Factorial design in regenerative medicine – high throughput screening of additives to promote survival of stored undifferentiated epidermal cells

Abstract

Purpose : Transplantation of cultured cell sheets containing a high percentage of undifferentiated epidermal cells is a promising strategy for treating limbal stem cell deficiency. For transport of cultured sheets from specialized culture units to hospitals all over the world there is a need to optimize conditions to preserve cell characteristics during storage. We aimed to identify additives that increase viability of stored undifferentiated epidermal cells using multifactorial design and an automated screening procedure for simultaneous testing of multiple additives.

Methods : Human epidermal cells were cultured to confluence and stored for 7-11 days at 12°C in storage media supplemented with various additives. The effect of storage additives was evaluated by comparing the number of live cells by calcein staining as well as morphology. Twentysix additives were tested using two experimental designs: 1) a two level factorial design in which 10 additives were added or omitted in 64 different combinations. 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.

Results : Addition of glycerol to the MEM basic storage medium increased the number of surviving cells by up to 35±6%, depending on the donor and storage time. 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 in combination with 8.8 μM fenoldopam mesylate. The beneficial effect of glycerol was shown in storage of cultured epidermal cell sheets from three different donors in two different storage media combinations for two different factorial designs.

Conclusions : We have developed a high throughput screening system enabling robust monitoring of live cells and identified glycerol as a beneficial additive for cell survival in epidermal cell sheets during storage at 12°C. The high throughput screening procedure enabled efficient monitoring of live cells. Our method should be useful also in other cell culture optimization strategies where a large number of factors are compared for their effect on cell viability.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.