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Cryopreservation and long-term culture of transformed murine corneal endothelial cells

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Author(s)
Christoph EnglerClare KelliherSungdong ChangHuan MengAlbert S. Jun
Keimyung Author(s)
Chang, Sung Dong
Department
Dept. of Ophthalmology (안과학)
Journal Title
Graefe's Archive for Clinical and Experimental Ophthalmology
Issued Date
2012
Volume
250
Issue
1
Abstract
Purpose To characterize the morphology and gene expression
of transformed murine corneal endothelial cells.
Methods Primary immortomouse corneal endothelial cells
were continuously cultured before and after cryopreservation.
Morphologic assessment, real time-reverse transcriptase polymerase
chain reaction ((RT)-PCR) and immunofluorescence
studies were performed using newly cultured cells, cells that
had been continuously in culture for 1 year, and cryopreserved
cells, to assess for structural and functional integrity. The
expression of corneal endothelial markers zonula occludens-1
(ZO1), NaK-ATPase and collagen VIII (α2) (COL8A2), and
myofibroblast markers Desmin, alpha smooth muscle actin
(αSMA), and Vimentin was assessed and compared by both
RT-PCR and immunofluorescence.
Results Cells in culture formed a monolayer, and exhibited
a polygonal shape after reaching confluence. Cells retained
this morphology during the full observation time of
12 months and when reused after cryopreservation. Immunofluorescence
experiments exhibited positive staining for
NaK-ATPase and COL8A2 with low variability between the
three groups. In RT-PCR experiments, ZO1, COL8A2 and
Desmin were increased in fresh and thawed cells, αSMA
was decreased, and NaK-ATPase and Vimentin remained
unchanged, compared to 12-month-old cells. Comparing
fresh and thawed cells, COL8A2 was increased in thawed
cells, while Desmin was increased in fresh cells.
Conclusions Using the immortomouse strain, murine
corneal endothelial cells can be propagated over a long
time period and be used after cryopreservation. Cells
retain the expression of NaK-ATPase, but show some
decline in ZO1 and COL8A2 over time and after
cryopreservation. The expression of myofibroblast
markers suggests an endothelial-to-mesenchymal transformation
process in culture.
Keywords Murine corneal endothelial cell culture . Gene
expression . Cryopreservation . Cornea
Keimyung Author(s)(Kor)
장성동
Publisher
School of Medicine
Citation
Christoph Engler et al. (2012). Cryopreservation and long-term culture of transformed
murine corneal endothelial cells. Graefe’s Archive for Clinical and Experimental Ophthalmology, 250(1), 103–110. doi: 10.1007/s00417-011-1805-7
Type
Article
ISSN
0721-832X
Source
http://lps3.link.springer.com.proxy.dsmc.or.kr/article/10.1007%2Fs00417-011-1805-7
DOI
10.1007/s00417-011-1805-7
URI
https://kumel.medlib.dsmc.or.kr/handle/2015.oak/35781
Appears in Collections:
1. School of Medicine (의과대학) > Dept. of Ophthalmology (안과학)
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