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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.

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Related in: MedlinePlus

The growth curve of NRCE cells at passage 191. The lag phase (Lag), logarithmic phase (Log), stationary phase (Sta), and decline phase (Dec) were shown.
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f2: The growth curve of NRCE cells at passage 191. The lag phase (Lag), logarithmic phase (Log), stationary phase (Sta), and decline phase (Dec) were shown.

Mentions: There were numerous pure NRCE cells attached to the bottom of the wells after the corneal fragments were detached (Figure 1A). These cells were in a typical polygonal morphology and many of them were still in the non-extended state. About 25 days later, the primary cultured NRCE cells grew into a confluent monolayer (Figure 1B). Most of the NRCE cells were plump and in polygonal cell morphology. During subsequent subculture, the polygonal morphology of NRCE cells began to elongate to some extent after the culture medium was replaced with 20% BCS-containing DMEM/F12 medium but no growth factors (Figure 1C). The NRCE cells grew and proliferated at a steady rate, and their doubling time was calculated to be 40.32 h at passage 191 (Figure 2). At present, the NRCE cells have been subcultured to passage 227 (Figure 1D), and a novel continuous untransfected NRCE cell line has been established.


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)

The growth curve of NRCE cells at passage 191. The lag phase (Lag), logarithmic phase (Log), stationary phase (Sta), and decline phase (Dec) were shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: The growth curve of NRCE cells at passage 191. The lag phase (Lag), logarithmic phase (Log), stationary phase (Sta), and decline phase (Dec) were shown.
Mentions: There were numerous pure NRCE cells attached to the bottom of the wells after the corneal fragments were detached (Figure 1A). These cells were in a typical polygonal morphology and many of them were still in the non-extended state. About 25 days later, the primary cultured NRCE cells grew into a confluent monolayer (Figure 1B). Most of the NRCE cells were plump and in polygonal cell morphology. During subsequent subculture, the polygonal morphology of NRCE cells began to elongate to some extent after the culture medium was replaced with 20% BCS-containing DMEM/F12 medium but no growth factors (Figure 1C). The NRCE cells grew and proliferated at a steady rate, and their doubling time was calculated to be 40.32 h at passage 191 (Figure 2). At present, the NRCE cells have been subcultured to passage 227 (Figure 1D), and a novel continuous untransfected NRCE cell line has been established.

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