Phenotypic characterization of culture expanded rabbit limbal corneal keratocytes

Norzana Abd. Ghafar, Chua Kien Hui, Wan Zurinah Wan Ngah, Jemaima Che Hamzah, Fauziah Othman, Ropilah Abd Rahman, Ruszymah Idrus

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

The in vivo quiescent corneal stroma keratocytes need to be transformed to activated state in order to obtain sufficient number of cells either for monolayer evaluation or corneal stroma reconstruction. This study aimed to investigate the phenotypic characterization of corneal stromal cells during culture expansion from the limbal region of the cornea. Isolated corneal keratocytes from limbal tissue of New Zealand White Strain rabbits' corneas (n = 6) were culture expanded until three passages. Keratocytes morphology was examined daily with viability, growth rate, number of cell doubling and population doubling time were recorded at each passage. The expression of collagen type 1, aldehyde dehydrogenase (ALDH), lumican and alpha smooth muscle actin (α-SMA) were detected by RT-PCR. Immunocytochemistry was also used to detect ALDH, α-SMA, collagen type I and Cytokeratin-3 (CK3). Growth kinetic study revealed that the growth rate was low at the initial passage but increase to about two folds with concomitant reduction in population doubling time in later passages. Freshly isolated and cultured keratocytes expressed collagen type 1, ALDH and lumican but α-SMA expression was absent. However, α-SMA was expressed along with the other genes during culture expansion. Keratocytes at P1 expressed all the proteins except CK3. These results suggest that cultured keratocytes maintained most of the gene expression profile of native keratocytes while the emergence of α-SMA in serial passages showed a mix population of various phenotypes. The phenotypic characterization of monolayer keratocytes provides useful information before reconstruction of bioengineered tissue or in vitro pharmaceutical applications.

Original languageEnglish
Pages (from-to)25-34
Number of pages10
JournalCell and Tissue Banking
Volume15
Issue number1
DOIs
Publication statusPublished - 2014

Fingerprint

Corneal Keratocytes
Aldehyde Dehydrogenase
Keratin-3
Collagen Type I
Aldehydes
Collagen
Corneal Stroma
Rabbits
Cell culture
Cornea
Monolayers
Tissue
Population
Serial Passage
Growth kinetics
Stromal Cells
Growth
Transcriptome
Gene expression
Drug products

Keywords

  • Alpha smooth muscle actin
  • Corneal keratocytes
  • Culture expansion
  • Limbal tissue
  • Rabbit

ASJC Scopus subject areas

  • Transplantation
  • Biomaterials
  • Biomedical Engineering
  • Cell Biology
  • Medicine(all)

Cite this

Phenotypic characterization of culture expanded rabbit limbal corneal keratocytes. / Abd. Ghafar, Norzana; Kien Hui, Chua; Wan Ngah, Wan Zurinah; Che Hamzah, Jemaima; Othman, Fauziah; Abd Rahman, Ropilah; Idrus, Ruszymah.

In: Cell and Tissue Banking, Vol. 15, No. 1, 2014, p. 25-34.

Research output: Contribution to journalArticle

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