TY - JOUR
T1 - Growing human corneal epithelium on collagen shield and subsequent transfer to denuded cornea in vitro
AU - He, Yu Guang
AU - McCulley, James P.
N1 - Funding Information:
ACKNOWLEDGEMENTS We appreciate Dr. Michael Berman for his helpful suggestions and criticisms during the preparation of this manuscript, and Ms. Vee Shine and Ms. Ying Wang for their excellent technical assistance. The manuscript was carefully prepared by Ms. Patricia Clarke. Supported in part by an unrestricted research grant from Research to Prevent Blindness, Inc., New York, New York.
PY - 1991
Y1 - 1991
N2 - Three fundamental in vitro experiments have been done in the present report: 1) comparison of three different nutrient media on their abilities to culture and passage the human corneal epithelial cells; 2) evaluation of the ability of extracellular matrix material to promote the growth of cultured human corneal epithelium on collagen corneal shields; and 3) determination of the feasibility of the shield to serve as a carrier for the transfer of cultured cells to allogeneic, denuded corneal surface in vitro. Primary cultures of human corneal epithelium were established from explants which were obtained from limbal and peripheral corneal tissue by three different nutrient media respectively: KGM (Keratinocyte Growth Medium), SHEM (Supplemental Hormonal Epithelial Medium), and one combination of the two media (KGM/SHEM). We found the KGM/SHEM combination to be more favorable because morphology was better preserved, the proliferation rate increased five-fold over the 14 days observed time course, and we were able to subculture the tissue for at least three passages. With this combined medium, a suspension of cultured corneal epithelial cells (5 × K5/ml) was seeded onto either the concave surface of collagen corneal shields or onto shields which had been coated with extracellular matrix materials (Matrigel™ or type IV collagen). The cells attached readily to all the coated shields (20/20) but to only a few of the uncoated shields (3/10), and formed a stratified tissue (2 to 3 layers) within seven days once the cells attached. However, the cells on the shields coated with Matrigel™ failed to become confluent under these conditions. The stratified tissue on type IV collagen coated shields could then be subsequently transferred to denuded human corneal stroma in organ culture by placing them together and incubating for 2-7 days. After that, histologic examinations showed that the epithelial cells had attached tightly to the recipient stromal surface, even after the removal of the collagen shield.
AB - Three fundamental in vitro experiments have been done in the present report: 1) comparison of three different nutrient media on their abilities to culture and passage the human corneal epithelial cells; 2) evaluation of the ability of extracellular matrix material to promote the growth of cultured human corneal epithelium on collagen corneal shields; and 3) determination of the feasibility of the shield to serve as a carrier for the transfer of cultured cells to allogeneic, denuded corneal surface in vitro. Primary cultures of human corneal epithelium were established from explants which were obtained from limbal and peripheral corneal tissue by three different nutrient media respectively: KGM (Keratinocyte Growth Medium), SHEM (Supplemental Hormonal Epithelial Medium), and one combination of the two media (KGM/SHEM). We found the KGM/SHEM combination to be more favorable because morphology was better preserved, the proliferation rate increased five-fold over the 14 days observed time course, and we were able to subculture the tissue for at least three passages. With this combined medium, a suspension of cultured corneal epithelial cells (5 × K5/ml) was seeded onto either the concave surface of collagen corneal shields or onto shields which had been coated with extracellular matrix materials (Matrigel™ or type IV collagen). The cells attached readily to all the coated shields (20/20) but to only a few of the uncoated shields (3/10), and formed a stratified tissue (2 to 3 layers) within seven days once the cells attached. However, the cells on the shields coated with Matrigel™ failed to become confluent under these conditions. The stratified tissue on type IV collagen coated shields could then be subsequently transferred to denuded human corneal stroma in organ culture by placing them together and incubating for 2-7 days. After that, histologic examinations showed that the epithelial cells had attached tightly to the recipient stromal surface, even after the removal of the collagen shield.
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U2 - 10.3109/02713689109013881
DO - 10.3109/02713689109013881
M3 - Article
C2 - 1790715
AN - SCOPUS:0025908920
SN - 0271-3683
VL - 10
SP - 851
EP - 863
JO - Current Eye Research
JF - Current Eye Research
IS - 9
ER -