DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral
DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral
35 al., 2009). Then, the umbilical cord matrix fragments were removed from the tissue culture plate or hydrogels and cells were detached using Trypsin-EDTA 0,05% (or 0,1% for the hydrogels)(Sigma), centrifuged, counted and seeded at the density of 3.000 cells/cm 2 in MSCs proliferation medium. II.2.2 - Cell culture MSCs were maintained as described by Leite, 2011. In detail, cells were passaged by washing with PBS 1x solution followed by dissociation and detachment using 0.05% trypsin-EDTA for 5 min at 37 degrees in the incubator. After, inhibition of trypsin using MSCs proliferation medium (with FBS), cells were centrifuged (at 290g for 5 min), resuspended, counted and seeded on tissue culture dishes at a density of 3000 cells/cm 2 in MSCs proliferation medium. The cells were maintained in a CO2 incubator at 37 o C, 5% CO2/95% air and 95% humidity. The medium was changed every 3 days. When cultured in the hydrogels, cells were left to adhere for 1 hour in a small Proliferation Medium (PM) volume (500µl on 24x50 hydrogels and 80µl on 15x15 hydrogels) and only after, the PM was added until the hydrogels and cells became submersed by PM. II.2.3 - Cryopreservation of MSCs Cells were trypsinized and collected to a conical tube. In order to determine the number of cells to freeze per vial, cells were counted and then centrifuged (at 290g for 5 minutes). The medium was then removed and the pellet was resuspended using 1 ml of FBS with 10% DMSO and transferred into cryopreservation vials. The vials were frozen at -80 o C overnight in an isopropanol cryo cooler (VWR) and then transferred into a liquid nitrogen tank. II.2.4 - Phenotypic characterization of UCM-MSCs The immunophenotypic characterization of UCM-MSCs was performed at passage 2. Cells were treated with accutase, labelled with antibodies against the indicated antigens and analyzed by flow cytometry (FACS Canto II, Becton-Dickinson). The following antibodies were used for the labeling: mouse FITC anti-human CD49e IgG2b,
36 clone SAM1; mouse Pe-Cy7 anti-human CD13 IgG1, clone WM15; mouse PE antihuman CD73 IgG1, clone AD2; mouse APC anti-human CD90 IgG1, clone 5E10; mouse PO anti-human CD45 IgG1, clone HI30; mouse PB anti-human CD11b IgG1, clone ICRF44; mouse APC-H7 anti-human HLA-DR IgG2a, clone G46-6. The staining and image acquisition and analyze II.2.5 - Preparation of polyacrylamide hydrogels Since polyacrylamide hydrogels were polymerized on top of autoclaved glass coverslips (15x15 cm or 24x50 cm), a treatment to allow the establishment of chemical covalent links between the coverslip and the hydrogel using a silane agent was necessary (adapted from Hoffecker et al., 2011). A dilution of 3-(Trimethoxysilyl) propyl methacrylate in ethanol of 1:200 was made and just before use was added 3% of diluted acetic acid (1:10 glacial acetic acid: water). This solution was placed on top of cleaned coverslips, allowed to react for 3 minutes and then the reactive coverslips were rinsed with ethanol to remove the residual reagent and dried (Hoffecker, et al. 2011). After the glass coverslips treatment with the silane agent, a saturated solution of N- Acryloxysuccinimide (NHS) was prepared in toluene and kept covered to prevent toluene evaporation. The NHS solution was used in the hydrogel composition to allow the functionalization of the hydrogel surface with covalently bound proteins to allow for cell adherence. Then, the solution of acrylamide, bis-acrylamide, water and TEMED (Tetramethylethylenediamine) was prepared according to Table I. The solution was degassed for 30 min (using a Vacuum Aspiration System from INTEGRA Biosciences). Afterwards, the solutions of NHS and ammonium persulfate (APS) were added to the hydrogel solutions and briefly mixed (adapted from Cretu, et al 2010).
- Page 5 and 6: iv isolation of hUCM-MSCs was ever
- Page 7 and 8: vi Conseguimos optimizar um novo pr
- Page 9 and 10: viii Table of contents Acknowledgem
- Page 11 and 12: x III. 6 - Influence on MSCs specif
- Page 13 and 14: xii Figure 2| Proteins related to t
- Page 15 and 16: xiv the change in distribution with
- Page 17 and 18: xvi Cells were stained with anti-B-
- Page 19 and 20: xviii List of abbreviations AA - Ac
- Page 22: 1 Chapter I Introduction
- Page 25 and 26: 4 There are many advantages in usin
- Page 27 and 28: 6 only ones MSCs can differentiate
- Page 29 and 30: Figure 1. Mechanotransduction in a
- Page 31 and 32: 10 1C)(Dogterom et al., 2005). Not
- Page 33 and 34: 12 extracellular ligand and the cyt
- Page 35 and 36: 14 Figure 3 - The Rigidity Sensing
- Page 37 and 38: 16 Figure 5 - Images depicted on A)
- Page 39 and 40: 18 Figure 7 - A) Immunofluorescence
- Page 41 and 42: 20 (GFAP) (is a class-III intermedi
- Page 43 and 44: 22 can specify lineage towards cell
- Page 45 and 46: 24 I.3 - Project rationale and expe
- Page 47 and 48: 26 Figure 12 - MSCs cultured on 1 a
- Page 49 and 50: 28 than that in soft substrate. Thi
- Page 52: 31 Chapter II - Materials and Metho
- Page 55: 34 Covance and DakoCytomation, resp
- Page 59 and 60: 38 To functionalize the complete su
- Page 62: 41 Chapter III Results
- Page 65 and 66: 44 2008) and also as previously est
- Page 67 and 68: 12.5% Ac PBS Fold increase of hMSCs
- Page 69 and 70: ≈12 kPa / COL-1 +FN ≈12 kPa / C
- Page 71 and 72: 50 Figure 19 - Immunophenotype of U
- Page 73 and 74: 52 coated with COL-1 (similar to wh
- Page 75 and 76: 54 hydrogels of 1 and 7 kPa (for ce
- Page 77: Figure 23 - hMSCs cultured on TCP (
- Page 81 and 82: 60 Discussion MSCs are widely used
- Page 83 and 84: 62 isolating MSCs on the PA hydroge
- Page 85 and 86: 64 2011) and expanded on TCPs for 5
- Page 88: 67 Conclusion In conclusion, we opt
- Page 91 and 92: 70 Cretu, A., Castagnino, P. & Asso
- Page 93 and 94: 72 McBeath, R. et al., 2004. Cell s
- Page 95: 74 Wang, N., Butler, J.P., and Ingb
35<br />
al., 2009). Then, the umbilical cord matrix fragments were removed from the tissue<br />
culture plate or hydrogels and cells were detached using Trypsin-EDTA 0,05% (or 0,1%<br />
for the hydrogels)(Sigma), centrifuged, counted and seeded at the density of 3.000<br />
cells/cm 2 in MSCs proliferation medium.<br />
II.2.2 - Cell culture<br />
MSCs were maintained as described by Leite, 2011. In detail, cells were passaged by<br />
washing with PBS 1x solution followed by dissociation and detachment using 0.05%<br />
trypsin-EDTA for 5 min at 37 degrees in the incubator. After, inhibition of trypsin using<br />
MSCs proliferation medium (with FBS), cells were centrifuged (at 290g for 5 min),<br />
resuspended, counted and seeded on tissue culture dishes at a density of 3000<br />
cells/cm 2 in MSCs proliferation medium. The cells were maintained in a CO2 incubator<br />
at 37 o C, 5% CO2/95% air and 95% humidity. The medium was changed every 3 days.<br />
When cultured in the hydrogels, cells were left to adhere for 1 hour in a small<br />
Proliferation Medium (PM) volume (500µl on 24x50 hydrogels and 80µl on 15x15<br />
hydrogels) and only after, the PM was added until the hydrogels and cells became<br />
submersed by PM.<br />
II.2.3 - Cryopreservation of MSCs<br />
Cells were trypsinized and collected to a conical tube. In order to determine the<br />
number of cells to freeze per vial, cells were counted and then centrifuged (at 290g for<br />
5 minutes). The medium was then removed and the pellet was resuspended using 1 ml<br />
of FBS with 10% DMSO and transferred into cryopreservation vials. The vials were<br />
frozen at -80 o C overnight in an isopropanol cryo cooler (VWR) and then transferred<br />
into a liquid nitrogen tank.<br />
II.2.4 - Phenotypic characterization of UCM-MSCs<br />
The immunophenotypic characterization of UCM-MSCs was performed at passage 2.<br />
Cells were treated with accutase, labelled with antibodies against the indicated<br />
antigens and analyzed by flow cytometry (FACS Canto II, Becton-Dickinson). The<br />
following antibodies were used for the labeling: mouse FITC anti-human CD49e IgG2b,
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