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

Localization of AQP5 during development of the mouse submandibular salivary gland

Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5–18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs

Superb Winter fur insulation in small Siberian musk deer (Moschus moschiferus)

We compared the morphology and thermal characteristics of winter pelage from two Siberian musk deer Moschus moschiferus (aged 5 and 41 mo.; 5.7 and 9.5 kg) and two Eurasian reindeer Rangifer tarandus tarandus (aged >48 mo.; 73 and 79 kg).  The depth of the fur over the back of musk deer was less (approximately 30 mm) than in reindeer (approximately 40 mm).  Guard hairs of musk deer were longer (mean = 50.0 mm) and had greater diameter at half-length (mean = 314.4 μm) than those of reindeer (mean = 38.6mm and = 243.9 μm, respectively).  The thermal characteristics (thermal conductivity and resistance) of the winter pelage of the two species were nevertheless similar (0.057 W·m-1·K-1 and 0.79 K·m2·W-1; and 0.037 W·m-1 ·K-1 and 1.00 K·m2·W-1, respectively) despite a tenfold difference in their body mass

Optimization of Storage Temperature for Cultured ARPE-19 Cells

Purpose. The establishment of future retinal pigment epithelium (RPE) replacement therapy is partly dependent on the availability of tissue-engineered RPE cells, which may be enhanced by the development of suitable storage methods for RPE. This study investigates the effect of different storage temperatures on the viability, morphology, and phenotype of cultured RPE. Methods. ARPE-19 cells were cultured under standard conditions and stored in HEPES-buffered MEM at nine temperatures (4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C, and 37°C) for seven days. Viability and phenotype were assessed by a microplate fluorometer and epifluorescence microscopy, while morphology was analyzed by scanning electron microscopy. Results. The percentage of viable cells preserved after storage was highest in the 16°C group (48.7%±9.8%; P<0.01 compared to 4°C, 8°C, and 24°C–37°C; P<0.05 compared to 12°C). Ultrastructure was best preserved at 12°C, 16°C, and 20°C. Expression of actin, ZO-1, PCNA, caspase-3, and RPE65 was maintained after storage at 16°C compared to control cells that were not stored. Conclusion. Out of nine temperatures tested between 4°C and 37°C, storage at 12°C, 16°C, and 20°C was optimal for maintenance of RPE cell viability, morphology, and phenotype. The preservation of RPE cells is critically dependent on storage temperature

Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes

Purpose Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed. Materials and Methods Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR. Results: Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C. Conclusion: HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture

Metabolic measurements taken from the cell viability experiment after two weeks storage.

<p>(A) Lactate/glucose values increased with temperature (<i>r</i>  =  0.927; <i>p</i> < 0.001 between 4°C and 28°C). (B) Glucose and lactate values were significantly grouped between 8°C and 20°C (* =  significantly grouped - (glucose: <i>p</i> > 0.05; lactate: <i>p</i> > 0.05)) and values were approximately double between 24°C and 37°C (§  =  significantly grouped - (glucose: <i>p</i> > 0.05; lactate: <i>p</i> > 0.05)). (C) Oxygen tension values were inversely correlated with temperature (<i>r</i>  =  - 0.939; <i>p</i> <0.001). (D) pH fluctuated between pH 7.1 and pH 7.2 from 4°C to 20°C. Decreased pH fluctuating around pH 7.0 reflected higher lactate production between 24°C and 37°C. Values represent the average seen in n  =  4 wells of a 12 well plate at each temperature.</p

Cell viability and cell death after two weeks storage.

<p>(A) Cell viability (CAM fluorescence). Cell viability was clearly best maintained at 24°C with a value of 95.2 ± 9.91% compared to non-stored control cells (<i>p</i>  =  0.984). All other temperatures had significantly reduced cell viability compared to non-stored control cells (<i>p</i> < 0.001). (B) Dead (EthD-1 fluorescence) values (n = 4). *  =  significantly lower compared to control (<i>p</i> < 0.05) §  =  significantly higher than control, (<i>p</i> < 0.001) (n  =  4).</p

Metabolic measurements after two weeks storage showing different volumes.

<p>(A) (B) (C) Glucose and lactate concentration. (D) (E) (F) Glucose used and lactate produced (calculated from (A) (B) (C)). (A) and (D) 24 well plate volume 2mL, area 1.9 cm² (n  =  3). (B) and (E) 12 well plate volume 5.6mL, area 3.5 cm² (n  =  7). (C) and (F) 6 well plate volume 16 mL, area 9.6 cm² (n  =  3).</p