title: Phenotypic characterization of culture expanded rabbit limbal corneal keratocytes.
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.
authors.affiliation: Department of Anatomy, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300, Kuala Lumpur, Malaysia, norzanafi9506@yahoo.com.
authors.forename: Norzana
authors.initials: N
authors.lastname: Abd Ghafar
authors.forename: Kien Hui
authors.initials: KH
authors.lastname: Chua
authors.forename: Wan Zurinah
authors.initials: WZ
authors.lastname: Wan Ngah
authors.forename: Jemaima
authors.initials: J
authors.lastname: Che Hamzah
authors.forename: Fauziah
authors.initials: F
authors.lastname: Othman
authors.forename: Ropilah
authors.initials: R
authors.lastname: Abd Rahman
authors.forename: Ruszymah
authors.initials: R
authors.lastname: Hj Idrus
mesh_terms: D000199:Actins / Q000096:biosynthesis; D000444:Aldehyde Dehydrogenase / Q000096:biosynthesis; D000818:Animals; D057005:Bioengineering; D064987:Cell- and Tissue-Based Therapy; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis; D024042:Collagen Type I / Q000096:biosynthesis; D060527:Corneal Keratocytes / Q000166:cytology* / Q000637:transplantation; D003319:Corneal Stroma / Q000166:cytology*; D005347:Fibroblasts; D015870:Gene Expression; D007632:Keratan Sulfate / Q000096:biosynthesis; D053558:Keratin-3 / Q000096:biosynthesis; D000071234:Lumican; D010641:Phenotype; D011817:Rabbits
chemical_list: D000199:Actins; D011508:Chondroitin Sulfate Proteoglycans; D024042:Collagen Type I; D053558:Keratin-3; D000071234:Lumican; D007632:Keratan Sulfate; D000444:Aldehyde Dehydrogenase