May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
New animal compound free medium for human corneal organ culture
Author Affiliations & Notes
  • G. Thuret
    Ophthalmology, Bellevue Hospital, Saint–Etienne, France
    Lab 'Cell adherence and survival in cancers and grafts' EA3063, St–Etienne, France
  • C. Manissolle
    Lab 'Cell adherence and survival in cancers and grafts' EA3063, St–Etienne, France
  • O. Garraud
    Eye Bank, French Blood Center, Saint–Etienne, France
  • S. Acquart
    Eye Bank, French Blood Center, Saint–Etienne, France
  • L. Campos
    Lab 'Cell adherence and survival in cancers and grafts' EA3063, St–Etienne, France
  • D. Guyotat
    Lab 'Cell adherence and survival in cancers and grafts' EA3063, St–Etienne, France
  • J. Maugery
    Ophthalmology, Bellevue Hospital, Saint–Etienne, France
  • P. Gain
    Ophthalmology, Bellevue Hospital, Saint–Etienne, France
    Lab 'Cell adherence and survival in cancers and grafts' EA3063, St–Etienne, France
  • Footnotes
    Commercial Relationships  G. Thuret, None; C. Manissolle, None; O. Garraud, None; S. Acquart, None; L. Campos, None; D. Guyotat, None; J. Maugery, None; P. Gain, None.
  • Footnotes
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Investigative Ophthalmology & Visual Science May 2004, Vol.45, 4868. doi:
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      G. Thuret, C. Manissolle, O. Garraud, S. Acquart, L. Campos, D. Guyotat, J. Maugery, P. Gain; New animal compound free medium for human corneal organ culture . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4868.

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

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Abstract

Abstract: : Purpose: Since the very beginning of corneal organ culture (OC), the replacement of foetal calf serum (FCS), vital to endothelial cells (EC), has been a challenge. Aim: to present the in vitro evaluation of a prototype of animal compound free (ACF) medium compared to the commercial media (with 2% of FCS) available in France. This medium contained also a new macromolecule instead of classical dextran for the deswelling at the end of storage. The new medium was produced by Stem Alpha (St–Clément les Places, France), a company specializing in serum free media for haematology. Because of proprietary interest of the company the full composition is not available Methods: Fifty paired scientific human corneas were randomly allocated at procurement to either the ACF medium or medium with FCS 2%. They were assessed blind to the medium type, at D2 and D30 of OC at 31°C (with medium renewal at D14), and after D2 of deswelling. Each time, the endothelial cell density (ECD) was determined using a Sambacornee endothelial analyser (Samba Technologies, Meylan, France), the quality of EC visualisation (good, average, not visible) was assessed (25 pairs). For the last two assessments a pachymetry was added and we finally compared on paired corneas: ECD, transparency, keratocytes density and number of epithelial layers on cross sections, endothelial apoptosis labeled by in situ TUNEL, and aspect of EC by scanning electron microscopy (3 pairs) Results: ECD were similar at D2. Daily EC loss during OC (–0.09±–0.89% vs –0.92±0.25%, p=0.021) and especially EC loss during deswelling was dramatically reduced with the ACF medium (+0.19±9.8% vs –18.4±9.1%, p= 0.011) although the deswelling capacities were comparable (–38±7 vs –39±3 % of deswelling, p=0.683). ECD ‘at delivery’ were dramatically higher by 40% in ACF medium (2547±520 vs 1775±355 cells/mm², p<0.001). Corneal transparencies, keratocytes densities as well as the number of epithelial layers did not differ between the two media. The ACF medium facilitated considerably the EC visualisation at each time point (100 vs 51% of good or average visualisation, p<0.001), TUNEL positives ECs were less frequent and EC surface appeared smoother with the ACF medium Conclusions: Our prototype of ACF medium proves its superiority over commercial medium with 2% FCS, regarding its ability to preserve EC viability. The innovative deswelling molecule appears far less toxic than dextran. Animal experimentation is ongoing before application to human

Keywords: cornea: endothelium • cornea: storage • clinical laboratory testing 
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