title: Ex vivo expansion of conjunctival and limbal epithelial cells using cord blood serum-supplemented culture medium.
abstract: OBJECTIVE
  Conventional cell culture methods use fetal bovine serum (FBS) as a growth supplement. The purpose of this study was to develop a xenobiotic-free culture system using umbilical cord blood serum (CBS) as an alternative growth supplement for the cultivation of human conjunctival and limbal epithelial cells.
  
  
  METHODS
  Human conjunctival and limbal epithelial cells were cultivated in varying concentrations of CBS-supplemented medium and compared with FBS-supplemented medium. Bromodeoxyuridine (BrdU) ELISA proliferation assay, colony-forming efficiency (CFE), and a number of cell generations were analyzed. Cytokeratin expression of cultured cells was evaluated (K3, K4, K12, K13, K14, K15, K19, and PanCK). The authors compared the cytokine and growth factor levels in CBS, FBS, and adult serum using antibody array assays.
  
  
  RESULTS
  Conjunctival and limbal cells cultivated in 0.25% CBS- and 0.5% CBS-supplemented culture media demonstrated the highest proliferative capacity in terms of BrdU proliferation assay, CFE, and number of cell generations. These results were comparable to FBS-supplemented medium. Cultured epithelial cells retained their normal cytokeratin expression. Cytokines brain-derived neurotrophic factor, growth-related oncogene, and leptin and growth factors EGF, HGF, FGF-6, IGF-1, PDGF, and IGFBP were present in higher concentrations in CBS than in FBS and adult serum.
  
  
  CONCLUSIONS
  CBS-supplemented culture medium supported the proliferation and differentiation of conjunctival and limbal epithelial cells. CBS contained a higher concentration of growth factors and cytokines than FBS and adult serum. CBS may be a viable and safer alternative to FBS as a growth supplement in the culture medium for culturing epithelial cells, which may have important clinical implications when bioengineering tissues for clinical use.
authors.affiliation: Eye & Cornea Transplant Center, Singapore. leopk@pacific.net.sg
authors.forename: Leonard Pek-Kiang
authors.initials: LP
authors.lastname: Ang
authors.forename: Tien Phuc
authors.initials: TP
authors.lastname: Do
authors.forename: Zaw M
authors.initials: ZM
authors.lastname: Thein
authors.forename: Hasan Mahmud
authors.initials: HM
authors.lastname: Reza
authors.forename: Xiao-Wei
authors.initials: XW
authors.lastname: Tan
authors.forename: Christine
authors.initials: C
authors.lastname: Yap
authors.forename: Donald T H
authors.initials: DT
authors.lastname: Tan
authors.forename: Roger W
authors.initials: RW
authors.lastname: Beuerman
mesh_terms: D001973:Bromodeoxyuridine / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism; D003470:Culture Media; D016207:Cytokines / Q000378:metabolism; D004261:DNA Replication; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005312:Fetal Blood / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007633:Keratins / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism
chemical_list: D003470:Culture Media; D016207:Cytokines; D007633:Keratins; D001973:Bromodeoxyuridine