Ambient temperature storage of encapsulated cells in alginate microspheres and core–shell capsules

Abstract

The development of reliable short-term storage strategies is essential for the clinical translation of cell-based therapies in regenerative medicine, particularly in settings where cryogenic infrastructure is limited or transport distances are substantial. The aim of the research was to evaluate the influence of three-dimensional culture systems (alginate microspheres and core–shell capsules with different core compositions) and fibroblast growth factor (FGF) supplementation on the preservation of viability and metabolic activity of human bone marrow–derived mesenchymal stromal cells and HeLa cells during ambient temperature storage. MSCs and HeLa cells were encapsulated in alginate microspheres or in core–shell capsules containing culture medium, gelatin, or porcine plasma as the core component using electrospraying and coaxial electrospraying techniques. After 17 days of cultivation, constructs were stored at +22 °C for up to 7 days. Cell viability was assessed using trypan blue exclusion and fluorescent FDA/EthD-1 staining. Metabolic activity was determined by the Alamar Blue assay and normalised to baseline values at the onset of ambient storage. Statistical evaluation was performed using two-way ANOVA and mixed-effects modelling. A significant time-dependent decrease in viability and metabolic activity was observed across all groups during ambient storage (p < 0.0001); however, the extent of decline varied depending on cell type and microenvironmental conditions. Three-dimensional encapsulation markedly improved cellular stability relative to monolayer culture, particularly by Day 7. The most consistent preservation of functional parameters across both cell types was achieved in alginate microspheres and in core–shell capsules with a culture-medium core. Capsules containing gelatin or plasma cores showed greater variability and more pronounced reductions in functional readouts, especially under FGF-deprived conditions. The beneficial effect of FGF supplementation was more evident in MSCs, whereas HeLa cells exhibited limited responsiveness. The type of 3D carrier and capsule core composition are critical determinants of cell preservation during ambient storage. Alginate microspheres and culture medium–based core–shell capsules represent promising platforms for short-term storage of cell-based products under non-cryogenic conditions. FGF supplementation enhances MSC resistance to storage-associated stress and may serve as a modulatory component in optimised ambient preservation strategies.