Abstract
Purpose: :
To develop techniques to culture the corneal endothelial cells and to identify the cultured human corneal endothelial cells maintain the property of stem cells by ex vivo expansion on amniotic membrane
Methods: :
Human donor corneas (from US Eye Bank) were selected for studies. Human corneal endothelium was obtained from the inner part of human limbal corneal rim. Human corneal endothelium was cut into a small pieces and cultured on the different kind of substrate including amniotic membrane. Series passages were done. To further understand the possibility of human corneal endothelium contains stem cells, we set up organ culture technique for human donor cornea. Human donor cornea was cultured in culture medium with BrdU for labeling 2 weeks then chashing for another 2 weeks. BrdU labeling retention cells will be studied under immunomicroscopy. To explore the human corneal endothelial stem cells can be preserved and cultured on amniotic membrane, cultured endothelial cells was studied with markers of Na/K ATPase, P63, and ABCG2. Cultured human corneal endothelial cells finally were growth on the human corneal disc.
Results: :
Age of human donor corneas was from 37 y/o to 70 y/o with mean 63+11 y/o. The death to cultured period was from 6 days to 10 days with mean 8.4 +1.8 days. The cultured human corneal endothelial cells (HCEC) could be growth on the substrate with basement membrane substance. Serial passages from each HCEC were performed and results with 4.5 + 2.3 (2 to 9 passages) had been achieved from the aged human corneas. The markers for Na/K ATPase, P63, and ABCG2 have shown on the system. We took one of p2 HCEC, and p0 HCEC cultured on the human corneal disc. About one month of culture the HCEC was confluent growth on the human corneal disc. TEM demonstrated mosaic pattern of HCEC on the cornea disc
Conclusions: :
The ex vivo expansion of human corneal endothelial cells systems have been established. The HCEC may maintain the stem cells property on AM. Also, the cultured HCEC could be transplanted on the human corneal disc. These techniques will allow us to further study the engineering of cornea.
Keywords: cornea: endothelium • cornea: basic science