DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

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ix I.4 – Objectives 29 Chapter II 31 II – Materials and Methods 33 II.1 - Materials 33 II.1.1 - Cell culture 33 II.1.2 – Polyacrylamide hydrogels 33 II.1.3 - Immunocytochemistry 33 II.1.4 – Biological material 34 II.1.4.1 - Umbilical Cord Samples 34 II.1.4.2 - human Mesenchymal Stem Cells (hMSCs) 34 II.2 - Methods 34 II.2.1 – Isolation of Mesenchymal Stem Cells (MSCs) from Umbilical Cord Fragments (UCFs) in Tissue Culture Plates (TCPs) and Polyacrylamide hydrogels 34 II.2.2 - Cell culture 35 II.2.3 - Cryopreservation of MSCs 35 II.2.4 - Phenotypic characterization of UCM-MSCs 35 II.2.5 - Preparation of polyacrylamide hydrogels 36 II.2.6 Differentiation protocols 38 II.2.6.1 adapted from Engler, et al. 2006 38 II.2.7 - Rheological characterization of polyacrylamide hydrogels 38 II.2.8 - Immunocytochemistry 39 II.2.9 - Statistical analysis 39 Chapter III 41 III – Results 43 III.1 – Rheological Characterization of Polyacrylamide Hydrogels 43 III.2 – Cell Adhesion to Polyacrylamide Hydrogels functionalized with Collagen I (COL-1) 44 III. 3 – hMSCs Proliferation Assay 46 III.4 – Isolation and Proliferation of MSCs from Human Umbilical Cord Isolation of hMSC from the Wharton Jelly on Polyacrylamide Hydrogels 47 III.5 - Immunophenotypic characterization of UCM-MSC 48
x III. 6 – Influence on MSCs specification by matrix elasticity 51 Chapter IV 58 IV – Discussion and Conclusion 60 Discussion 60 Conclusion 67 References 69
Page 1 and 2: DEPARTAMENTO DE CIÊNCIAS DA VIDA F
Page 3 and 4: ii Queria agradecer à minha namora
Page 5 and 6: iv isolation of hUCM-MSCs was ever
Page 7 and 8: vi Conseguimos optimizar um novo pr
Page 9: viii Table of contents Acknowledgem
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 and 56: 34 Covance and DakoCytomation, resp
Page 57 and 58: 36 clone SAM1; mouse Pe-Cy7 anti-hu
Page 59 and 60: 38 To functionalize the complete su
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41 Chapter III Results
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44 2008) and also as previously est
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12.5% Ac PBS Fold increase of hMSCs
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≈12 kPa / COL-1 +FN ≈12 kPa / C
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50 Figure 19 - Immunophenotype of U
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52 coated with COL-1 (similar to wh
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54 hydrogels of 1 and 7 kPa (for ce
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Figure 23 - hMSCs cultured on TCP (
Page 81 and 82:
60 Discussion MSCs are widely used
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62 isolating MSCs on the PA hydroge
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64 2011) and expanded on TCPs for 5
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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
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DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

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