DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

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Covance and DakoCytomation, respectively. DAPI (4',6-Diamidino-2-phenylindole
dihydrochloride) was from Sigma Aldrich.
II.1.4 – Biological material
II.1.4.1 - Umbilical Cord Samples
Cryopreserved Umbilical Cord Fragments were obtained from pre-existing frozen
samples from Crioestaminal, S.A and from the Cell Biology Lab (Biocant). All samples
were obtained after the informed consent of the donors.
II.1.4.2 - human Mesenchymal Stem Cells (hMSCs)
hMSCs were obtained both by isolation from cryopreserved umbilical cord fragments
or from pre-existing lines from the Cell Biology Lab (Biocant), from the previous work
of Leite, 2011.
II.2 - Methods
II.2.1 – Isolation of Mesenchymal Stem Cells (MSCs) from Umbilical Cord Fragments
(UCFs) in Tissue Culture Plates (TCPs) and Polyacrylamide hydrogels
The isolation of MSCs was adapted from a protocol described by Cristiana O. Leite,
2011. The UCFs normally had to be cut from 5-3 mm long to 1-2 mm long with the help
of a scalpel and forceps. Groups of 15 to 30 fragments were transferred to 21 cm 2
tissue culture plate (TCP) or to 24x50 cm polyacrylamide hydrogels (12cm 2 ) and left to
dry for the necessary time (10 to 25 minutes) to promote the attachment of the
fragments to the plastic or hydrogel surface. Once the cord fragments were properly
attached, MSCs proliferation medium [Alpha-MEM supplemented with 10% MSCqualified
Fetal Bovine Serum (FBS), 1% Penicillin/Streptomycin and 1% Amphotericin
B], was added to the culture plate (the hydrogels were also inside a culture plate), until
all the fragments were totally immersed. The fragments were then cultured for 15 to
20 days in an incubator at 37°C with 5% CO2/95% air and 95% humidity, until MSCs
were migrating out of the UCM pieces and forming well defined colonies (Reinisch et