Cryopreservation and long-term culture of transformed murine corneal endothelial cells.
Graefes Arch Clin Exp Ophthalmol
; 250(1): 103-10, 2012 Jan.
Article
em En
| MEDLINE
| ID: mdl-21881843
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.
Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Endotélio Corneano
/
Criopreservação
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Células Endoteliais
Limite:
Animals
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article