Effect of storage temperature on cultured epidermal cell sheets stored in xenobiotic-free medium

Cultured epidermal cell sheets (CECS) are used in regenerative medicine in patients with burns, and have potential to treat limbal stem cell deficiency (LSCD), as demonstrated in animal models. Despite widespread use, short-term storage options for CECS are limited. Advantages of storage include: flexibility in scheduling surgery, reserve sheets for repeat operations, more opportunity for quality control, and improved transportation to allow wider distribution. Studies on storage of CECS have thus far focused on cryopreservation, whereas refrigeration is a convenient method commonly used for whole skin graft storage in burns clinics. It has been shown that preservation of viable cells using these methods is variable. This study evaluated the effect of different temperatures spanning 4°C to 37°C, on the cell viability, morphology, proliferation and metabolic status of CECS stored over a two week period in a xenobiotic–free system. Compared to non-stored control, best cell viability was obtained at 24°C (95.2±9.9%); reduced cell viability, at approximately 60%, was demonstrated at several of the temperatures (12°C, 28°C, 32°C and 37°C). Metabolic activity was significantly higher between 24°C and 37°C, where glucose, lactate, lactate/glucose ratios, and oxygen tension indicated increased activation of the glycolytic pathway under aerobic conditions. Preservation of morphology as shown by phase contrast and scanning electron micrographs was best at 12°C and 16°C. PCNA immunocytochemistry indicated that only 12°C and 20°C allowed maintenance of proliferative function at a similar level to non-stored control. In conclusion, results indicate that 12°C and 24°C merit further investigation as the prospective optimum temperature for short-term storage of cultured epidermal cell sheets

Transcription and microRNA Profiling of Cultured Human Tympanic Membrane Epidermal Keratinocytes

The human tympanic membrane (TM) has a thin outer epidermal layer which plays an important role in TM homeostasis and ear health. The specialised cells of the TM epidermis have a different physiology compared to normal skin epidermal keratinocytes, displaying a dynamic and constitutive migration that maintains a clear TM surface and assists in regeneration. Here, we characterise and compare molecular phenotypes in keratinocyte cultures from TM and normal skin. TM keratinocytes were isolated by enzymatic digestion and cultured in vitro. We compared global mRNA and microRNA expression of the cultured cells with that of human epidermal keratinocyte cultures. Genes with either relatively higher or lower expression were analysed further using the biostatistical tools g:Profiler and Ingenuity Pathway Analysis. Approximately 500 genes were found differentially expressed. Gene ontology enrichment and Ingenuity analyses identified cellular migration and closely related biological processes to be the most significant functions of the genes highly expressed in the TM keratinocytes. The genes of low expression showed a marked difference in homeobox (HOX) genes of clusters A and C, giving the TM keratinocytes a strikingly low HOX gene expression profile. An in vitro scratch wound assay showed a more individualised cell movement in cells from the tympanic membrane than normal epidermal keratinocytes. We identified 10 microRNAs with differential expression, several of which can also be linked to regulation of cell migration and expression of HOX genes. Our data provides clues to understanding the specific physiological properties of TM keratinocytes, including candidate genes for constitutive migration, and may thus help focus further research

Paracrine Activity from Adipose-Derived Stem Cells on in Vitro Wound Healing in Human Tympanic Membrane Keratinocytes

Stem cell therapies for tympanic membrane repair have shown initial experimental success using mesenchymal stem cells in rat models to promote healing; however, the mechanisms providing this benefit are not known. We investigated in vitro the paracrine effects of human adipose-derived stem cells (ADSCs) on wound healing mechanisms for human tympanic membrane-derived keratinocytes (hTM) and immortalized human keratinocytes (HaCaT). ADSC conditioned media (CMADSC) were assessed for paracrine activity on keratinocyte proliferation and migration, with hypoxic conditions for ADSC culture used to generate contrasting effects on cytokine gene expression. Keratinocytes cultured in CMADSC showed a significant increase in cell number compared to serum-free cultures and further significant increases in hypoxic CMADSC. Assessment of ADSC gene expression on a cytokine array showed a range of wound healing cytokines expressed and under stringent hypoxic and serum-free conditions was upregulated (VEGF A, MMP9, Tissue Factor, PAI-1) or downregulated (CXCL5, CCL7, TNF-α). Several of these may contribute to the activity of conditioned media on the keratinocytes with potential applications in TM perforation repair. VEGFA protein was confirmed by immunoassay to be increased in conditioned media. Together with gene regulation associated with hypoxia in ADSCs, this study has provided several strong leads for a stem cell–derived approach to TM wound healing

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

Cell viability comparison with PCNA, glucose use, and lactate production.

<p>(A) Correlation between cell viability and PCNA expression shows a distinct separation between high and low temperatures within the dataset (<i>r</i>  =  0.316; <i>p</i> < 0.05). (B) Cell viability was also correlated with glucose use (<i>r</i>  =  0.782; <i>p</i> < 0.001). (C) Cell viability compared to metabolic values by temperature. Values represent the average seen in n  =  4 wells of a 12 well plate at each temperature.</p

Proliferation as indicated by PCNA positive cells.

<p>(A) Representative images of PCNA staining (TRITC - red) combined with nuclear staining (DAPI – blue) showing 4°C, 12°C, 24°C and 37°C compared to non-stored control. Expression was significantly reduced at all temperatures (*  =  significantly reduced, <i>p</i> < 0.05) except at 12°C and 20°C (B); significantly higher PCNA expression was seen at 12°C with 50 ± 15% and 20°C with 46 ± 10%, where values were comparable to the control value of 57 ± 14% (<i>p</i>  =  0.983 and 0.752, respectively).</p

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