Cryopreservation and lentiviral-mediated genetic modification of human primary cultured corneal endothelial cells

Leejee H. Suh, Cheng Zhang, Roy S. Chuck, Walter J. Stark, Stuart Naylor, Katie Binley, Shukti Chakravarti, Albert S. Jun

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3056-3061
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Volume48
Issue number7
DOIs
StatePublished - Jul 2007
Externally publishedYes

Fingerprint

Cryopreservation
Medical Genetics
Endothelial Cells
Equine infectious anemia virus
Flow Cytometry
Collagen
Zonula Occludens-1 Protein
Gene Expression
Intercellular Junctions
Organ Culture Techniques
Dimethyl Sulfoxide
Fluorescence Microscopy
Cornea
Population
Culture Media
Cultured Cells
Antibodies
Genes

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Cryopreservation and lentiviral-mediated genetic modification of human primary cultured corneal endothelial cells. / Suh, Leejee H.; Zhang, Cheng; Chuck, Roy S.; Stark, Walter J.; Naylor, Stuart; Binley, Katie; Chakravarti, Shukti; Jun, Albert S.

In: Investigative Ophthalmology and Visual Science, Vol. 48, No. 7, 07.2007, p. 3056-3061.

Research output: Contribution to journalArticle

Suh, Leejee H. ; Zhang, Cheng ; Chuck, Roy S. ; Stark, Walter J. ; Naylor, Stuart ; Binley, Katie ; Chakravarti, Shukti ; Jun, Albert S. / Cryopreservation and lentiviral-mediated genetic modification of human primary cultured corneal endothelial cells. In: Investigative Ophthalmology and Visual Science. 2007 ; Vol. 48, No. 7. pp. 3056-3061.
@article{f2ac076ad1be486c86254546dbc166a1,
title = "Cryopreservation and lentiviral-mediated genetic modification of human primary cultured corneal endothelial cells",
abstract = "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.",
author = "Suh, {Leejee H.} and Cheng Zhang and Chuck, {Roy S.} and Stark, {Walter J.} and Stuart Naylor and Katie Binley and Shukti Chakravarti and Jun, {Albert S.}",
year = "2007",
month = "7",
doi = "10.1167/iovs.06-0771",
language = "English (US)",
volume = "48",
pages = "3056--3061",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "7",

}

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.

UR - http://www.scopus.com/inward/record.url?scp=34548064094&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34548064094&partnerID=8YFLogxK

U2 - 10.1167/iovs.06-0771

DO - 10.1167/iovs.06-0771

M3 - Article

VL - 48

SP - 3056

EP - 3061

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 7

ER -