DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

12.5% Ac PBS
Fold increase of hMSCs
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III. 3 – hMSCs Proliferation Assay
Mesenchymal stem cells (MSCs) are known to attach and proliferate on polyacrylamide
hydrogels functionalized with COL-1 (Engler et al., 2004). Hence, we decided to screen
hMSC proliferation on polyacrylamide hydrogels bearing distinct degrees of stiffness
(Figure 16). For that, hMSCs were plated at a density of 3000 cells/cm 2 on hydrogels
previously functionalized with 50 µg/ml COL-1 diluted in 1x PBS (PBS) or in acetic acid
(AA). Cells were fixed after 1 or 5 days in culture, stained with DAPI and counted using
a fluorescence microscope. We observed that proliferation was generally higher on the
≈10 kPa hydrogels, especially when COL-1 was diluted in PBS. Although there were no
statistically significant differences, this condition showed slightly increased cell
proliferation when compared to plastic (Figure 16). We also observed that in general,
proliferation in the ≈7kPa and ≈12kPa hydrogels was normally lower than on plastic,
being the lowest in the ≈7kPa hydrogels. In order to proliferate hMSCs in
polyacrylamide hydrogels, the ≈10 kPa was selected for subsequent experiments.
Day 1 Day 5
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5
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1
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Proliferation Assay of hMSCs in Polyacrylamide
Hydrogels from day 1 to day 5
≈12kPa PBS
≈12kPa AA
≈10kPa PBS
≈10kPa AA
≈7kPa PBS
≈7kPa AA
Plastic
Figure 16 – Left and right upper images: Representative fluorescence microscopy images of hMSCs plated on
polyacrylamide hydrogels with 12.5% acrylamide at day 1 (left) and 5 (right) after being fixed and stained with
DAPI (in blue). Size bar corresponds to 200µm. Bottom graphic: Proliferation assay of hMSCs in
polyacrylamide hydrogels, showing the fold increase of the number of cells from day 1 to day 5 (n=3).