3D bioprinting of bicellular liver lobule-mimetic structures via microextrusion of cellulose nanocrystal-incorporated shear-thinning bioink

Abstract

Abstract3D 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.

Review on Multicomponent Hydrogel Bioinks Based on Natural Biomaterials for Bioprinting 3D Liver Tissues

“…Compared with the conventional manufacturing process for tissue engineering, 3D bioprinting enables the fabrication of tissue-mimetic constructs with the desired shapes and biofunctionalities ( Kyle et al, 2017 ; Mao et al, 2020a ; Wu et al, 2020 ). This technique enables the precise distribution of cell-laden bioinks in a layer-by-layer manner in the predefined design; thus, the printed structure can involve cell–cell and cell–matrix interactions, which are not encountered in 2D culture systems ( Seol et al, 2014 ; Mao et al, 2020a ; Wu et al, 2020 ). Additionally, the high process flexibility of 3D bioprinting affords advantages in fabricating multiscale tissue structures with various designs ( Kolesky et al, 2016 ; Gao et al, 2017 ; Skylar-Scott et al, 2019 ).…”

“…Alginate is a biopolymer found in brown seaweed or algae ( Duin et al, 2019 ; Ye et al, 2019 ; Piras and Smith, 2020 ; Sarkar et al, 2021 ). As alginate is a negatively charged polysaccharide, alginate hydrogel can be readily polymerized by reacting with multivalent cations, such as ( Lee et al, 2000 ; Kong et al, 2002 ; Jang et al, 2018 ; Duin et al, 2019 ; Ye et al, 2019 ; Piras and Smith, 2020 ; Wu et al, 2020 ; Sarkar et al, 2021 ). Moreover, alginate is biocompatible and controllable in terms of its mechanical properties and printability, thus making it applicable in several bioprinting techniques ( Suntornnond et al, 2017 ; Tai et al, 2019 ; Davoodi et al, 2020 ; Piras and Smith, 2020 ) and for fabricating various forms of tissue constructs ( Huang et al, 2012 ; Onoe et al, 2013 ; Andersen et al, 2015 ; Piras and Smith, 2020 ).…”

“…We believe that this review article can serve as an attractive reference guide for readers who aim to utilize multicomponent bioinks for liver tissue engineering. Compared with the conventional manufacturing process for tissue engineering, 3D bioprinting enables the fabrication of tissue-mimetic constructs with the desired shapes and biofunctionalities (Kyle et al, 2017;Mao et al, 2020a;Wu et al, 2020). This technique enables the precise distribution of cell-laden bioinks in a layer-by-layer manner in the predefined design; thus, the printed structure can involve cell-cell and cell-matrix interactions, which are not encountered in 2D culture systems (Seol et al, 2014;Mao et al, 2020a;Wu et al, 2020).…”

Polymeric Bioinks for 3D Hepatic Printing

“…Therefore, the developed structure served as a dual [79]. (B) Adjacent NIH/3T3 in 1% alginate, 3% nanocrystalline cellulose and 5% GelMA (135ACG) and HepG2 in 4% GelMA on day 7 (green: live cells; red: dead cells; Scale bar: 100 µm) Image reproduced with permission from [87]. (C) The macroscopic images of the digital light process printed GelMA/dECM and GelMA scaffolds in inner gear-like design (Scale bar: 500 µm).…”

“…NIH/3T3 and HepG2 spheroids were confined to their respective spaces, thereby maintaining both homotypic and heterotypic interactions (Figure 4B). The hepatic cells exhibited arrest in proliferation with enhanced liver functionality [87]. In a recent study, a GelMA-dECM polymer blend was used to print hepatic structure using a digital light process based on a bioprinting device.…”