title: Cryopreservation and long-term culture of transformed murine corneal endothelial cells.
abstract: OBJECTIVE
  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.
authors: Wilmer Eye Institute, The Johns Hopkins Medical Institutions, Smith 5011 400 N. Broadway, Baltimore, MD 21231, USA.
authors: Christoph
authors: C
authors: Engler
authors: Clare
authors: C
authors: Kelliher
authors: Sungdong
authors: S
authors: Chang
authors: Huan
authors: H
authors: Meng
authors: Albert S
authors: AS
authors: Jun
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015415:Biomarkers; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D024102:Collagen Type VIII / Q000235:genetics / Q000378:metabolism; D015925:Cryopreservation; D003893:Desmin / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism; D014746:Vimentin / Q000235:genetics / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
chemical_list: C558396:Acta2 protein, mouse; D000199:Actins; D015415:Biomarkers; C507463:Col8a2 protein, mouse; D024102:Collagen Type VIII; D003893:Desmin; D008565:Membrane Proteins; D010750:Phosphoproteins; C569043:Tjp1 protein, mouse; D014746:Vimentin; D062826:Zonula Occludens-1 Protein; D000254:Sodium-Potassium-Exchanging ATPase