Poster T6 - Fabrication of Microvessels in High Swelling Synthetic Hydrogels

Introduction: An essential functional feature of most native tissues and the tumor microenvironment (TME) is the microvasculature. Several groups have engineered in vitro microvasculature within low swelling, natural hydrogels, using either cell-driven remodeling or via generating intricate vessel networks using microfabrication or bioprinting. But natural matrices like collagen and fibrin, face lot-to-lot variability issues, and offer limited modularity and tunability that can hinder experimental design using these models. To overcome these limitations, synthetic hydrogels have emerged as popular in vitro tissue-engineered models of the TME [1]. However, the successful integration of functional, perfusable vasculature embedded within synthetic hydrogels remains a challenge, due to the high swelling nature of synthetic hydrogels that occurs upon equilibrium hydration in confined environments. This high-swelling nature of synthetic hydrogels leads to collapse of internally fabricated structures, when cultured in a confined microfluidic environment. Efforts from other groups have focused on modifying the macromers in the synthetic hydrogels to neutralize swelling, however this may lead to limitations in tuning the stiffness and degradability of the gel [2,3]. Here, we demonstrate a new biofabrication method to generate simple microvessels embedded within a high swelling, poly (ethlyene glycol) (PEG)- based hydrogel to function as an in vitro model of microvasculature in the TME. We also demonstrate the ability to encapsulate cancer cells in the hydrogel surrounding the microvessel thereby opening the possibility to investigate the influence of hydrogel properties on cancer cell fate and subsequent influences of cancer cell fate on endothelial cell properties such as angiogenesis.

Materials and

Methods: The proteolytically-degradable PEG-diacrylate macromer, PEG-PQ-PEG (PEG-PQ), and the integrin-ligating mono-acrylated macromer, PEG-RGDS (RGDS), used in this study were synthesized as previously described [1]. All hydrogel formulations used in this study contained 5% weight/volume (w/v) PEG-PQ, 2.1 mM RGDS and were photo-crosslinked using lithium phenyl-2,4,6-trimethylbenzoylphosphinate at 3.0 mg/mL via exposure to broad spectrum UV (wavelength: 365 nm, intensity: 10 mW/cm2) for 1 min. The triple-negative breast cancer cell line, MDA-MB-231 (p231) was used for hydrogel encapsulation, while human brain microvascular endothelial cell line (hCMEC/D3) was used to form the vascular network. Fabricating the micro-vessel on device consists of the following steps: [a] pipetting the p231-laden (50,000 cells per mL) pre-polymer solutions onto the central well of a custom poly(dimethyl siloxane) (PDMS) mold and placing a 3-(trimethoxysilyl)propyl methacrylate (TMSPMA) functionalized coverslip on top; [b] threading a low-friction, medical grade GlideMed™ coated stainless steel wire through the guide channels on PDMS; [c] photo-crosslinking the p231-laden hydrogel around the wire template in the central well under UV; [d] removing the wire with device submerged in cell media to generate microchannels; [e] functionalizing the microchannels with 100 µg/mL PhotoCol® (methacrylated type-1 collagen) and allowing the hydrogel to swell to equilibrium in culture media over 24h before finally perfusing the microchannels with hCMEC/D3 cells (28 million cells/mL). The vascularized hydrogel was cultured in 24 well plates, under rocker driven flow for four days until a confluent vessel was obtained before fixation and immunostaining. Figure 1, shows the schematic for the fabricated device.

Results, Conclusions, and Discussions: Template-wire based micromolding was used to fabricate cylindrical microchannels within photopolymerizable PEG-PQ. Figure 1 (inset) shows the phase contrast image of the microchannel embedded inside the cell-laden hydrogel. We first examined multiple materials such as, uncoated or coated stainless steel, fluorocarbon and nylon for wire-templates to assess the fidelity of micromolding synthetic hydrogels. The GlideMed™ coated stainless steel resulted in the most robust fabrication of microchannels. We were able to counter the effect of high gel swelling ratio on internally fabricated structures by allowing the hydrogel to swell to equilibrium in an unconfined environment (data not shown). To evaluate the ability to engineer micro-vascular networks in our platform, we cultured human brain microvascular endothelial cells (hCMECs) within the fabricated microchannels under a rocker-driven flow. Rocker-driven flow is convenient, simple, inexpensive, compact, and high-throughput — especially compared to pump-driven systems. For the conditions used herein, the rocker produces a maximum amplitude of 1.25 dyne/cm2 and an average shear of 0.80 dyne/cm2 every 30 sec. Figure 2 depicts the microveseel stained for nuclei, F-actin and VE-cadherin (endothelial cell marker). We were able to aid the adhesion of hCMECs to the hydrogel walls via the addition of the cell-adhesive peptide (RGDS) and methacrylated collagen (PhotoCol®) functionalization. hCMECs successfully formed a confluent endothelial layer that remained viable for a minimum of 4 days. Current efforts include characterizing the patency and permeability of the microvessel as well as enabling co-culture of different cells types that are involved with microvessels such as pericytes. With this method, we have demonstrated the ability to form an engineered in vitro microvessel embedded within a cell-laden synthetic hydrogel, without having to modify the macromer backbone. This method paves the way for incorporating perfusable hydrogels into microfluidic devices via tubing, opening up exciting possibilities in fields such as organ-on-a-chip and 3D cancer modeling for drug development applications.

Acknowledgements (Optional): References
1. Farino Reyes, C. J., Pradhan, S. & Slater, J. H. The Influence of Ligand Density and Degradability on Hydrogel Induced Breast Cancer Dormancy and Reactivation. Adv. Healthc. Mater. 10, 2002227 (2021)
2. Brown, A. et al. Engineering PEG-based hydrogels to foster efficient endothelial network formation in free-swelling and confined microenvironments. Biomaterials 243, 119921 (2020).
3. Trappmann, B. et al. Matrix degradability controls multicellularity of 3D cell migration. Nat. Commun.8,371(2017).