DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

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To functionalize the complete surface of the hydrogels its surface were covered with
protein solution, using 500 μL or 70μL for an area of 12 cm 2 (24x50 cm coverslips) or
2.25 cm 2 (15x15 cm coverslips), respectively. The hydrogels were incubated ON at 4 o C
to promote the crosslinking of the proteins with NHS and then were washed once with
PBS. The unreacted NHS was blocked with 1mg/mL heat-inactivated fatty-acid free BSA
(bovine serum albumin) in MEM-α for 30 minutes. BSA solution in PBS (20 mg/ml) was
inactivated in a 68°C water bath for 30 min. Hydrogels were rinsed once with PBS and
placed in a plate with MEM-α 4h to equilibrate. Hydrogels were washed with PBS once
before the cells were seeded (Adapted from Cretu, et al. 2010).
II.2.6 Differentiation protocols
II.2.6.1 adapted from Engler, et al. 2006
In order to see if the cells isolated in TCPs or in the tunable hydrogels had different
plasticity or not, hMSCs were cultured on 10% and 3% acrylamide hydrogels and in
TCP. To inhibit proliferation, cells were exposed to mitomycin C (10 mg/ml) for 2 hr
and washed three times with media prior to plating. Cells were plated at a 3000
Cells/cm 2 and let in culture for 7 days. After it, cells were fixated and labelled with anti-
O4 and anti-Nestin mouse antibodies and anti-Beta-III-Tubulin and anti-GFAP rabbit
antibodies.
II.2.7 - Rheological characterization of polyacrylamide hydrogels
The stiffness of hydrogels was determined by rheology using Kinexus Pro rheometer
and rSpace software (Malvern). The hydrogels were prepared and polymerized using a
vertical electrophoresis system with a 1mm spacer (Mini-Protean 3, BioRad) and
equilibrated overnight in PBS, following a similar protocol as for those used for cell
culture, except that the gels were not linked to coverslips neither functionalized with
protein. After zeroing the rheometer, each gel was loaded and trimmed on the bottom
plate. Then, the gap (distance between the top and bottom plates) was defined as
1mm and fine-tuned to a distance at which the gel was applied a normal force of 0.1N.