# Three-Dimensional-Bioprinted Liver Chips and Challenges
## 1 Introduction
Linking multiple organ models via their vascular channels also creates human body-on-a-chip systems that enable studies of drug pharmacokinetics (PK) and pharmacodynamics (PD) in vitro, providing advantageous alternatives to animal models [36; 37; 38]. By using flexible side chambers, OOC is also able to simulate cyclic strains, fluid shear stresses, and mechanical compression that cells experience in vivo during heartbeat, peristalsis, and respiration [33; 39]. Three-dimensional bioprinting (a subset of 3D printing [40; 41]) is a promising method to produce 3D biomimetic cell cultures on microfluidic chips by the precise layer-by-layer placement of single/multiple cell type(s) [42; 43; 44; 45]. After bioprinting, the printed material should be able to effectively adhere to the microfluidic substrate and maintain its mechanical properties and structure (either using an external scaffold or by establishing its own extracellular matrix) during the incubation period under culture conditions (e.g., the temperature and pressure required for culture) [47]. Three common approaches for 3D bioprinting are (Figure 1A): light-induced printing (e.g., stereolithography, a high-resolution (10 \(\upmu\)m), non-contact printing method), inkjet-based printing (a fast multi-material printing method with higher cell viability compared to extrusion-based methods due to lower shear stress on the cells during printing [48]), and extrusion-based printing (where multiple print heads can be used to print the bioink and microfluidic chip simultaneously at 100 \(\upmu\)m resolution) [47; 49; 50]. While light-induced methods are costly, slow, and cause thermal damage to the cells, inkjet printing suffers from poor functionality for vertical printing causing clogging of the nuzzle, while extrusion-based bioprinting supports limited bio-material (high-viscosity material with shear-thinning capability is required) (Table 1) [48]. Fabrications methods of liver-on-chip platforms do not limit to 3D bioprinted methods, e.g., photolithography and micromolding [51]. Although these methods are well-established approaches with high resolution, they usually need a cleanroom for production and high skills in micromanufacturing. However, a discussion of the advantages and limitations of these methods, as well as materials used for these fabrication methods, are out of the scope of this perspective paper. Further information can be found elsewhere [51; 52; 53]. The liver is the main organ for filtering, detoxification, biotransformation of drugs, bile acid production, and protein synthesis in the body [71,72]. Ingested drugs hit the liver first after entering the bloodstream. Since drug-induced damage to the liver is ubiquitous, there is an urgent need for the development of in vitro liver models for preclinical and clinical testing. Liver-on-chip methods are beneficial for monitoring drug efficacy/toxicity, screening foodborne pathogens/diseases, inspecting the effects of cosmetics and dietary supplements on human organs, and understanding liver function and disease impairment (Table 2) [73,74]. Although the liver from human biopsies has a high regenerative and proliferative capacity, in conventional 2D and 3D cultures, in vitro hepatocyte functionality (e.g., albumin secretion and enzymatic activity) diminishes after being removed from the natural environment of the liver (within one week), which is due to the lack of an efficient perfusion system for proper nutrient delivery and waste product removal [6]. However, liver-on-chip platforms provide continuous perfusion through microfluidic channels, allowing a long-term culture of liver cells [75].