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

• Published in: PloS one 2019-10, Vol.14 (10), p.e0222597-e0222597
• Main Authors: Freitas-Ribeiro, Sara, Carvalho, Andreia Filipa, Costa, Marina, Cerqueira, Mariana Teixeira, Marques, Alexandra Pinto, Reis, Rui Luís, Pirraco, Rogério Pedro
• Format: Article
• Language: English
• Subjects: Adipocytes - cytology; Adipocytes - drug effects; Adipocytes - metabolism; Adipose Tissue - cytology; Adipose Tissue - metabolism; Adipose Tissue - surgery; Apoptosis; Biology and Life Sciences; Biomedical materials; Bone marrow; Cell culture; Cell cycle; Cell death; Cell differentiation; Cell Differentiation - drug effects; Cell- and Tissue-Based Therapy; Clinical trials; Differentiation; Extracellular matrix; Extracellular Matrix - drug effects; Extracellular Matrix - metabolism; Fabrication; Health aspects; Humans; Hypothermia; Integrity; Laboratories; Medical treatment; Methylene blue; Organ Preservation Solutions - pharmacology; Organs; Osteoblasts - cytology; Osteoblasts - drug effects; Osteoblasts - metabolism; Precision medicine; Preservation; Refrigeration - methods; Regenerative medicine; Research and Analysis Methods; Stem cells; Stem Cells - cytology; Stem Cells - drug effects; Stem Cells - metabolism; Stromal Cells - cytology; Stromal Cells - drug effects; Stromal Cells - metabolism; Structure-function relationships; Supplements; Tissue Culture Techniques - methods; Tissue engineering; Tissue Preservation - methods; Transplants & implants
• ISSN: 1932-6203; 1932-6203
• 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.