title: The expression of TIMPs in cryo-preserved and freeze-dried amniotic membrane.
abstract: OBJECTIVE
  To investigate the change of tissue inhibitor of metalloproteinase (TIMP) in cryopreserved amniotic membranes (AM) according to preservation time, and to evaluate the expression of TIMP in freeze-dried AM.
  
  
  METHODS
  Cryopreserved or fresh AMs were incubated in dispase II for two hours at 37 degrees C and their epithelial cells were scraped with a cell scraper. Remaining stromal AM was minced and frozen in liquid nitrogen, and then treated with 0.1% diethyl pyrocarbonate. The mRNA levels of TIMP-1 and -2 were determined by reverse transcription-polymerase chain reaction (RT-PCR) in epithelial and stromal cells of fresh AM, AMs cryopreserved for 6 and 12 months, and freeze dried AM, respectively. Western blot analysis and immunohistochemical staining were performed to assess the expression of TIMP-1 in fresh, cryopreserved, and freeze dried AMs.
  
  
  RESULTS
  RT-PCR revealed that mRNAs of TIMP-1 and -2 were expressed in the amniotic epithelial cells of both fresh and cryopreserved AMs, while the stromal cells of fresh or cryopreseved AMs and freeze-dried AM showed higher expression of TIMP-1 than TIMP-2 mRNA. On Western blot analysis, the level of TIMP-1 was more in fresh AMs than in cryopreserved or freeze-dried AM, but it was not statistically significant.
  
  
  CONCLUSIONS
  TIMP-1 was expressed in cryopreserved AMs until 12 months, and the amount of expression was comparable to that in fresh AMs.
authors.affiliation: Department of Ophthalmology, Chosun University College of Medicine, Gwangju, and Seoul National University Hospital Clinical Research Institute, Korea.
authors.forename: Jae Woong
authors.initials: JW
authors.lastname: Koh
authors.forename: Young Joo
authors.initials: YJ
authors.lastname: Shin
authors.forename: Joo Youn
authors.initials: JY
authors.lastname: Oh
authors.forename: Mee Kum
authors.initials: MK
authors.lastname: Kim
authors.forename: Jung Hwa
authors.initials: JH
authors.lastname: Ko
authors.forename: Jeong Min
authors.initials: JM
authors.lastname: Hwang
authors.forename: Won Ryang
authors.initials: WR
authors.lastname: Wee
authors.forename: Jin Hak
authors.initials: JH
authors.lastname: Lee
mesh_terms: D000650:Amnion / Q000378:metabolism*; D015153:Blotting, Western; D015925:Cryopreservation; D004847:Epithelial Cells / Q000378:metabolism; D005612:Freeze Drying; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D007124:Immunoenzyme Techniques; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000378:metabolism; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics* / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics* / Q000378:metabolism; D014021:Tissue Preservation
chemical_list: D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2