Abstract
3D bioprinting of living cellular constructs with heterogeneity in cell types and extra cellular matrices (ECMs) matching those of biological tissues remains challenging. Here, we demonstrate that, through bioink material design, microextrusion-based (ME) bioprinting techniques have the potential to address this challenge. A new bioink employing alginate (1%), cellulose nanocrystal (CNC) (3%), and gelatin methacryloyl (GelMA) (5%) (namely 135ACG hybrid ink) was formulated for the direct printing of cell-laden and acellular architectures. The 135ACG ink displayed excellent shear-thinning behavior and solid-like properties, leading to high printability without cell damage. After crosslinking, the ACG gel can also provide a stiff ECM ideal for stromal cell growth. By controlling the degree of substitution and polymer concentration, a GelMA (4%) bioink was designed to encapsulate hepatoma cells (hepG2), as GelMA gel possesses the desired low mechanical stiffness matching that of human liver tissue. Four different versions of to-scale liver lobule-mimetic constructs were fabricated via ME bioprinting, with precise positioning of two different cell types (NIH/3T3 and hepG2) embedded in matching ECMs (135ACG and GelMA, respectively). The four versions allowed us to exam effects of mechanical cues and intercellular interactions on cell behaviors. Fibroblasts thrived in stiff 135ACG matrix and aligned at the 135ACG/GelMA boundary due to durotaxis, while hepG2 formed spheroids exclusively in the soft GelMA matrix. Elevated albumin production was observed in the bicellular 3D co-culture of hepG2 and NIH/3T3, both with and without direct intercellular contact, indicating that improved hepatic cell function can be attributed to soluble chemical factors. Overall, our results showed that complex constructs with multiple cell types and varying ECMs can be bioprinted and potentially useful for both fundamental biomedical research and translational tissue engineering.
Highlights
Three-dimensional (3D) printing is an additive manufacturing process that fabricates 3D architectures layer by layer with various materials, such as plastics, metal, ceramics, and p olymers[1,2,3]
To mimic an array of liver lobules to-scale, a honeycomb lattice was first printed with a bioink composed of 1% alginate, 3% cellulose nanocrystal (CNC), and 5% gelatin methacryloyl (GelMA) (135ACG)
Four different variations were 3D printed in this work, referred to as S1; S2; S3; and S4
Summary
Three-dimensional (3D) printing is an additive manufacturing process that fabricates 3D architectures layer by layer with various materials, such as plastics, metal, ceramics, and p olymers[1,2,3]. We incorporated alginate, cellulose nanocrystal (CNC), and gelatin methacryloyl (GelMA) to create a hybrid bioink (ACG) and investigated its rheological and mechanical properties. Our previous study showed that CNC/alginate hydrogel cannot provide a suitable microenvironment for cell proliferation. Naturally-derived proteins, such as gelatin, collagen, and fibrinogen, are common materials that have been utilized in tissue engineering to promote cell g rowth[20,21,22]. Among these materials, gelatin has the advantages of low cost and high p urity[23]. Our results showed that, through bioink material design, ME bioprinting techniques have enormous potential for creating 3D heterogeneous and physiologically-relevant tissue constructs
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