The expression of TIMPs in cryo-preserved and freeze-dried amniotic membrane.
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.
Koh
Jae Woong
JW
Department of Ophthalmology, Chosun University College of Medicine, Gwangju, and Seoul National University Hospital Clinical Research Institute, Korea.
Shin
Young Joo
YJ
Oh
Joo Youn
JY
Kim
Mee Kum
MK
Ko
Jung Hwa
JH
Hwang
Jeong Min
JM
Wee
Won Ryang
WR
Lee
Jin Hak
JH
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
D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2