Limits...
Establishment and characterization of a novel untransfected corneal endothelial cell line from New Zealand white rabbits.

Fan T, Wang D, Zhao J, Wang J, Fu Y, Guo R - Mol. Vis. (2009)

Bottom Line: The results of gene expression patterns of marker proteins and membrane transport proteins, combined with immunofluorescent localization patterns of cell junction proteins, indicated that NRCE cells retained normal corneal endothelial characteristics and normal expression pattern of functional proteins.Furthermore, these cells, without any tumorigenic potency, had excellent biocompatibility to denuded amnions in 20% BCS-containing DMEM/F12 medium, and formed confluent cell sheets attached tightly to denuded amnions.These results suggest that a novel untransfected NRCE cell line established here maintains normal corneal endothelial characteristics and potencies to form normal cell junctions and perform normal functions of transmembrane transport.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Corneal Tissue Engineering, Ocean University of China, Shandong, China. tjfan@ouc.edu.cn

ABSTRACT

Purpose: To establish and characterize a novel untransfected corneal endothelial cell line from New Zealand white rabbits (NRCE cell line) for studies on corneal endothelial cells.

Methods: Primary culture was initiated with a pure population of NRCE cells from corneal endothelia by successive detachment and reattachment procedure of different durations, and cultured in 20% fetal bovine serum-containing DMEM/F12 media with several supplements. The cell line was characterized by chromosome analysis, fluorescence immunoassay and reverse transcription PCR. The tumorigenic potency of the cell line was examined by subcutaneous inoculation to nude mice. The biocompatibility of the cell line to denuded amnions was examined with routine microscopic and electron microscopic techniques.

Results: NRCE cells in primary culture proliferated to confluency in 25 days and has been subcultured to passage 227 to date. The novel NRCE cell line, with a steady growing rate in 20% bovine calf serum (BCS)-containing DMEM/F12 medium and a population doubling time of 40.32 h at passage 191, has been established. NRCE cells exhibited chromosomal aneuploidy but their modal chromosome number was still 44. The results of gene expression patterns of marker proteins and membrane transport proteins, combined with immunofluorescent localization patterns of cell junction proteins, indicated that NRCE cells retained normal corneal endothelial characteristics and normal expression pattern of functional proteins. Furthermore, these cells, without any tumorigenic potency, had excellent biocompatibility to denuded amnions in 20% BCS-containing DMEM/F12 medium, and formed confluent cell sheets attached tightly to denuded amnions.

Conclusions: These results suggest that a novel untransfected NRCE cell line established here maintains normal corneal endothelial characteristics and potencies to form normal cell junctions and perform normal functions of transmembrane transport.

Show MeSH

Related in: MedlinePlus

Evaluation of biocompatibility of the passage 191 NRCE cells on denuded amnion. A: NRCE cells grew into a confluent cell sheet on denuded amnion 90 h later. Scale bar: 40μm. B: SEM image showing the cell morphology of a NRCE cell sheet on denuded amnion. Scale bar: 10 μm. C: TEM image of a NRCE cell from a live rabbit corneal endothelium on Descement's membrane (DM). Scale bar: 0.2 μm. D: TEM image of a NRCE cell on denuded amnion. The tight attachment status of the NRCE cell to denuded amnion (dAM), almost identical to that of the NRCE cell on Descement's membrane in (C), was shown. Scale bar: 0.2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2690960&req=5

f7: Evaluation of biocompatibility of the passage 191 NRCE cells on denuded amnion. A: NRCE cells grew into a confluent cell sheet on denuded amnion 90 h later. Scale bar: 40μm. B: SEM image showing the cell morphology of a NRCE cell sheet on denuded amnion. Scale bar: 10 μm. C: TEM image of a NRCE cell from a live rabbit corneal endothelium on Descement's membrane (DM). Scale bar: 0.2 μm. D: TEM image of a NRCE cell on denuded amnion. The tight attachment status of the NRCE cell to denuded amnion (dAM), almost identical to that of the NRCE cell on Descement's membrane in (C), was shown. Scale bar: 0.2 μm.

Mentions: Passage 191 NRCE cells grew very well on denuded amnions, and confluent cell sheets formed 90 h later (Figure 7A,B). In addition, the cell sheets established tight attachments to denuded amnions, almost identical to that between NRCE cells and Descement's membranes in vivo (Figure 7C,D). These results indicated that the established NRCE cell line has excellent biocompatibility with denuded amnions.


Establishment and characterization of a novel untransfected corneal endothelial cell line from New Zealand white rabbits.

Fan T, Wang D, Zhao J, Wang J, Fu Y, Guo R - Mol. Vis. (2009)

Evaluation of biocompatibility of the passage 191 NRCE cells on denuded amnion. A: NRCE cells grew into a confluent cell sheet on denuded amnion 90 h later. Scale bar: 40μm. B: SEM image showing the cell morphology of a NRCE cell sheet on denuded amnion. Scale bar: 10 μm. C: TEM image of a NRCE cell from a live rabbit corneal endothelium on Descement's membrane (DM). Scale bar: 0.2 μm. D: TEM image of a NRCE cell on denuded amnion. The tight attachment status of the NRCE cell to denuded amnion (dAM), almost identical to that of the NRCE cell on Descement's membrane in (C), was shown. Scale bar: 0.2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2690960&req=5

f7: Evaluation of biocompatibility of the passage 191 NRCE cells on denuded amnion. A: NRCE cells grew into a confluent cell sheet on denuded amnion 90 h later. Scale bar: 40μm. B: SEM image showing the cell morphology of a NRCE cell sheet on denuded amnion. Scale bar: 10 μm. C: TEM image of a NRCE cell from a live rabbit corneal endothelium on Descement's membrane (DM). Scale bar: 0.2 μm. D: TEM image of a NRCE cell on denuded amnion. The tight attachment status of the NRCE cell to denuded amnion (dAM), almost identical to that of the NRCE cell on Descement's membrane in (C), was shown. Scale bar: 0.2 μm.
Mentions: Passage 191 NRCE cells grew very well on denuded amnions, and confluent cell sheets formed 90 h later (Figure 7A,B). In addition, the cell sheets established tight attachments to denuded amnions, almost identical to that between NRCE cells and Descement's membranes in vivo (Figure 7C,D). These results indicated that the established NRCE cell line has excellent biocompatibility with denuded amnions.

Bottom Line: The results of gene expression patterns of marker proteins and membrane transport proteins, combined with immunofluorescent localization patterns of cell junction proteins, indicated that NRCE cells retained normal corneal endothelial characteristics and normal expression pattern of functional proteins.Furthermore, these cells, without any tumorigenic potency, had excellent biocompatibility to denuded amnions in 20% BCS-containing DMEM/F12 medium, and formed confluent cell sheets attached tightly to denuded amnions.These results suggest that a novel untransfected NRCE cell line established here maintains normal corneal endothelial characteristics and potencies to form normal cell junctions and perform normal functions of transmembrane transport.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Corneal Tissue Engineering, Ocean University of China, Shandong, China. tjfan@ouc.edu.cn

ABSTRACT

Purpose: To establish and characterize a novel untransfected corneal endothelial cell line from New Zealand white rabbits (NRCE cell line) for studies on corneal endothelial cells.

Methods: Primary culture was initiated with a pure population of NRCE cells from corneal endothelia by successive detachment and reattachment procedure of different durations, and cultured in 20% fetal bovine serum-containing DMEM/F12 media with several supplements. The cell line was characterized by chromosome analysis, fluorescence immunoassay and reverse transcription PCR. The tumorigenic potency of the cell line was examined by subcutaneous inoculation to nude mice. The biocompatibility of the cell line to denuded amnions was examined with routine microscopic and electron microscopic techniques.

Results: NRCE cells in primary culture proliferated to confluency in 25 days and has been subcultured to passage 227 to date. The novel NRCE cell line, with a steady growing rate in 20% bovine calf serum (BCS)-containing DMEM/F12 medium and a population doubling time of 40.32 h at passage 191, has been established. NRCE cells exhibited chromosomal aneuploidy but their modal chromosome number was still 44. The results of gene expression patterns of marker proteins and membrane transport proteins, combined with immunofluorescent localization patterns of cell junction proteins, indicated that NRCE cells retained normal corneal endothelial characteristics and normal expression pattern of functional proteins. Furthermore, these cells, without any tumorigenic potency, had excellent biocompatibility to denuded amnions in 20% BCS-containing DMEM/F12 medium, and formed confluent cell sheets attached tightly to denuded amnions.

Conclusions: These results suggest that a novel untransfected NRCE cell line established here maintains normal corneal endothelial characteristics and potencies to form normal cell junctions and perform normal functions of transmembrane transport.

Show MeSH
Related in: MedlinePlus