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Effects of Organ Culture and Optisol-GS Storage on Structural Integrity, Phenotypes, and Apoptosis in Cultured Corneal Epithelium

¹From the Center for Eye Research, Department of Ophthalmology, the ²Department of Pathology, and the ⁵Center for Clinical Research, Ulleval University Hospital, University of Oslo, Norway; the ³Department of Oral Biology, Faculty of Dentistry, University of Oslo, Norway; and the ⁴The Apoptosis and Cell Death Research Program, Burnham Institute for Medical Research, La Jolla, California.

PURPOSE. A previous report has described the use of eye bank storage of cultured human limbal epithelial cells (HLECs) to provide a reliable source of tissue for treating limbal stem cell deficiency. In the present study, conventional organ culture (OC) storage and Optisol-GS (Bausch & Lomb, Irvine, CA) storage of cultured HLECs were compared.

METHODS. Three-week HLEC cultures were either organ cultured at 31°C or 23°C or stored in Optisol-GS at 5°C in a closed container for 1 week. Morphology was studied by light microscopy and transmission electron microscopy, and phenotypic characterization was assessed by immunohistochemistry. Apoptosis was evaluated by real-time RT-PCR microarray analysis, caspase-3 immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL).

RESULTS. The ultrastructure was preserved at 23°C, while storage at 31°C and 5°C was associated with enlarged intercellular spaces, separation of desmosomes, and detachment of epithelial cells. Cultured HLECs remained undifferentiated in all storage conditions. The expression of the antiapoptotic gene BCL2 was prominently upregulated in storage at 23°C and 5°C. Downregulation of BCL2A1, BIRC1, and TNF and upregulation of CARD6 in 23°C and 5°C storage conditions suggests a reduction in nuclear factor-B activity. No significant increase in cleaved caspase-3 and TUNEL staining was observed in response to eye bank storage, and the labeling indices of cleaved caspase-3 (range, 0.0%–4.7%) and TUNEL (range, 0.0%–7.8%) were low.

CONCLUSIONS. These data indicate that OC storage of cultured HLECs at ambient temperature is superior to OC storage at 31°C and Optisol-GS storage at 5°C and that apoptosis is minimal after eye bank storage of cultured HLECs.

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				T P Utheim, S Raeder, O A Utheim, M de la Paz, B Roald, and T Lyberg Sterility control and long-term eye-bank storage of cultured human limbal epithelial cells for transplantation Br. J. Ophthalmol., July 1, 2009; 93(7): 980 - 983. [Abstract] [Full Text] [PDF]