Aqueous two-phase deposition and fibrinolysis of fibroblast-laden fibrin micro-scaffolds

Abstract

This paper describes printing of microscale fibroblast-laden matrices using an aqueous two-phase approach that controls thrombin-mediated enzymatic crosslinking of fibrin. Optimization of aqueous two-phase formulations enabled polymerization of consistent sub-microliter volumes of cell-laden fibrin. When plasminogen was added to these micro-scaffolds, the primary normal human lung fibroblasts converted it to plasmin, triggering gradual degradation of the fibrin. Time-lapse live-cell imaging and automated image analysis provided readouts of time to degradation of 50% of the scaffold as well as maximum degradation rate. The time required for degradation decreased linearly with cell number while it increased in a dose-dependent manner upon addition of TGF-β1. Fibroblasts isolated from idiopathic pulmonary fibrosis patients showed similar trends with regards to response to TGF-β1 stimulation. Addition of reactive oxygen species (ROS) slowed fibrinolysis but only in the absence of TGF-β1, consistent with published studies demonstrating that pro-fibrotic cellular phenotypes induced by TGF-β1 are mediated, at least in part, through increased production of ROS. FDA-approved and experimental anti-fibrosis drugs were also tested for their effects on fibrinolysis rates. Given the central role of fibrinolysis in both normal and pathogenic wound healing of various tissues, the high-throughput cell-mediated fibrinolysis assay described has broad applicability in the study of many different cell types and diseases. Furthermore, aqueous two-phase printing of fibrin addresses several current limitations of fibrin bio-inks, potentially enabling future applications in tissue engineering and in vitro models.

Advanced 3D In Vitro Lung Fibrosis Models: Contemporary Status, Clinical Uptake, and Prospective Outlooks

“…In this approach, fibrinogen was concentrated in the dextran phase, and after droplet formation, the PEG phase served as a reservoir of thrombin, allowing slow diffusion into the dextran phase. The fibrin degradation rate was increased with increasing cell density, while the addition of TGF-β1 decreased it in a dose-dependent manner . Their main finding was that therapeutic agents did not demonstrate a notable influence on degradation, indicating that the mechanism of action does not cure the damaged structure.…”

“…The fibrin degradation rate was increased with increasing cell density, while the addition of TGF-β1 decreased it in a dose-dependent manner. 144 Their main finding was that therapeutic agents did not demonstrate a notable influence on degradation, indicating that the mechanism of action does not cure the damaged structure. From this, we could assume that the shape of the fibrin network significantly influences the fibrinolysis rate; scaffolds with tight fibrin conformations degrade more slowly than those with loose-end conformations and thick fibers.…”

Emerging delivery systems based on aqueous two-phase systems: A review

“…Moreover, ATPSs were capable of helping treat skin wounds by simulating cells with an extracellular matrix 143 . In such a cell–matrix simulation system, the ratio of substances such as enzymes and collagen fibers could be artificially regulated and thus applied to different scenarios 144 . However, ATPSs still have many untapped potential and challenges in other organizational applications.…”

In Situ Endothelialization of Free‐Form 3D Network of Interconnected Tubular Channels via Interfacial Coacervation by Aqueous‐in‐Aqueous Embedded Bioprinting

“…[ 30–36 ] Recently, we and others demonstrate a reconfigurable aqueous‐in‐aqueous embedded bioprinting method [ 37 ] by using cyto‐mimic aqueous two‐phase systems (ATPS). [ 38–45 ] In this approach, the ultra‐low interfacial tension between ATPS, the ink and medium phase, permit the usage of low‐viscosity cell‐laden inks. [ 37 ] As such, free‐form complex architectures of cell‐laden liquid structures are facilely constructed in a single step, offering the possibility for creating tissue‐like constructs of truly arbitrary architectures.…”