Fig 1.
Schematic overview of the overall methodology followed along the work.
Fig 2.
Metabolic activity and amount of dsDNA.
Confluent (A, B) and non-confluent (C, D) hASCs were preserved at 4°C, in the presence and absence (Culture medium) of hypothermic storage solutions for 3 and 7 days. A) Metabolic activity of confluent cultures in the different conditions as given by fluorescence intensity after alamar blue assay. B) Amount of dsDNA in confluent hASCs cultured in the different conditions. C) Metabolic activity of non-confluent cultures in the different conditions as given by fluorescence intensity after alamar blue assay. D) Amount of dsDNA in non-confluent hASCs cultured in the different conditions. Metabolic activity and amount of dsDNA data are presented as mean±stddev of the % of the values for, respectively, the 37°C and 0h controls and were analyzed using one-way ANOVA and Tukey’s post-tests (*p < 0.05, **p < 0.01 and ***p < 0.001).
Table 1.
Flow cytometry analysis of mesenchymal and viability markers.
Flow cytometry analysis of confluent hASCs cultured at 4°C, in the presence and absence (culture medium) of hypothermic storage solutions for 3 and 7 days. A control culture at 37°C was also performed. Values for 0 hours correspond to cells before 4°C incubation. Mesenchymal markers CD105, CD90, CD73 as well as the viability marker 7-AAD were screened.
Fig 3.
Brightfield and phalloidin staining images.
Representative images of confluent hASCs preserved at 4°C, in the presence and absence (Culture medium) of hypothermic storage solutions for 3 and 7 days. Images of control culture at 37°C was also included. Values for 0 hours correspond to cells before 4°C incubation A) Morphology assessment at the end of preservation period, and after a respective recovery period of 24 hours. B) Phalloidin staining for the actin filaments (orange) performed after cultures being subjected to a recovery period of 24 hours. Cell nuclei were counterstained with DAPI (blue). Scale bar: 50μm.
Fig 4.
Immunocytochemistry images of expression of extracellular matrix proteins.
Representative immunocytochemistry images of expression of extracellular matrix laminin, fibronectin and type I collagen (all in green) in confluent hASCs preserved at 4°C, in the presence and absence (Culture medium) of hypothermic storage solutions for 3 and 7 days. A control culture at 37°C was also included. Immunocytochemistry was performed after cultures were subjected to a respective recovery period of 24 hours. Values for 0 hours correspond to cells before 4°C incubation. Cell nuclei were counterstained with DAPI (blue). Scale bar: 50μm.
Fig 5.
Western blot analysis of extracellular matrix and caspase 3 proteins.
Western blot analysis of confluent hASCs preserved at 4°C, in the presence and absence (Culture medium) of hypothermic storage solutions for 3 and 7 days. A control culture at 37°C was also performed. The expression of extracellular matrix proteins fibronectin, type I collagen and laminin, as well as, caspase 3 activation expression was assessed. Samples were collected after cultures being subjected to a respective recovery period of 24 hours. For extracellular matrix proteins 15μl of each sample were loaded, whereas for caspase 3, 30μg were analyzed. Negative and positive controls for apoptosis, without and with mitomycin respectively, were also included to assess caspase activation. A) Ponceau S and B) β-Tubulin are shown as loading controls for fibronectin, type I collagen and laminin expression and caspase 3 activation, respectively.
Fig 6.
Differentiation potential and stained area quantification.
Representative images of the differentiation potential and stained area quantification in confluent hASCs preserved at 4°C, in the presence and absence (Culture medium) of hypothermic storage solutions for 3 and 7 days. A control culture at 37°C was also included. A) Alizarin Red S staining for mineralization during osteogenic differentiation. B) Oil Red O staining for lipid accumulation during adipogenic differentiation. Scale bar: 100μm. C) Quantification of alizarin red S stained area by ImageJ software. D) Quantification of Oil red O stained area given by ImageJ. Stained area values are presented as mean±stddev and were analyzed using one-way ANOVA and Tukey’s post-tests (*p < 0.05 and **p < 0.01).