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Purpose: To evaluate the effect of storage temperature on the morphology, viability, cell number and metabolism of cultured human conjunctival epithelial cells (HCjEs)., Materials and Methods: Three-day cultured HCjEs were stored at nine different temperatures between 4 °C and 37 °C for four and seven days. Phenotype was assessed by immunofluorescence microscopy, morphology by scanning electron microscopy, viability and cell number by a microplate fluorometer and glucose metabolism by a blood gas analyzer., Results: Cultured cells not subjected to storage expressed the conjunctival cytokeratins 7 and 19 and the proliferation marker proliferating cell nuclear antigen. Cell morphology was best maintained following four-day storage between 12 °C and 28 °C and following 12 °C storage after seven days. Assessed by propidium iodide uptake, the percentage of viable cells after four-day storage was maintained only between 12 °C and 28 °C, whereas it had decreased in all other groups (p < 0.05; n = 4). After seven days this percentage was maintained in the 12 °C group, but it had decreased in all other groups, compared to the control (p < 0.05; n = 4). The total number of cells remaining in the cultures after four-day storage, compared to the control, had declined in all groups (p < 0.05; n = 4), except 12 °C and 20 °C groups. Following seven days this number had decreased in all groups (p < 0.01; n = 4), except 12 °C storage. Four-day storage at 12 °C demonstrated superior preservation of the number of calcein-stained viable cells (p < 0.05) and the least accumulation of ethidium homodimer 1-stained dead cells (p < 0.001), compared to storage at 4 °C and 24 °C (n = 6). The total metabolism of glucose to lactate after four-day storage was higher in the 24 °C group compared to 4 °C and 12 °C groups, as well as the control (p < 0.001; n = 3)., Conclusions: Storage at 12 °C appears optimal for preserving the morphology, viability and total cell number in stored HCjE cultures. The superior cell preservation at 12 °C may be related to temperature-associated effects on cell metabolism.

The use of amniotic membrane (AM) represents one of the major developments in ocular surface reconstruction. However, in a study on patients with primary pterygium, transplantation of AM with ex vivo expanded human conjunctival epithelial cells (HCjE) promoted earlier epithelialization than AM alone. We previously showed that cultured human limbal epithelial cells maintain their morphology, phenotype, and viability for one week when stored at 23°C. The current study investigates the feasibility of storing HCjE in HEPES-MEM and Optisol-GS at 23°C for 4 and 7 days, respectively. The five experimental groups were analyzed by light microscopy, immunohistochemistry, transmission electron microscopy, and a viability assay. The ultrastructural integrity of cultured HCjE was well preserved following 4 days of storage, however, 7 days of storage resulted in some loss of cell-cell contacts and epithelial detachment from the amniotic membrane. The number of microvilli in cultured HCjE not subjected to storage was 2.03±0.38 microvilli/μm. In comparison, after 4 and 7 days of HEPES-MEM storage this number was 1.69±0.54 microvilli/μm; P=0.98 and 0.89±1.0 microvilli/μm; P=0.28, respectively. After Optisol-GS storage for 4 and 7 days, the mean number of microvilli was 1.07±1.0 microvilli/μm; P=0.47 and 0.07±0.07 microvilli/μm; P=0.03, respectively. The number of cell layers in cultured HCjE not subjected to storage was 4.4±0.3 cell layers, as opposed to 4.0±0.9 cell layers; P=0.89 after 4 days of HEPES-MEM storage and 2.8±0.6 cell layers; P=0.01 after 7 days of storage in HEPES-MEM. The number of cell layers after 4 and 7 days of storage in Optisol-GS was 3.7±0.2 cell layers; P=0.46 and 3.4±0.4 cell layers; P=0.18, respectively. The expression of markers for undifferentiated cells (ΔNp63α, ABCG2 and p63), proliferating cells (Ki67 and PCNA), goblet cells (Ck7 and MUC5AC), stratified squamous epithelial cells (Ck4), and apoptotic cells (caspase-3) in cultured HCjE appeared to be unchanged after 4 and 7 days of HEPES-MEM and Optisol-GS storage. The percentage of viable cells in cultured HCjE not subjected to storage (91.4%±3.2%) was sustained after 4 and 7 days of storage in HEPES-MEM (94.1%±4.5%; P=0.99 and 85.1%±13.7%; P=0.87, respectively) as well as after 4 and 7 days of storage in Optisol-GS (87.7%±15.2%; P=0.97 and 79.8%±15.7%; P=0.48, respectively). We conclude that cultured HCjE may be stored for at least 4 days in serum-free conditions at 23°C while maintaining the phenotype and viability. HEPES-MEM appears to be comparable to Optisol-GS for serum-free storage with preservation of the ultrastructure for at least 4 days., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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 degrees C or 23 degrees C or stored in Optisol-GS at 5 degrees 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 degrees C, while storage at 31 degrees C and 5 degrees 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 degrees C and 5 degrees C. Downregulation of BCL2A1, BIRC1, and TNF and upregulation of CARD6 in 23 degrees C and 5 degrees C storage conditions suggests a reduction in nuclear factor-kappaB 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 degrees C and Optisol-GS storage at 5 degrees C and that apoptosis is minimal after eye bank storage of cultured HLECs.