DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

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2008) and also as previously established in the laboratory (Lourenço, 2012). The
percentage of acrylamide and bis-acrylamide is correlated with the stiffness of the
hydrogels and its increase enhances the Young’s moduli (Table I). The hydrogels
produced had a range of stiffness between ≈12kPa (15% Ac + 0.45% Bac) and ≈1kPa
(3% Ac + 0.2% Bac) (Table II). Being the soft ones compatible with the range of stiffness
described for the brain tissue (Moore et al., 2010) and the stiff ones for better
proliferation (Figure 16), as intended.
III.2 – Cell Adhesion to Polyacrylamide Hydrogels functionalized with
Collagen I (COL-1)
Collagen I (COL-1) is the most abundant type of collagen found in the ECM of
mammalian organisms and it is often used to coat or functionalize substrates for the
culture of a variety of cell types (Badylak, 2005). In order to culture human umbilical
cord matrix mesenchymal stem cells (hUCM-MSCs) on Polyacrylamide hydrogels
functionalized with COL-1, several concentrations of protein were tested to check in
which condition cells would show better adherence. Small protein spots (≈ 2.5mm 2 )
were created on the ≈7kPa hydrogels using 2 µl of COL-1 solutions at the following
concentrations: 3.125 µg/ml, 6.25 µg/ml, 12.5 µg/ml, 25 µg/ml and 50 µg/ml. Phasecontrast
microscopy images were taken at day 1 and day 6 after seeding the cells (at a
density of 3000 cells/cm 2 ) to assess the adhesion of MSCs during a period of 6 days
(Figure 15). We can clearly see at day 1 that the less the concentration of COL-1 used,
the less MSCs initially adhere and that more cells get a round morphology, indicating
that these cells did not attach very well to the hydrogels and consequently will detach
and/or die. At day 6 we can see that at 3.125 µg/ml there are almost no cells attached;
at 6.25 µg/ml there is an area with almost no adherent cells; at 12.50 µg/ml the spot is
more homogeneous in terms of adhesion and cells are spread and adherent, but still a
lot of free space in the spot when compared with the 25 and 50 µg/ml spots, where we
can easily see almost all the spot filled with adherent cells that present a fibroblastoidlike
healthy morphology. We selected 50 µg/ml as the most suitable protein
concentration, since it promoted the best adhesion from day 1 to day 6.