TY - JOUR
T1 - Cryopreservation and lentiviral-mediated genetic modification of human primary cultured corneal endothelial cells
AU - Suh, Leejee H.
AU - Zhang, Cheng
AU - Chuck, Roy S.
AU - Stark, Walter J.
AU - Naylor, Stuart
AU - Binley, Katie
AU - Chakravarti, Shukti
AU - Jun, Albert S.
PY - 2007/7
Y1 - 2007/7
N2 - PURPOSE. To determine the viability and potential usefulness of cryopreserved human primary cultured corneal endothelial cells by characterizing their morphology, gene expression, and ability for genetic modification by the lentiviral vector equine infectious anemia virus (EIAV). METHODS. Primary cultured endothelial cells were dissociated from human corneas and grown in organ culture medium. Corneal endothelial cell origin was confirmed by morphology and immunostaining with polyclonal anti- collagen VIII antibodies. Cells of different passages were cryopreserved in medium containing dimethyl sulfoxide and were assessed after thawing for morphology, proliferative capacity, gene expression, and ability to form cell-cell junctions. EIAV encoding enhanced green fluorescent protein (eGFP) was used to transduce cryopreserved human corneal endothelial cells. Transduced cells were then sorted by fluorescence-activated cell sorting (FACS) and imaged with fluorescence microscopy. RESULTS. Cryopreserved, primary, cultured human corneal endothelial cells are viable and retain their ability to proliferate, produce collagen VIII, and express ZO-1, a tight-junction protein. EIAV-based gene transfer of eGFP is highly efficient and nontoxic to cryopreserved human primary cultured corneal endothelial cells. These genetically modified cells can be selected to nearly pure populations with FACS sorting. CONCLUSIONS. Human primary cultured corneal endothelial cells retain their phenotypic properties after cryopreservation. The ability to store, genetically modify, and sort these cells through FACS to pure populations has the potential to greatly expand their future therapeutic application to treat corneal endothelial disorders.
AB - PURPOSE. To determine the viability and potential usefulness of cryopreserved human primary cultured corneal endothelial cells by characterizing their morphology, gene expression, and ability for genetic modification by the lentiviral vector equine infectious anemia virus (EIAV). METHODS. Primary cultured endothelial cells were dissociated from human corneas and grown in organ culture medium. Corneal endothelial cell origin was confirmed by morphology and immunostaining with polyclonal anti- collagen VIII antibodies. Cells of different passages were cryopreserved in medium containing dimethyl sulfoxide and were assessed after thawing for morphology, proliferative capacity, gene expression, and ability to form cell-cell junctions. EIAV encoding enhanced green fluorescent protein (eGFP) was used to transduce cryopreserved human corneal endothelial cells. Transduced cells were then sorted by fluorescence-activated cell sorting (FACS) and imaged with fluorescence microscopy. RESULTS. Cryopreserved, primary, cultured human corneal endothelial cells are viable and retain their ability to proliferate, produce collagen VIII, and express ZO-1, a tight-junction protein. EIAV-based gene transfer of eGFP is highly efficient and nontoxic to cryopreserved human primary cultured corneal endothelial cells. These genetically modified cells can be selected to nearly pure populations with FACS sorting. CONCLUSIONS. Human primary cultured corneal endothelial cells retain their phenotypic properties after cryopreservation. The ability to store, genetically modify, and sort these cells through FACS to pure populations has the potential to greatly expand their future therapeutic application to treat corneal endothelial disorders.
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U2 - 10.1167/iovs.06-0771
DO - 10.1167/iovs.06-0771
M3 - Article
C2 - 17591873
AN - SCOPUS:34548064094
SN - 0146-0404
VL - 48
SP - 3056
EP - 3061
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
IS - 7
ER -