The expression of TIMPs in cryo-preserved and freeze-dried amniotic membrane.

PURPOSE

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.

CONCLUSION

TIMP-1 was expressed in cryopreserved AMs until 12 months, and the amount of expression was comparable to that in fresh AMs.