title: Increased proliferation and replicative lifespan of isolated human corneal endothelial cells with L-ascorbic acid 2-phosphate.
abstract: OBJECTIVE
  To explore an alternative culture method for human corneal endothelial cells (HCECs) and to examine the effect of l-ascorbic acid 2-phosphate (Asc-2P) on the growth of these cells.
  
  
  METHODS
  The influence of various mitogens, extracellular matrices (ECMs), and Asc-2P on growth of cultured HCECs was examined. HCECs were obtained from donors ranging in age from 12 to 74 years, and primary cultures and subcultures were performed with or without Asc-2P. Expanded HCECs were characterized with immunostaining and reverse transcription polymerase chain reaction (RT-PCR) and evaluated for generation of 8-hydroxy-2-deoxyguanosine (8-OHdG) with immunostaining and an enzyme-linked immunosorbent assay (ELISA).
  
  
  RESULTS
  Culture with Asc-2P and bFGF on atelocollagen promoted the proliferation of HCECs in both primary cultures and subcultures as efficiently as conventional culture using ECM derived from bovine corneal endothelial cells. Zonula occludens-1, N-cadherin, connexin 43, and Na+/K+-ATPase were localized at plasma membranes of cultured HCECs. mRNAs of the voltage-dependent anion channels (VDAC2 and VDAC3), sodium bicarbonate cotransporter member 4 (SLC4A4), and chloride channel proteins (CLCN2 and CLCN3) were detected by RT-PCR. During multiple passages, cultures without Asc-2P showed a decrease in growth and irregular cell morphology, whereas cultures with Asc-2P sustained cell growth and maintained the characteristic polygonal morphology. ELISA for 8-OHdG showed that the levels in mitochondrial DNA significantly decreased when HCECs were subcultured with Asc-2P.
  
  
  CONCLUSIONS
  Combination of Asc-2P and bFGF on atelocollagen allows successful culture for HCECs. Asc-2P extends the lifespan of cultured HCECs, partly due to protection against oxidative DNA damage.
authors: Corneal Regeneration Research Team, Foundation for Biomedical Research and Innovation, Kobe, Japan.
authors: Nobuyuki
authors: N
authors: Shima
authors: Miwa
authors: M
authors: Kimoto
authors: Masahiro
authors: M
authors: Yamaguchi
authors: Satoru
authors: S
authors: Yamagami
mesh_terms: D000328:Adult; D000368:Aged; D000970:Antineoplastic Agents / Q000494:pharmacology; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects*; D016922:Cellular Senescence / Q000187:drug effects*; D002648:Child; D004249:DNA Damage / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects; D019573:Epithelium, Corneal / Q000166:cytology*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006801:Humans; D008875:Middle Aged; D018384:Oxidative Stress / Q000187:drug effects; D061251:Primary Cell Culture / Q000379:methods*
chemical_list: D000970:Antineoplastic Agents; D016222:Fibroblast Growth Factor 2; C011669:ascorbate-2-phosphate; D001205:Ascorbic Acid