Effect of Storage Temperature on Structure and Function of Cultured Human Oral Keratinocytes

Purpose/Aims To assess the effect of storage temperature on the viability, phenotype, metabolism, and morphology of cultured human oral keratinocytes (HOK). Materials and Methods Cultured HOK cells were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium (MEM) at nine temperatures in approximately 4°C increments from 4°C to 37°C for seven days. Cells were characterized for viability by calcein fluorescence, phenotype retention by immunocytochemistry, metabolic parameters (pH, glucose, lactate, and O2) within the storage medium by blood gas analysis, and morphology by scanning electron microscopy and light microscopy. Results: Relative to the cultured, but non-stored control cells, a high percentage of viable cells were retained only in the 12°C and 16°C storage groups (85%±13% and 68%±10%, respectively). Expression of ABCG2, Bmi1, C/EBPδ, PCNA, cytokeratin 18, and caspase-3 were preserved after storage in the 5 groups between 4°C and 20°C, compared to the non-stored control. Glucose, pH and pO2 in the storage medium declined, whereas lactate increased with increasing storage temperature. Morphology was best preserved following storage of the three groups between 12°C, 16°C, and 20°C. Conclusion: We conclude that storage temperatures of 12°C and 16°C were optimal for maintenance of cell viability, phenotype, and morphology of cultured HOK. The storage method described in the present study may be applicable for other cell types and tissues; thus its significance may extend beyond HOK and the field of ophthalmology

List of the antibodies.

<p>List of the antibodies.</p

Calcein-acetoxymethyl ester (CAM), which exclusively stains live cells, was used to analyze cell survival.

<p>(A) Control cells were CAM positive (green). Original magnification: 200x. (B) HOK were seeded in multidishes at different concentrations and incubated for two hours to ensure attachment. The CAM reagent was added to the cells for one hour, and CAM fluorescence was measured with a microplate fluorometer (excitation/emission filter pair 485/538nm).</p

Photomicrographs showing immunostaining of (A) ABCG2 (red), (B) Bmi-1 (green), (C) C/EBPδ (red), (D) PCNA (red), (E) CK18 (green), and (F) cleaved caspase-3 (red) in HOK cultured for five days without subsequent storage (control).

<p>Photomicrographs are representative of four independent samples. Cell nuclei were counterstained with DAPI (blue). Original magnification: 200x.</p

Photomicrographs of cultured HOK stored for seven days at nine temperatures were captured with an inverted light microscope with 400x magnification (A-J).

<p>Photomicrographs are representative of two independent samples. White cross (B) shows an intercellular space.</p

List of the antibodies.

<p>The immunoreactivity was graded as 0 (undetectable), + (detectable in <1/4 of the cells), ++ (detectable in 1/4–1/2 of the cells), +++ (detectable in 1/2-3/4 of the cells) and ++++ (detectable in >3/4 of the cells). All scores were assigned at a magnification of 200x by experienced investigators.</p><p>List of the antibodies.</p

To assess the effect of storage temperature on HOK metabolism the (A) pH in the storage medium and (B) partial pressure of oxygen (pO₂ kPa) was measured by a blood gas analyzer following seven days of storage.

<p>(N = 8 for stored samples and N = 5 for samples not subjected to storage (control)). * P < 0.05 compared to control. Error bars indicate standard error of the mean.</p

Photomicrographs of cultured HOK stored for seven days at nine temperatures were captured with a scanning electron microscope with 6000x magnification (A-J).

<p>Cultured HOK cells not subjected to storage served as control. Photomicrographs are representative of two independent samples. The white arrow in (C) shows the apoptotic body. White arrow (I) shows artifacts due to sample preparation.</p

Cultured HOK were stored for seven days at nine different temperatures and viability was assessed by measuring calcein-acetoxymethyl ester (CAM) fluorescence with a microplate fluorometer.

<p>The bar chart illustrates the percentage of viable cells remaining after storage compared to control cells. (N = 5) * <i>P</i> < 0.05 compared to the control. Error bars indicate standard error of the mean.</p

Photomicrographs of cultured HOK stored for seven days at nine temperatures were captured with a scanning electron microscope with 550x magnification (A-J).

<p>Cultured HOK cells not subjected to storage served as control. Photomicrographs are representative of two independent samples. White arrow (B) shows cell shrinkage and the white arrow in (J) shows the detachment of the cells.</p