1. Developing an Engineered Synthetic Microenvironment
for 3D Vasculogenesis
Catherine E. Oliver1, Jorge Valdez2, Linda Griffith2
1Department of Biomedical Engineering, University of Connecticut
2Department Bioengineering, Massachusetts Institute of Technology
Research completed as part of the MIT Emergent Behaviors of Integrated Cellular Systems REU program.
Introduction
2D Assay:
o Performed to learn how the individual peptides and peptide combinations affect
endothelial cell attachment
Chemistry of Bioactive Gel Solution:
o Synthetic PEG hydrogels covalently crosslinked via Michael-type addition chemistry
Preparation:
o Fixed using 4% PFA
o Stained with DAPI and phalloidin to visualize nuclei and actin, respectively
o Imaged using epi-fluorescent microscopy and quantified using Image J software
3D Assay:
o Performed to assess the overall matrix remodeling within a 3D microenvironment due to
vasculogenesis through proteolysis with encapsulated endothelial cells
Chemistry of Bioactive Gel Solution:
o Synthetic PEG hydrogels crosslinked using thiol-norbornene photochemistry
The continual enhancement of standard experimental methods to expedite the
evaluation process of new drug agents is critical to the progression of cancer therapy and
disease treatment. The scope of this project, therefore, involves developing an engineered
synthetic microenvironment for 3D vasculogenesis with an overall long-term goal of
developing physiologically relevant in vitro systems incorporating endothelial networks to
be used for drug screenings. Synthetic PEG hydrogels were used in this study due to their
advantage of modularity, a characteristic which allows for the independent control of
variables such as adhesion and stiffness. The PEG hydrogels were decorated with the
widely used peptide ligand, Arginine-Glycine-Aspartic RGD, and the novel peptide ligands,
fibronectin-derived Synergy-RGD and collagen-mimetic GFOGER, which are believed to
target α5β1 and α2β1, respectively—two integrins implicated in angiogenesis. By engaging
these specific integrins, which RGD has very low affinity towards, we hypothesize that
Synergy-RGD and GFOGER will demonstrate greater adhesive qualities leading to increased
endothelial cell attachment and vasculogenesis. Thus, the following experimentation
involved the use of Synergy-RGD and GFOGER as well as potentially synergistic peptide
combinations to study how their effects in 2D and 3D compare to RGD.
Methods
Results and Conclusions
Acknowledgements
This research was supported by the National Science Foundation Science and Technology Center
Emergent Behaviors of Integrated Cellular Systems (EBICS) Grant Number CBET-0939511.
HS-
PEG-Acrylate
HS-
HS-
-SH
-SH
Thiol cross-linker Thiol ligand
Preparation:
o Fixed using 4% PFA
o Stained with DAPI and phalloidin to visualize nuclei and actin, respectively
o Imaged using confocal microscopy and quantified using Image J software
PEG-Norbornene
HS-
-SH
Thiol cross-linker Thiol ligand
HS-
2D Results:
Figure 1. Plot of number of attached cells per cm2 versus ligand concentration (μM) comparing individual effect of RGD, Syn-RGD and
GFOGER on IPS-endothelial cell attachment.
o GFOGER has greatest effect on IPS-endo cell attachment compared to RGD and Syn-RGD
Figure 2. Plot of number of attached cells per cm2 versus ligand concentration (μM) comparing individual effect of RGD and GFOGER
to its combined effect on IPS-endothelial cell attachment.
Figure 3. Plot of number of attached cells per cm2 versus ligand concentration (μM) comparing individual effect of RGD, Syn-RGD and
GFOGER to their combined effect with a heparin-binding ligand on IPS-endothelial cell attachment.
o Combining RGD and GFOGER showed increased cell attachment compared to when used
independently
o Heparin-binding peptide shows potential to increase cell attachment when used
synergistically with RGD, Syn-RGD and GFOGER
3D Results:
Figure 4, 5 and 6. Brightfield images of RGD, Syn-RGD and GFOGER, respectively, on Day 2 of incubation within a 3D synthetic PEG-
hydrogel microenvironment at a concentration of 6M cells/ml.
o GFOGER shows highly developed vascular structure formation by Day 2 compared
to RGD and Syn-RGD
Figure 7, 8, 9 and 10. Confocal images of GFOGER on Day 2 of incubation within a 3D synthetic PEG-hydrogel microenvironment at
concentrations of 1, 4, 6 and 9M cells/ml.
o GFOGER shows most prominent vascular structure formation at 6 and 9M cells/ml
Future Work
o Development of quantitative metrics to assess vasculogenesis in 3D assays
o Study synergistic peptide combinations in 3D
o Create functional assays using microfluidic devices to show perfusability of the
networks
UV
5-7 hours
2&4. 3.1.
Inert Gel Precursor
Solution + Cells
Well Plate
5. Fix
6. Stain
7. Image
5 hours
1. 2.
Precursor
Solution
Cells
Well Plate
3. Fix
4. Stain
5. Image