Effect of Storage Temperature on Cultured Epidermal Cell Sheets Stored in Xenobiotic-Free Medium

Abstract

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.

Optimization of Storage Temperature for Retention of Undifferentiated Cell Character of Cultured Human Epidermal Cell Sheets

“…The slight differences between the two methods might be explained by the more qualitative estimate of trypan blue exclusion versus quantification of fluorescent signal emitted by CAM. Viability was improved compared to results in our two-week storage study 17 where cell viability values in temperature groups between 4 °C and 37 °C typically ranged between ~20% and ~60%. Cell viability was comparable at 24 °C between the two studies (~90% normalized to control).…”

“…We have previously shown that temperature has a significant impact on the quality of stored cultured cells from a variety of tissues 13 – 16 . Based on analyses of phenotype (best at 12 °C) and viability (best at 24 °C) of CES in our two-week storage study 17 , 18 , we hypothesized that 12 °C may be most promising for retention of proliferative capacity and undifferentiated cell phenotype in CES following one-week of storage. Therefore, in-depth analyses were carried out herein to compare one-week storage of CES stored at temperatures 4 °C, 8 °C, 12 °C, 16 °C, and 24 °C with non-stored control cell sheets.…”

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

“…Considering the challenges that are inherent to cryopreservation, our goal was to develop an easily implementable and inexpensive short-term storage method for cultured HOK. Previous publications on storage of cultured epithelial cells [ 16 – 21 ] demonstrated that storage temperature profoundly affects the viability, phenotype, metabolism and morphology of these cells. We hypothesized that storage temperature will similarly affect cultured HOK and conducted the present study to define the optimal temperature for storage of HOK.…”

To Serum or Not to Serum: Reduced-serum based methods for contact-based co-culture of human dermal fibroblasts (HDFa) and epidermal keratinocytes (HaCaT) for wound bed studies

“…However, the use of serum poses a discrepancy in the context of broblast and keratinocyte co-culture. The presence of serum is required to support the proliferation of broblasts, which is in contrast to keratinocytes, which grow poorly or fail to attach and grow in the presence of serum, and are therefore cultivated in serum-free media (16,(25)(26)(27)(28). To overcome this, a large number of protocols for contact-based co-culture of broblasts and keratinocytes, start with growing con uent layers of broblasts in media supplemented with high concentrations of animal serum (up to 10%), after which keratinocytes are seeded on broblast layers in serum-free media (10,11,15,16,19).…”