Scalable Networks of Engineered Extracellular Matrix as Biomimetic Tissue Culture Models with Defined Heterogeneity

Abstract

The extracellular matrix (ECM) is a complex amalgam of proteins and polysaccharides that actively influences cell fate via biochemical and biophysical cues. Fibronectin (Fn) is the bedrock of many native interstitial ECM, existing as a fibrillar protein network that sequesters cell signaling factors and ECM macromolecules and serves an indispensable role in ECM assembly. Despite its ubiquity in mammalian biology, it remained an outstanding challenge to recapitulate critical aspects of Fn in native tissue such as its fibrillar morphology with defined, native bioactivity in a scalable, three-dimensional construct. Our lab recently pioneered the creation of 3D fibrillar Fn networks suspended across hyper-porous polymer scaffolds on the millimeter length scale. This is achieved by shearing a Fn solution across a polymeric scaffold at the solution/air interface, which promotes the formation of robust 3D networks through hydrodynamic processes.

Native Fn fibrillogensis occurs through Fn-Fn interactions following integrin mediated stretching of solute Fn by cells. First, I investigated in vitro hydrodynamic assembly looking toward hallmarks of cell-based Fn fibrillogenesis for comparison. I found that hydrodynamically induced fibrillogenesis revealed domains of Fn that were only conformationally active in a fibrillar state and not when statically adsorbed onto a synthetic surface. Furthermore, the engineered Fn networks exhibited other notable hallmarks of cell-assembled fibronectin including stability, co-assembly with collagen-I, tissue-mimetic mechanical properties, cell-like fibrillar morphology, and native-requisites for assembly. These native like constructs are referred to as engineered extracellular matrices (EECMs).

Second, I demonstrated that the fluid shear-interface can be engineered during fibril assembly to tailor flow profiles, enabling the creation of precisely aligned or non-aligned EECMs (aEECMs and naEECMs, respectively). The aEECMs significantly influenced fibroblast fate by guiding cell orientation, increasing nuclear and cytoplasmic aspect ratio and promoting a dramatic increase in directionally persistent cell motility.

Third, by taking advantage of Fn’s conformational sensitivity, I employed a site-specific conjugation strategy to create well defined glycan-Fn conjugates using hyaluronan (HA). I demonstrated these to be tumor-mimetic and to maintain relevant bioactivity as demonstrated with domain specific Fn mAbs as well as a link-module based HA binding protein stain. Fn-HA EECMs were leveraged to study tumor cell regulation and appeared to uncover a unique molecular weight dependent cooperative and antagonist role of HA in the presence of conformationally active, fibrillar Fn. Finally, I reflect on collaborative pursuits and side projects to look toward future applications of EECMs where there is huge potential in possible industrial collaborations and new opportunities for the creation of advanced composites. EECMs are readily compatible with conventional cell culture techniques, various analysis modalities and show great promise as they are practical, definable, scalable and highly efficacious in a broad array of applications.

To date, EECMs have been shown to successfully facilitate bone regeneration, reliably expand patient tumor cells ex vivo, serve as a defined substrate for stem-cell engineered heart/brain organoids, and govern tumor cell phenotype – all whilst having great translational promise. These EECMs are continuing to be leveraged to address challenging problems in tissue regeneration and tumor microenvironment engineering, where they elucidate the broader value of engineered, proteinaceous biomaterials with defined heterogeneity.