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Serum-free medium and hydroxyethyl starch supports cell survival better than Minimal Essential Medium and dextran in organ-cultured mouse corneas
  1. Lilla Knels1,
  2. Doris Goetze1,
  3. Katrin Engelmann2,3,
  4. Monika Valtink1
  1. 1Medical Faculty, Institute of Anatomy, TU Dresden, Dresden, Germany
  2. 2CRTD/DFG-Center for Regenerative Therapies Dresden—Cluster of Excellence, TU Dresden, Dresden, Germany
  3. 3Department of Ophthalmology, Klinikum Chemnitz gGmbH, Chemnitz, Germany
  1. Correspondence to Dr Monika Valtink, Medical Faculty, Institute of Anatomy, TU Dresden, Fetscherstr. 74, Dresden 01307, Germany; monika.valtink{at}tu-dresden.de

Abstract

Background/aims In a previous study, we observed a deleterious effect of serum-supplemented Minimal Essential Medium (MEM) on human corneal endothelial cell survival in a cell culture model. Consequently, here we studied the effects of conventional, serum-supplemented MEM and a serum-free medium in combination with two different deswelling substances on cell survival in whole corneas in a mouse model.

Methods Murine corneas were cultured for 4 days in MEM+2% fetal calf serum (FCS) or serum-free Human Endothelial-SFM (SFM), both supplemented with either 6% dextran T500 or 7.5% hydroxyethyl starch (HES) 130/0.4. Cells were examined by differential interference contrast microscopy, H&E staining, immunocytochemistry for cleaved caspase-3, Bcl-2, haem oxygenase-1 and immunoblotting for cleaved caspase-3.

Results In MEM, endothelial cells were almost completely lost after 4 days and the number of epithelial cells was markedly reduced. The remaining cells showed fragmented nuclei and were positive for cleaved caspase-3 and strongly positive for Bcl-2. Corneas cultured in SFM retained an almost closed layer of endothelial cells. Fewer cells were positive for Bcl-2, and only a few cells were positive for cleaved caspase-3 even under staurosporine administration. HES supplementation was well tolerated by corneal cells over 4 days, while a 4-day supplementation with dextran resulted in the loss of endothelial and epithelial cells.

Conclusions Serum-free medium, Human Endothelial-SFM, promoted cell survival during corneal organ culture better than MEM+2% FCS. HES 130/0.4 appeared to be tolerated better by the cells than dextran T500.

  • Cornea
  • Apotosis
  • Eye (Tissue) Banking

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