High Throughput Screening of Additives Using Factorial Design to Promote Survival of Stored Cultured Epithelial Sheets

There is a need to optimize storage conditions to preserve cell characteristics during transport of cultured cell sheets from specialized culture units to distant hospitals. In this study, we aimed to explore a method to identify additives that diminish the decrease in the viability of stored undifferentiated epidermal cells using multifactorial design and an automated screening procedure. The cultured cells were stored for 7–11 days at 12°C in media supplemented with various additives. Effects were evaluated by calcein staining of live cells as well as morphology. Twenty-six additives were tested using (1) a two-level factorial design in which 10 additives were added or omitted in 64 different combinations and (2) a mixture design with 5 additives at 5 different concentrations in a total of 64 different mixtures. Automated microscopy and cell counting with Fiji enabled efficient processing of data. Significant regression models were identified by Design-Expert software. A calculated maximum increase of live cells to 37 ± 6% was achieved upon storage of cell sheets for 11 days in the presence of 6% glycerol. The beneficial effect of glycerol was shown for epidermal cell sheets from three different donors in two different storage media and with two different factorial designs. We have thus developed a high throughput screening system enabling robust assessment of live cells and identified glycerol as a beneficial additive that has a positive effect on epidermal cell sheet upon storage at 12°C. We believe this method could be of use in other cell culture optimization strategies where a large number of conditions are compared for their effect on cell viability or other quantifiable dependent variables.