines; D016326:Extracellular Matrix Proteins; D019188:Organ Preservation Solutions