Fabrication of perfusable 3D hepatic lobule-like constructs through assembly of multiple cell type laden hydrogel microstructures

Abstract

The in vitro reproduction of three-dimensional (3D) cellular constructs to physiologically mimic human liver is highly desired for drug screening and clinical research. However, the fabrication of a liver-mimetic 3D model using traditional bottom-up technologies is challenging owing to the complex architecture and specific functions of real liver tissue. This work proposes a versatile strategy for spatially assembling gear-like microstructures encapsulating multiple cell types, and reorganizing them into 3D lobule-like micro-architecture with physiological relevance to native liver tissue. Gear-like microstructures were fabricated by photo-crosslinking poly(ethylene glycol) diacrylate (PEGDA) hydrogel mixed with hepatocytes and fibroblasts, in a digital micromirror device (DMD)-based microfluidic channel. The microstructures were assembled through coordinated micromanipulation based on local fluid force, and spatially self-aligned through hydrophilic–hydrophobic interactions into a 3D integrated construct with lobule-like morphology and a perfusable central lumen. The resulting 3D lobule-like constructs allowed long-term co-culture of hepatocytes and fibroblasts with high cell viability. The co-cultured constructs enhanced hepatocyte proliferation and spreading, as well as liver functions including a 50% increase in albumin secretion and urea synthesis. For hepatotoxicity assessment, the 3D lobule-like construct enabled drug perfusion through its built-in lumen for simulation of drug diffusion in the liver, which could improve the response sensitivity and efficiency to hepatotoxic drug. These results demonstrated that this method provides a valuable 3D co-culture model with perfusable lobule-like architecture and physiological functions, which has potential applications in drug discovery and tissue engineering applications.

Multicellular Co-Culture in Three-Dimensional Gelatin Methacryloyl Hydrogels for Liver Tissue Engineering

“…These results demonstrated that co-culture of HepG2 cells with NIH/3T3 cells in the 3D microtissues enhanced albumin secretion and urea synthesis than mono-culture of HepG2 cells in the 3D microtissues. Moreover, during the culture period, the albumin secretion and urea synthesis of the co-cultured 3D microtissues maintained higher level than that of the PEGDA-based 3D constructs in our previous work [20]. The PEGDA-based precursor solution is thicker than the GelMA one, which means cells undergo a harsh environment during the process for fabricating cellular PEGDA micromodules, thus may cause cells injury and influence cell recovery and function expression.…”

“…However, these studies are focused on cellular sheets constructions with simple structures, which could not mimic complex 3D tissue architectures at micro scale. In our previous study, poly(ethylene glycol) diacrylate (PEGDA) hydrogel was used to fabricate 3D constructs encapsulating hepatocytes and fibroblasts to mimic liver lobules [20]. PEGDA hydrogel has strong mechanical properties for 3D assembly, but it is non-biodegradable which limits cell organization for 3D.…”

2D to 3D Manipulation and Assembly of Microstructures Using Optothermally Generated Surface Bubble Microrobots

“…For example, microstructures can be moved, rotated, and patterned by a bubble‐actuated microrobot in 2D, or by multiple light‐controlled surface bubble robots . To assemble 3D constructs, hydrodynamic forces are introduced by the floating bubble, which is then injected by a glass capillary under the cell‐laden microstructures . At the same time, a glass rod can be employed as a holder for collecting annular microcomponents, resulting in the formation of a vascular‐like microtube.…”

Magnetic Micromachine Using Nickel Nanoparticles for Propelling and Releasing in Indirect Assembly of Cell-Laden Micromodules

“…Particularly, PEGDA was modified by Arg-Gly-Asp-Ser (RGDs) to improve cell adhesion and viability. The specific procedure was introduced in References [32,33]. The fabrication of micromodules is shown in Figure 2.…”