September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Modified supplemented hormonal epithelial medium in combination with 3T3 fibroblasts allows long term culture and expansion of limbal epithelial stem cells
Author Affiliations & Notes
  • Marina Schock
    Ophthalmology, University Hospital Essen, Essen, Germany
  • Bernadette Brockmann-Ahmed
    Ophthalmology, University Hospital Essen, Essen, Germany
  • Henning Thomasen
    Ophthalmology, University Hospital Essen, Essen, Germany
  • Klaus-Peter Steuhl
    Ophthalmology, University Hospital Essen, Essen, Germany
  • Daniel Meller
    Ophthalmology, University Hospital Jena, Jena, Germany
  • Footnotes
    Commercial Relationships   Marina Schock, None; Bernadette Brockmann-Ahmed, None; Henning Thomasen, None; Klaus-Peter Steuhl, None; Daniel Meller, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4904. doi:
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      Marina Schock, Bernadette Brockmann-Ahmed, Henning Thomasen, Klaus-Peter Steuhl, Daniel Meller; Modified supplemented hormonal epithelial medium in combination with 3T3 fibroblasts allows long term culture and expansion of limbal epithelial stem cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4904.

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

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Abstract

Purpose : Limbal stem cell deficiency is one of the most common causes for blindness worldwide. It is characterized by the lack of limbal stem cells which leads to ingrowth of the conjunctiva into the cornea. In order to characterize the limbal stem cells for a better clinical application we have compared culture media and conditions to obtain a sufficient amount of cells for analysis.

Methods : Human limbal tissue obtained from enucleated patients was dissolved into single cells by collagenase digestion overnight. Four different culture conditions were compared: Cells grown with 1) supplemented hormonal epithelial medium (SHEM), 2) with a modification of SHEM (SHEMmod), 3) with mitomycin C treated 3T3 fibroblasts on SHEM and with 4) SHEMmod on mitomycin C treated 3T3 fibroblasts. The cells were passaged every week until senescence occurred. The expression of the differentiation markers K3, K12 and connexin 43 and the putative stem cell markers K15 and ΔNp63α was analysed over the passages using reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry.

Results : With culture condition 4) we were able to keep the limbal cells in culture for 25 passages. Under condition 1) cells rapidly became senescent and condition 2) and 4) allowed only up to 5 passages. Morphologically, the cells under condition 1) - 3) showed increased differentiated morphology with every passage while just the cells under condition 4) still showed a characteristic cobblestone pattern with a small cell size and a high nuclear/cytoplasmic ratio until passage 25. Only condition 4) showed notable expression of K3 and a much higher expression of K12 and connexin 43 than the other three conditions. With every passage the expression of the differentiation markers declined. K3 and K12 were almost undetectable in later passages whereas connexin 43 retained a low but stable expression level. Expression of ΔNp63α and K15 declined upon passaging for conditions 1) – 3) but was stable with variations from passage to passage for condition 4).

Conclusions : SHEMmod in combination with 3T3 fibroblasts enables the production of a large amount of cells with an undifferentiated phenotype. Further investigations on these cells will show if they could be used for research and transplantation to overcome the problem of limited donor cells.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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