Strategies for the hypothermic preservation of cell sheets of human adipose stem cells

Autoři: Sara Freitas-Ribeiro aff001;  Andreia Filipa Carvalho aff001;  Marina Costa aff001;  Mariana Teixeira Cerqueira aff001;  Alexandra Pinto Marques aff001;  Rui Luís Reis aff001;  Rogério Pedro Pirraco aff001
Působiště autorů: 3B's Research Group, I3Bs–Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimarães, Portugal aff001;  ICVS/3B’s–PT Government Associate Laboratory, Braga/Guimarães, Portugal aff002;  The Discoveries Centre for Regenerative and Precision Medicine, Headquarters at University of Minho, Guimarães, Portugal aff003
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222597


Cell Sheet (CS) Engineering is a regenerative medicine strategy proposed for the treatment of injured or diseased organs and tissues. In fact, several clinical trials are underway using CS-based methodologies. However, the clinical application of such cell-based methodologies poses several challenges related with the preservation of CS structure and function from the fabrication site to the bedside. Pausing cells at hypothermic temperatures has been suggested as a valuable method for short-term cell preservation. In this study, we tested the efficiency of two preservation strategies, one using culture medium supplementation with Rokepie and the other using the preservation solution Hypothermosol, in preserving human adipose stromal/stem cells (hASC) CS-like confluent cultures at 4°C, during 3 and 7 days. Both preservation strategies demonstrated excellent ability to preserve cell function during the first 3 days in hypothermia, as demonstrated by metabolic activity results and assessment of extracellular matrix integrity and differentiation potential. At the end of the 7th day of hypothermic incubation, the decrease in cell metabolic activity was more evident for all conditions. Nonetheless, hASC incubated with Rokepie and Hypothermosol retained a higher metabolic activity and extracellular matrix integrity in comparison with unsupplemented cells. Differentiation results for the later time point showed that supplementation with both Rokepie and Hypothermosol rescued adipogenic differentiation potential but only Rokepie was able to preserve hASC osteogenic potential.

Klíčová slova:

Alizarin staining – Apoptosis – Cell differentiation – Cell metabolism – Cell staining – Collagens – Flow cytometry – Necrotic cell death


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