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ENCONTRO COM A CIÊNCIA EM PORTUGAL - CIÊNCIA’16July 5, Centro de Congressos de Lisboa, Lisbon, Portugal
Pedro L. [email protected] Editor in Chief
DESIGNER HYDROGELS AS
3D CELL CARRIERS
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Institute for Research and Innovation in Health
Integrative ProgramsNeurobiology & Neurologic DisordersHost Interaction & ResponseCancer
400 PhDs50 Research groups
www.i3s.up.pt
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The extracellular matrix
Tobias Meckel (2014)
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3D cell carriers
3D cell carriers for regenerative
therapies
3D systems for mechanistic studies and disease models
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The promise of stem cells
Adult mesenchymal stem cells
(bone marrow, adipose tissue, cord blood, …)
Expansionpotential
Differentiation potential
+: homing to sites of injury, paracrine effects, immunomodulatory properties, …
Healthyhippie.net
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The relevance of carriers for cell therapies
Biomaterial-based deployment enhances efficacy of endothelial progenitor cells
Silva, Mooney +, PNAS (2008)
Bolus cell injection:
• Vast majority (ca. 90%) rapidly die• No control over cell fate• Other tissues may be affected• High doses required: expensive• Low efficiency of vehicles
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ECM-inspired multifunctional biomaterials
Fonseca, Granja, Barrias +, Prog Polym Sci (2014)
Natural ECM regulating cell behavior
• Biochemical features
• Cell adhesion (integrin binding)
• Proteolytic susceptibility (matrix remodeling)
• Direct and indirect signaling (chemokine binding)
• Biophysical features
• Hydration (crosslinking)
• Mesh size (pore size)
• Mechanical properties (fiber strength)
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Biomimetic cellular environmentsCell adhesion as a key feature of the ECM
Engineering extracellular matrix ligands, such as the RGD-sequence, into artificial matrices enhances functionality in terms of cell behavior
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Cell entrapment in RGD-alginate
Day 8
Day 28
High metabolic activity (MTT)
Alginate RGD-Alginate
MC3T3 cells
Day 16Day 23
F-actin
DNA
Evangelista, Granja +, Biomaterials (2007)
ECM synthesis
Cell protrusions
7750x
46400x
Collagen fibers (Day 6)
TEM
Osteocalcin secretion
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Biomimetic cellular environmentsProteolytic degradation as key feature of the ECM
Fonseca, Granja +, Prog Polym Sci (2014)
Matrix metalloproteinases (MMPs) secreted by MSC can cleave components of ECM (space for cell movement)
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Cell entrapment in RGD/PVGLIG-alginate
Alginate
15 μm
15 μm
RGD-Alginate
400 μm
F-actin Nucleus
Fonseca, Granja, Barrias +, Acta Biomater (2011)
RGD/PVGLIG-Alginate
Protease-sensitive hydrogels provide cell-driven degradation
Contrary to 2D, cell migration in 3D depends on topography, steric hindrance, matrix mechanics and proteolytic sensitivity
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In vivo matrix remodelingPVGLIG/RGD-alginate degradation and host tissue invasion
Fonseca, Granja, Barrias +, Biomacromolecules (2014)
Alginate degradation Host tissue invasion
Alginate
4 weeks subcutaneous implantation in SCID mice (2% alginate)
H&E staining: 100x Safranin-O staining; 100x
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Biomimetic cellular environmentsCell signaling as an essential feature of the ECM
Presentation of osteogenic peptides to increase the proliferation of MSCs and guide their differentiation along the osteoblastic lineage
Bioactive sequence of Osteogenic Growth Peptide (OGP): NH2-GGGYGFGG-COOH
Maia, Granja +, Acta Biomater (2013)
Immobilization within hydrogels
Name Aminoacidsequence
OGP1 GGGYGFGG Permanentshort
OGP2 GGPVG¯LIGGYGFGG Temporary
OGP3 GGGIVGPLGYGFGG Permanentlong
RGD GGGGRGDSP RGD
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Cell entrapment in RGD/PVGLIG/OGP-alginateIn vivo degradation, host tissue invasion and vascularization
Maia, Granja, Barrias +, J Control Rel (2014)
Safranin-O/Fast green-staining
1 mm
Day 28
Transplanted MSCs in OGP/PVGLIG/RGD-alginate promoted hydrogel degradation, host tissue invasion, and vascularization
10 mm
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Biomimetic cellular environmentsMatrix stiffness as a key feature of the ECM
Mechanical properties of extracellular matrices influence cell behavior
http://www.mechanobiology.nl
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Cell-driven contraction of soft hydrogels
1wt%: Lower stiffness “deformable”
2wt%: higher stiffness “elastic”
Viscoelastic properties (DMA, compression)
hMSC-driven contraction of soft 3D hydrogel
matrices (RGD-alginate)
Maia, Granja +, Acta Biomater (2014)
24h1wt%
2wt%
1.5 mm
0h 1wt%
3D context allows cells to alter their
surrounding microenvironment
(contrary to 2D)
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Expression of fibronectin and its role in the stabilization of cell-cell contacts
4h 24h
1 wt-%
1 wt-%RGD-Alg(1-100)
2 wt-%RGD-Alg(2-100)
Alginate
Maia, Granja +, Acta Biomater (2014)
Self-assembly of hMSC-ECM
(self synthesized) microtissues
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Storage modulus of IPNs of alginate-collagen I can be modulated by the extent of Cacrosslinking, without affecting cell viability or integrin receptor expression
Matrix stiffness regulates fibroblast biology
F-actinDAPI
50Pa F-actinDAPI
1200Pa
Branco da Cunha, Granja, Mooney +, Biomaterials (2014)
Independent control of matrix
stiffness
collagen-I IPNalginate
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Effect of stiffness on wound healing response
Stiffer moduli promote different wound healing genetic programs, leading to upregulation of inflammation mediators IL-10 and COX-2
IL-10
COX-2
Branco da Cunha, Granja +, Biomaterials (2014)
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3D cell culture systems
3D cell carriers for regenerative
therapies
3D systems for mechanistic studies and disease models
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Branco da Cunha, Granja, Seruca +, Lab Invest (2010)
CD44v6 as biomarker of gastric cancer
v6-encoding CD44 forms expression is restricted to transformed gastric tissues
Normal gastric mucosa
CD44v6 expression is very faint
Incomplete intestinal metaplasia
CD44v6 expression is strong and observed in >75% of the columnar cells
Low- and high-grade dysplastic lesions
CD44v6 overexpression (>25% positive neoplasic cells) and strong positivity
Primary gastric carcinomas
CD44v6 overexpression was observed in 27/43 (62.8%) primary gastric carcinomas
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AGS cells quickly recover CD44st expression once re-seeded in 2D
Loss of CD44s in 3D was independent of cell and matrix type
AGS cells gradually de novo express CD44v6 expression in 3D
Branco da Cunha, Granja +, Biomaterials (2016)
Culture dimensionality controls the expression of CD44 receptors in GC cells
AGS cells gradually lose CD44st expression in 3D (collagen-I)
CD44 expression in 2D vs. 3D
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Branco da Cunha, Granja +, Biomaterials (2016)
Matrix stiffness affects cancer cell gene expression
CD44st expression by cells entrapped in matrices (48h) decreases in with increasing stiffness
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Dimensionality provided by 3D matrices approximates the spatial, mechanical and biochemical context of a living organ, and offers new insights into how cells might act in vivo.
Biofunctionalized 3D hydrogels constitute adequate cell carriers for cell regenerative therapies and models for mechanistic and disease studies, by providing:
• Three dimensionality
• Soluble gradients
• Cell-cell and cell-matrix interaction
• Mechanical stress
• Steric limitation to cell spreading, proliferation and migration
Complex co-cultures enhance biomimicry of 3D culture systems.
Overall conclusions
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Acknowledgements
Biocarrier GroupFeb 2015
Funding:
GroupCC BarriasB SarmentoAF SousaT SantosSJ BidarraSC NevesRB PereiraBN LourençoF Araújo
FCUPP Gomes
U MinhoJM OliveiraME GomesRL ReisM Gama
U LisbonJP Conde
UNESP (BR)MA Juliano
i3SMA BarbosaR SoaresR SerucaC OliveiraF Carneiro
Harvard U (US)DJ MooneyEA Silva
U Manchester (UK)P BártoloA Bayat
U Maastricht (NL)L Moroni
AlumniSG GuerreiroM GrellierC SalgadoCB CunhaFR MaiaKB FonsecaM EvangelistaF MunarinRC AlmeidaC Pereira
Poli Milano (IT)P Petrini
U Helsinki (FI)HA Santos
U Bordeaux/INSERM (FR)J AmedéeJ-C Fricain
U Mainz (DE)CJ KirkpatrickR UngerC Brochhausen
VU Med CtrAmsterdam (NL)J van Loon
