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Shin Hatou, Satoru Yoshida, Kazunari Higa, Hideyuki Miyashita, Kazuo Tsubota, Shigeto Shimmura; Tissue Engineered Corneal Endothelium Derived From Cornea-derived Precursor (COPs). Invest. Ophthalmol. Vis. Sci. 2011;52(14):6596.
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Corneal endothelial dysfunction remains common indication for keratoplasty, accounting for half of the total number of such procedures. Corneal endothelium as well as corneal stroma is originated from neural crest. We previously reported about isolation of Cornea-derived Precursor (COPs) which has characteristic of multipotent neural crest-derived stem cells from corneal storoma. In this presentation, we report tissue engineered corneal endothelium (TECE) derived from COPs.
We cultured COPs from mouse corneal stroma in the specific endothelium-deriving medium with 1% fetal bovine serum for one week. RT-PCR was performed to detect markers characterizing corneal endothelial function (Na,K-ATPase α1-subunit, carbonic anhydrase, Na,HCO3 co-transporter, collagen IV, collagen VIII). The pump function attributable to Na,K-ATPase activity of confluent monolayers of these cells on Snapwell inserts was measured with an Ussing chamber. The pump function was calculated as the difference in short-circuit current measured before and after the addition of ouabain. The pump function of these cells was compared with that of primary cultured mouse corneal endothelial cells (mCE) and 3T3 cells.
After one week culture, hexagonal mosaic pattern monolayer cells were obtained from COPs. RT-PCR revealed the expression of all of the above markers. Na,K-ATPase pump activity of these cells was 32.9±6.3µA/cm2, whereas mCE was 29.3±9.5µA/cm2 and 3T3 was 9.7±2.8µA/cm2.
We successfully derived TECE from mouse corneal stromal cells (COPs), which has equal pump function with mCE. If this protocol could be applied to human cornea, corneal endothelial grafts may be mass-produced efficiently from one donor cornea.
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