Cryopreservation and lentiviral-mediated genetic modification of human primary cultured corneal endothelial cells.
OBJECTIVE
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.
Suh
Leejee H
LH
Wilmer Eye Institute, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287-9238, USA.
Zhang
Cheng
C
Chuck
Roy S
RS
Stark
Walter J
WJ
Naylor
Stuart
S
Binley
Katie
K
Chakravarti
Shukti
S
Jun
Albert S
AS
D000328:Adult; D049109:Cell Proliferation; D002478:Cells, Cultured; D002675:Child, Preschool; D024102:Collagen Type VIII / Q000378:metabolism; D015925:Cryopreservation; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression / Q000502:physiology*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D004860:Infectious Anemia Virus, Equine / Q000235:genetics*; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009926:Organ Preservation; D010750:Phosphoproteins / Q000378:metabolism; D014019:Tissue Donors; D062826:Zonula Occludens-1 Protein
D024102:Collagen Type VIII; D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins