# Three-Dimensional-Bioprinted Liver Chips and Challenges
## 3 Three-Dimensional-Bioprinted Liver-on-Chip Platforms  
[PERSON] et al. generated a 3D-bioprinted, syringe-pump driven liver-on-chip setup with continuous perfusable bioreactor for long-term culture (30 days) and analysis of human HepG2/C3A spheroids for drug toxicity (Figure 1B,C) [75]. The bioreactor was comprised of multiple PDMS and poly(methyl methacrylate) (PMMA) layers (PDMS was cast around laser cut PMMA molds) to form three chambers and a set of channels with a total volume of 2.4 mL and flow rate of 200 \(\upmu\)L.h\({}^{-1}\). While most PDMS-based microfluidic chips are permanently sealed using plasma, in this setup, layers were compressed using nuts and screws, preventing leakage of compounds, so that the setup can be disassembled in any stage of the experiment for direct scrutiny of cell culture conditions. The bioprinting bioink was composed of HepG2/C3A spheroids (191 \(\pm\) 10 \(\upmu\)m) and gelatin methacryloyl (GelMA), crosslinked using ultraviolet (UV) light (850 mW for 15 s). The drug simulation performance was analyzed using 15 mm acetaminophen to trigger a toxic response in the hepatic culture, reporting analogous performance to animal tests. During the 30-day culture period, cell number increased tenfold, which is a typical cell density range for hydrogel encapsulation. The hepatic function of the proposed setups was examined by measuring the secretion of biomarkers (albumin, ceruoplasmin, A1 AT, and transferrin). The proposed setup was able to efficiently emulate key hepatic tissue cell types, such as Kupffer, stellate, hepatocytes, and endothelial cells, distribution of different ECM-like biomaterials, and their ratios [75]. [PERSON] et al. developed a pumpless (gravity-induced), single-step 3D-bioprinted liver-on-chip setup (0.03 \(\times\) 0.1 \(\times\) 1 mm\({}^{3}\)) with a 3D liver decellularized extracellular matrix (dECM) environment for cells, two reservoirs to ensure a continuous supply of nutrients through channels, and vascular/biliary channels for supplying nutrients (upper vascular channel) and removing waste products (lower biliary channel) (flow rate in the 3D liver-on-a-chip was 25 \(\upmu\)L min\({}^{-1}\)) (Figure 1D,E) [71]. Sterilized transparent PMMA and poly(ethylene/vinyl acetate) were used as printing substrates and structural materials for the 3D liver-on-a-chip, respectively. Cell printing was performed using liver dECM bioinks and gelatin. The setup was comprised of 2 channels: a lower channel, which was printed firstly, followed by the printing of a microporous structure above the lower channel (200 \(\upmu\)m \(\times\) 500 \(\upmu\)m pore size was selected for maximum bioink retention and waste product removal). Subsequently, a HepaRG-liver dECM bioink mixture was printed on the microporous structure. Finally, after printing the second channel and reservoir, a human umbilical vein endothelial cells (HUVEC)-gelatin bioink mixture was used to fill the gaps. The structural printing was conducted under 110 \({}^{\circ}\)C and 660 kPa conditions. The accuracy of cell positioning was examined by obtaining images of the cell layer, demonstrating successful delivery of HUVECs above the HepaRG cell-laden liver dECM biobank (Figure 1E, green-labeled cells are HepaRG and red-labeled cells are HUVECs). In order to evaluate the basic function of the proposed 3D-bioprinted platform, albumin/urea secretion was compared to that of a 2D culture, pointing out a higher level of secreted albumin and urea in the 3D-bioprinted model, whereas the secretion level decreased overtime continuously in the 2D model. Moreover, the drug response of the model was examined by applying 5 mm of acetaminophen (APAP) and measuring albumin secretion, highlighting the higher drug responsiveness and sensitivity of the 3D-bioprinted liver-on-chip setup compared to the 2D model. The presence of biliary channels and multiple cell types, as well as simulating a liver-like microenvironment, were the main aspects that enhanced the functionality of this setup [71].