April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Fibroin-Based Materials Support Cultivation of Limbal Stromal Cells
Author Affiliations & Notes
  • L. J. Sinfield
    Medical Sciences,
    Queensland University of Technology, Brisbane, Australia
  • K. George
    Medical Sciences,
    Queensland University of Technology, Brisbane, Australia
  • Z. Zainuddin
    Queensland Eye Institute, South Brisbane, Australia
  • T. V. Chirila
    Queensland Eye Institute, South Brisbane, Australia
  • D. Hutmacher
    Built Environment and Engineering,
    Queensland University of Technology, Brisbane, Australia
  • I. R. Schwab
    UC Davis Eye Center, University of California at Davis, Sacramento, California
  • D. G. Harkin
    Medical Sciences,
    Queensland University of Technology, Brisbane, Australia
  • Footnotes
    Commercial Relationships  L.J. Sinfield, None; K. George, None; Z. Zainuddin, None; T.V. Chirila, None; D. Hutmacher, None; I.R. Schwab, None; D.G. Harkin, None.
  • Footnotes
    Support  NH&MRC 553038
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 6211. doi:
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    • Get Citation

      L. J. Sinfield, K. George, Z. Zainuddin, T. V. Chirila, D. Hutmacher, I. R. Schwab, D. G. Harkin; Fibroin-Based Materials Support Cultivation of Limbal Stromal Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6211.

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

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Abstract

Purpose: : The silk protein fibroin provides a potential substrate for use in ocular tissue reconstruction. We have previously demonstrated that transparent membranes produced from fibroin support cultivation of human limbal epithelial cells (Tissue Eng A. 14(2008)1203-11). We presently extend this body of work to studies of human limbal stromal cell (HLS) growth on fibroin in the presence and absence of serum.

Methods: : Primary cultures of HLS cells were established in DMEM/F12 medium supplemented with either 10% fetal bovine serum (FBS) or 2% B27 supplement. Defined keratinocyte serum-free medium (DK-SFM, Invitrogen) was also tested. The resulting cultures were analysed by flow cytometry for expression of CD34, CD90, CD45, and CD141. Cultures grown under each condition were subsequently passaged either onto transparent fibroin membranes prepared from purified fibroin or within 3D scaffolds prepared from partially-solubilised fibroin.

Results: : HLS cultures were successfully established under each condition, but grew more slowly and passaged poorly in the absence of serum. Cultures grown in 10% FBS were <0.5% CD34+ (keratocytes) and >97% CD90+ (fibroblasts). Cultures established in 2% B27 formed floating spheres and contained >8% CD34+ cells and reduced CD90 expression. Cultures established in DK-SFM displayed traces of epithelial cell growth (CD141), but mostly consisted of CD90+ cells with <1% CD34+ cells. Cells of bone marrow lineage (CD45) were rarely observed under any conditions. Cultures grown in 10% FBS were able to adhere to and proliferate on silk fibroin 3-D scaffolds and transparent films while those grown serum-free could not. Adhesion of HLS cells to fibroin was initially poorer than that displayed on tissue culture plastic.

Conclusions: : HLS cultures containing cells of predominantly fibroblast lineage can be grown on fibroin-based materials, but this process is dependent upon additional ECM factors such as those provided by serum.

Keywords: cornea: stroma and keratocytes • transplantation • cornea: basic science 
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