DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

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Table I – Composition of the hydrogels solutions - volume added of each reagent (µL)
per one milliliter of solution.
Volume (µl)
Gel 1 - 15%
Acrylamide/
0,45% Bisacrylamide
Volume (µl)
Gel 2 - 12,5%
Acryl/ 0,37%
Bis-acryl (µl
Gel 3 - 10%
Acryl, 0,3%
Bis-acryl (µl)
Gel 4 - 3%
Acryl, 0,2% Bisacryl
(µl)
Acrylamide 40% 375 312,5 250 75
Bis-acrylamide (2%) 225 185 150 100
NHS 220 220 220 220
APS 10% 3 3 3 3
TEMED 99% 1 1 1 1
Water 176 278,5 376 601
For hydrogels polymerization, each solution (Table I) was poured between the reactive
coverslips (which had been previously adsorbed to a back glass with 1 mm spacers)
and the outer glass of the gel casting system (Mini protean 3, Bio-Rad). In this
platform, the outer glass was treated with a solution of dichlorodimethylsilane in order
to turn it hydrophobic and facilitate the polymerization of an hydrogel with a smooth
surface and its subsequent detachment from the outer glass (Engler et al., 2004). After
polymerization (30 minutes), hydrogels on treated coverslips were washed three times
with PBS, on a rocker, five minutes per wash. The sterilization was made by exposure
to UV light during 30 minutes in an air flow cabinet.
The hydrogels need to be functionalized with proteins that allow cell attachment, since
cells do not adhere to polyacrylamide hydrogels and to mimic the extracellular matrix
(ECM). The proteins were covalently bond to the surface of the hydrogels by a NHSester
crosslinking reaction (primary amines with the NHS) on the hydrogel. Collagen
type-I (COL-1) and Fibronectin (FN) were diluted in 1x PBS (when referred COL-1 was
diluted in 0.2N acid acetic, AA) at a concentration of 2.4μg/cm 2 and 10ug/ml
respectively. Combination of COL-1/FN was prepared maintaining the concentrations
as previously described.