Electrostatic Assembly of Multiarm PEG-Based Hydrogels as Extracellular Matrix Mimics: Cell Response in the Presence and Absence of RGD Cell Adhesive Ligands

Abstract

Synthetic hydrogels have been used widely as extracellular matrix (ECM) mimics due to the ability to control and mimic physical and biochemical cues observed in natural ECM proteins such as collagen, laminin, and fibronectin. Most synthetic hydrogels are formed via covalent bonding resulting in slow gelation which is incompatible with drop-on-demand 3D bioprinting of cells and injectable hydrogels for therapeutic delivery. Herein, we developed an electrostatically crosslinked PEG-based hydrogel system for creating high-throughput 3D in vitro models using synthetic hydrogels to mimic the ECM cancer environment. A 3-arm PEG-based polymer backbone was first modified with either permanent cationic charged moieties (2-(methacryloyloxy)ethyl trimethylammonium) or permanent anionic charged moieties (3-sulfopropyl methacrylate potassium salt). The resulting charged polymers can be conjugated further with various amounts of cell adhesive RGD motifs (0, 25, 75, and 98%) to study the influences of RGD motifs on breast cancer (MCF-7) spheroid formation. Formation, stability, and mechanical properties of hydrogels were tested with, and without, RGD to evaluate the cellular response to material parameters in a 3D environment. The hydrogels can be degraded in the presence of salts at room temperature by breaking the interaction of oppositely charged polymer chains. MCF-7 cells could be released with high viability through brief exposure to NaCl solution. Flow cytometry characterization demonstrated that embedded MCF-7 cells proliferate better in a softer (60 Pa) 3D hydrogel environment compared to those that are stiffer (1160 Pa). As the stiffness increases, the RGD motif plays a role in promoting cell proliferation in the stiffer hydrogel. Flow cytometry characterization demonstrated that embedded MCF-7 cells proliferate better in a softer (60 Pa) 3D hydrogel environment compared to those that are stiffer (1160 Pa). As the stiffness increases, the RGD motif plays a role in promoting cell proliferation in the stiffer hydrogel. Additionally, cell viability was not impacted by the tested hydrogel stiffness range between 60 to 1160 Pa. Taken together, this PEG-based tuneable hydrogel system shows great promise as a 3D ECM mimic of cancer extracellular environments with controllable biophysical and biochemical properties. The ease of gelation and dissolution through salt concentration provides a way to quickly harvest cells for further analysis at any given time of interest without compromising cell viability.

Programming temporal stiffness cues within extracellular matrix hydrogels for modelling cancer niches

“…To study the effects of static ECM mechanics on disease progression in in vitro models, most studies focus on tuning the initial hydrogel properties to achieve distinct hydrogels of variable static stiffness (through modulating the density of polymer or number of crosslinks formed), and then transferring cells between matrices or comparing cellular phenotype in side-by-side hydrogels [ 13 , 31 , [58] , [59] , [60] , [61] , [62] , [63] , [64] ]. Early work utilised hydrogel systems to understand the effect of matrix stiffness on integrin adhesions, cellular organisation and growth patterns [ 65 ].…”

Electrostatically Cross-Linked Bioinks for Jetting-Based Bioprinting of 3D Cell Cultures

“…By mixing equimolar amounts of the positively charged polymers with the negatively charged polymers, the polymers instantly gelled and were compatible with the jetting printing technique. This developed hydrogel system has been shown to have high biocompatibility, instant gelation, and chemical and mechanical tunability. , Herein, a typical microvalve-based jetting bioprinting system consisting of a two-axis movable robotic platform, with a printhead comprising an automated sample loading system and multiple electromechanical dispensing microvalves was used . A graphic illustration of a drop-on-demand printing system is shown in Figure b.…”

“…This combination of anionic and cationic polymer for the bioink was shown to gelate instantly upon contact with each other as shown by the bioinks rheological properties. 22,23 By mixing equimolar amounts of the positively charged polymers with the negatively charged polymers, the polymers instantly gelled and were compatible with the jetting printing technique. This developed hydrogel system has been shown to have high biocompatibility, instant gelation, and chemical and mechanical tunability.…”

“…Recently, we have developed a physically cross-linked synthetic hydrogel system of a diblock (AB-) copolymer hydrogel system using a three-arm PEG-based polymer containing permanently charged moieties [3-sulfopropyl methacrylate potassium salt, PSPMA, or 2-(methacryloyloxy)­ethyl trimethylammonium, PMAETMA], as shown in Figure a. This combination of anionic and cationic polymer for the bioink was shown to gelate instantly upon contact with each other as shown by the bioinks rheological properties. , …”