Primary culture of porcine nonpigmented ciliary epithelium

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Purpose: Primary culture of nonpigmented ciliary epithelium (NPE) has proved difficult in the past. Here we report development of a method of growing and maintaining primary cultures of NPE from porcine eye. Studies were conducted to confirm that the cultured NPE expressed proteins characteristic of native NPE. Methods: Intact rings of NPE were isolated from adult pig eyes. A mixture of hyaluronidase and collagenase was used to detach the cells from the basement membrane and vitreous. Dispersed cells were seeded at high density and grown in DMEM with 20% fetal bovine serum under 5% CO2 and 95% air. Protein expression was examined by immunohistochemistry and immunoblot analysis. Results: NPE cells were grown in primary culture and maintained up to 10th passage. Analysis of the ciliary body showed three Na, K-ATPase isoforms (α 1, α 2, α 3) and three nitric oxide synthase isoforms (eNOS, nNOS, iNOS) enriched in the NPE layer but weaker or absent in the PE layer. Each of these proteins as well as the tight junction-specific protein ZO-1 was detected in the cultured NPE. Conclusions: We developed a simple and reliable way to isolate, culture, and maintain NPE cells from porcine eyes. Success of the method hinged on our ability to isolate pure NPE in large number, detach the cells from the vitreous, and seed the cells at high density. The cultured cells express several proteins that are characteristic of native NPE. NPE cells cultured in this way may prove to be valuable for the study of ciliary body function.

Original languageEnglish (US)
Pages (from-to)511-522
Number of pages12
JournalCurrent Eye Research
Volume32
Issue number6
DOIs
StatePublished - Jun 2007

Fingerprint

Swine
Epithelium
Ciliary Body
Cultured Cells
Protein Isoforms
Proteins
Zonula Occludens-1 Protein
Hyaluronoglucosaminidase
Collagenases
Basement Membrane
Nitric Oxide Synthase
Seeds
Cell Count
Immunohistochemistry
Air
Cell Membrane
Serum

Keywords

  • Immunoblot
  • Immunohistochemistry
  • Nonpigmented ciliary epithelium
  • Porcine eye
  • Primary culture

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Primary culture of porcine nonpigmented ciliary epithelium. / Shahidullah, Mohammad -; Tamiya, Shigeo; Delamere, Nicholas A.

In: Current Eye Research, Vol. 32, No. 6, 06.2007, p. 511-522.

Research output: Contribution to journalArticle

@article{757c5a5b0fc3459188f41f14708cb42b,
title = "Primary culture of porcine nonpigmented ciliary epithelium",
abstract = "Purpose: Primary culture of nonpigmented ciliary epithelium (NPE) has proved difficult in the past. Here we report development of a method of growing and maintaining primary cultures of NPE from porcine eye. Studies were conducted to confirm that the cultured NPE expressed proteins characteristic of native NPE. Methods: Intact rings of NPE were isolated from adult pig eyes. A mixture of hyaluronidase and collagenase was used to detach the cells from the basement membrane and vitreous. Dispersed cells were seeded at high density and grown in DMEM with 20{\%} fetal bovine serum under 5{\%} CO2 and 95{\%} air. Protein expression was examined by immunohistochemistry and immunoblot analysis. Results: NPE cells were grown in primary culture and maintained up to 10th passage. Analysis of the ciliary body showed three Na, K-ATPase isoforms (α 1, α 2, α 3) and three nitric oxide synthase isoforms (eNOS, nNOS, iNOS) enriched in the NPE layer but weaker or absent in the PE layer. Each of these proteins as well as the tight junction-specific protein ZO-1 was detected in the cultured NPE. Conclusions: We developed a simple and reliable way to isolate, culture, and maintain NPE cells from porcine eyes. Success of the method hinged on our ability to isolate pure NPE in large number, detach the cells from the vitreous, and seed the cells at high density. The cultured cells express several proteins that are characteristic of native NPE. NPE cells cultured in this way may prove to be valuable for the study of ciliary body function.",
keywords = "Immunoblot, Immunohistochemistry, Nonpigmented ciliary epithelium, Porcine eye, Primary culture",
author = "Shahidullah, {Mohammad -} and Shigeo Tamiya and Delamere, {Nicholas A}",
year = "2007",
month = "6",
doi = "10.1080/02713680701434899",
language = "English (US)",
volume = "32",
pages = "511--522",
journal = "Current Eye Research",
issn = "0271-3683",
publisher = "Informa Healthcare",
number = "6",

}

TY - JOUR

T1 - Primary culture of porcine nonpigmented ciliary epithelium

AU - Shahidullah, Mohammad -

AU - Tamiya, Shigeo

AU - Delamere, Nicholas A

PY - 2007/6

Y1 - 2007/6

N2 - Purpose: Primary culture of nonpigmented ciliary epithelium (NPE) has proved difficult in the past. Here we report development of a method of growing and maintaining primary cultures of NPE from porcine eye. Studies were conducted to confirm that the cultured NPE expressed proteins characteristic of native NPE. Methods: Intact rings of NPE were isolated from adult pig eyes. A mixture of hyaluronidase and collagenase was used to detach the cells from the basement membrane and vitreous. Dispersed cells were seeded at high density and grown in DMEM with 20% fetal bovine serum under 5% CO2 and 95% air. Protein expression was examined by immunohistochemistry and immunoblot analysis. Results: NPE cells were grown in primary culture and maintained up to 10th passage. Analysis of the ciliary body showed three Na, K-ATPase isoforms (α 1, α 2, α 3) and three nitric oxide synthase isoforms (eNOS, nNOS, iNOS) enriched in the NPE layer but weaker or absent in the PE layer. Each of these proteins as well as the tight junction-specific protein ZO-1 was detected in the cultured NPE. Conclusions: We developed a simple and reliable way to isolate, culture, and maintain NPE cells from porcine eyes. Success of the method hinged on our ability to isolate pure NPE in large number, detach the cells from the vitreous, and seed the cells at high density. The cultured cells express several proteins that are characteristic of native NPE. NPE cells cultured in this way may prove to be valuable for the study of ciliary body function.

AB - Purpose: Primary culture of nonpigmented ciliary epithelium (NPE) has proved difficult in the past. Here we report development of a method of growing and maintaining primary cultures of NPE from porcine eye. Studies were conducted to confirm that the cultured NPE expressed proteins characteristic of native NPE. Methods: Intact rings of NPE were isolated from adult pig eyes. A mixture of hyaluronidase and collagenase was used to detach the cells from the basement membrane and vitreous. Dispersed cells were seeded at high density and grown in DMEM with 20% fetal bovine serum under 5% CO2 and 95% air. Protein expression was examined by immunohistochemistry and immunoblot analysis. Results: NPE cells were grown in primary culture and maintained up to 10th passage. Analysis of the ciliary body showed three Na, K-ATPase isoforms (α 1, α 2, α 3) and three nitric oxide synthase isoforms (eNOS, nNOS, iNOS) enriched in the NPE layer but weaker or absent in the PE layer. Each of these proteins as well as the tight junction-specific protein ZO-1 was detected in the cultured NPE. Conclusions: We developed a simple and reliable way to isolate, culture, and maintain NPE cells from porcine eyes. Success of the method hinged on our ability to isolate pure NPE in large number, detach the cells from the vitreous, and seed the cells at high density. The cultured cells express several proteins that are characteristic of native NPE. NPE cells cultured in this way may prove to be valuable for the study of ciliary body function.

KW - Immunoblot

KW - Immunohistochemistry

KW - Nonpigmented ciliary epithelium

KW - Porcine eye

KW - Primary culture

UR - http://www.scopus.com/inward/record.url?scp=34447118202&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34447118202&partnerID=8YFLogxK

U2 - 10.1080/02713680701434899

DO - 10.1080/02713680701434899

M3 - Article

VL - 32

SP - 511

EP - 522

JO - Current Eye Research

JF - Current Eye Research

SN - 0271-3683

IS - 6

ER -