Developing an Engineered Synthetic Microenvironment for 3D Vasculogenesis

Catherine E. Oliver1, Jorge Valdez2, Linda Griffith2

1Department of Biomedical Engineering, University of Connecticut2Department 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% PFAo 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 3Do 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