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Laboratory science - Extended reports
The use of human serum in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells
  1. L P K Ang1,2,3,
  2. D T H Tan1,2,3,
  3. C J Y Seah3,
  4. R W Beuerman3
  1. 1Singapore National Eye Center, 11 Third Hospital Avenue, Singapore 168751, Singapore
  2. 2Department of Ophthalmology, National University of Singapore, 5 Lower Kent Ridge Road, Singapore 119074, Singapore
  3. 3Singapore Eye Research Institute, 11 Third Hospital Avenue, Singapore 168751, Singapore
  1. Correspondence to: Leonard P K Ang MD, Singapore National Eye Center, 11 Third Hospital Avenue, Singapore 168751; leopkpacific.net.sg

Abstract

Aim: To evaluate the use of human serum (HS) in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells, and compare it with fetal bovine serum (FBS) and bovine pituitary extract (BPE).

Methods: Conjunctival epithelial cells were cultivated in media supplemented with HS (5%, 10%), FBS (5%, 10%), and BPE (70 μg/ml, 140 μg/ml). The colony forming efficiency (CFE), bromodeoxyuridine (BrdU) ELISA proliferation assay, and cell generations were analysed. Cells were evaluated for keratin (K4, K19, and K3) and MUC5AC expression by immunostaining and RT-PCR. Conjunctival equivalents constructed on amniotic membranes were transplanted onto severe combined immune deficient (SCID) mice for 10 days and analysed histologically.

Results: The proliferation assays of HS supplemented cultures (CFE, 6.7% (SD 1.8%); BrdU absorbance, 0.86 (0.16)) were comparable to FBS supplemented (CFE, 9.3% (1.8%); BrdU absorbance, 1.11 (0.18)) and BPE supplemented cultures (CFE, 5.9 (1.5); BrdU absorbance, 0.65 (0.12)). Goblet cell densities for HS, FBS, and BPE supplemented media were 52 cells/cm2, 60 cells/cm2, and 50 cells/cm2, respectively. HS supplemented cultures formed stratified epithelial sheets in vivo following transplantation.

Conclusions: The proliferative capacity of conjunctival epithelial cells cultivated in HS supplemented cultures was comparable to FBS and BPE supplemented cultures. The elimination of animal material from the culture system is advantageous when cultivating cells for clinical transplantation.

  • BPE, bovine pituitary extract
  • BrdU, bromodeoxyuridine
  • CFE, colony forming efficiency
  • FBS, fetal bovine serum
  • HAM, human amniotic membranes
  • HS, human serum
  • PAS, periodic acid Schiff
  • SCID, severe combined immune deficient
  • human serum
  • conjunctiva
  • epithelium
  • BPE, bovine pituitary extract
  • BrdU, bromodeoxyuridine
  • CFE, colony forming efficiency
  • FBS, fetal bovine serum
  • HAM, human amniotic membranes
  • HS, human serum
  • PAS, periodic acid Schiff
  • SCID, severe combined immune deficient
  • human serum
  • conjunctiva
  • epithelium

https://doi.org/10.1136/bjo.2004.055046

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    Footnotes

    • Financial support: Singapore Biomedical Research Council grants R268/12/2002 and 04/1/35/19/302.

    • The authors have no competing interest in any of the material discussed in the paper.

    • Meeting presentation: The material was presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology (ARVO), 4–8 May 2003, Fort Lauderdale, FL, USA.

      Ethical approval from the institutional review board of the Singapore National Eye Center was obtained for this study.

      Written informed consent was obtained from all patients who participated in this study.