March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Isolation and Propagation of Human Corneal Endothelial Cells Using a Dual Media Culture System
Author Affiliations & Notes
  • Gary S. Peh
    Singapore Eye Research Institute, Singapore, Singapore
  • Kah-Peng Toh
    Singapore Eye Research Institute, Singapore, Singapore
  • Deepashree Balehosur
    Singapore Eye Research Institute, Singapore, Singapore
  • Heng-Pei Ang
    Singapore Eye Research Institute, Singapore, Singapore
  • Man-Xin Lee
    Singapore Eye Research Institute, Singapore, Singapore
  • Donald T. Tan
    Singapore National Eye Centre, Singapore, Singapore
    Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore., Singapore, Singapore
  • Jodhbir Mehta
    Singapore National Eye Centre, Singapore, Singapore
    Department of Clinical Sciences, Duke-NUS Graduate Medical School, Singapore, Singapore
  • Footnotes
    Commercial Relationships  Gary S. Peh, None; Kah-Peng Toh, None; Deepashree Balehosur, None; Heng-Pei Ang, None; Man-Xin Lee, None; Donald T. Tan, None; Jodhbir Mehta, None
  • Footnotes
    Support  TCR 621/42/2008
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 6013. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Gary S. Peh, Kah-Peng Toh, Deepashree Balehosur, Heng-Pei Ang, Man-Xin Lee, Donald T. Tan, Jodhbir Mehta; Isolation and Propagation of Human Corneal Endothelial Cells Using a Dual Media Culture System. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6013.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: : A novel culture system for the in vitro propagation of isolated human corneal endothelial cells (hCECs) using a dual media approach.

Methods: : Cultures of primary hCECs isolated from pairs of donor corneas deemed unsuitable for transplantation were established and propagated using a novel dual media culture system. Isolated cells were expanded in a medium that supported the proliferation of hCECs, identified and coded in our previous study as M4 - a 1:1 mixture of the basal media Ham’s F12 and M199 (F99). The effect of incorporating a second medium, coded here as M5, into the abovementioned culture condition at least 24 hours before and after cellular passage, was examined in this study. Primary hCECs were established and cultured in each of these conditions for five passages: (A) M4 only, (B) M5 only, or (C) using M5 to stabilize and maintain the cultivated hCECs before and after the proliferative phase, with M4 as the medium utilized to promote the proliferation of hCECs. The cultured hCECs from each condition were analyzed for (1) their cellular morphology, specifically their circularity; (2) their propensity to proliferate in each of these conditions; and (3) their expression of markers indicative of corneal endothelium such as Na+K+ATPase and ZO-1.

Results: : Established primary hCECs cultures propagated using the dual media culture system could be serially passaged and were able to retain the unique polygonal morphology of the hCECs. Cultured cells also retained markers indicative of corneal endothelium. In contrast, most hCECs expanded in M4 alone appeared to have lost the unique polygonal morphology of hCECs and turned fibroblastic-like, some as early as the third passage, and the usage of M5 only were not able to support the active proliferation of hCECs.

Conclusions: : The unique morphology of cultured hCECs can be maintained using the novel dual media culture system described in this study. We propose the use of the proliferative medium M4 followed by the maintenance medium M5 for the extended propagation of hCECs.

Keywords: cornea: endothelium • cornea: basic science • cell adhesions/cell junctions 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×