# Three-Dimensional-Bioprinted Liver Chips and Challenges
## 3 Three-Dimensional-Bioprinted Liver-on-Chip Platforms
In spite of the substantial attention given to OOC platforms, this technology still suffers from poor selectivity of different cell types in the presence of spatial heterogeneity while facing challenges in forming different types of ECM environments for cell-ECM interactions and protein absorption, primarily owing to conventional fabrication methods (e.g., micromolding, microcontact printing, and soft lithography) [78]. To address these pitfalls, PCL can be a viable candidate. PCL is a biocompatible, biodegradable, and non-toxic polyester with a low melting point (-60 \({}^{\circ}\)C) that produces less cell damage during the bioprinting process (i.e., higher cell viability) [78]. Moreover, co-bioprinting enables simultaneous printing of the microchip and cells with no need for a low-throughput secondary cell seeding step. In the co-printing method, fabrication by multiple printing materials enables the embedding of microfluidic structures and self-healing materials into the structure, facilitating tissue engineering of organs containing multiple vasculatures, ECM, and cells [85]. As one of the first attempts to co-print liver tissue on a chip, a single-step co-3D-bioprinting approach (without a secondary cell seeding process) was developed to fabricate a PCL-based 3D-printed microchip for perfused liver-on-a-chip applications [78]. PCL (to produce microchannels) and hydrogels (containing encapsulated HUVEC and HepG2 cell lines) were printed by a pneumatic 3D bioprinter with a minimum line width of 175 \(\upmu\)m and channel size of 15 mm \(\times\) 1.5 mm \(\times\) 1.5 mm. The protein absorption rates of the PCL-based bioprinted 3D channels were compared with those of soft lithographic PDMS-based channels), confirming the better performance of PCL-based channels on heterotypic cell types and biomaterials for more accurate drug screening (protein absorption depth in the PCL-based channel wall was 50 \(\upmu\)m, while absorption depth in the PDMS-based channel wall was 400 \(\upmu\)m). Nevertheless, PCL-based microfluidic channels are less optically transparent compared to PDMS channels, confining the assessment of the cell proliferation/viability rate and real-time analysis of cell interactions as well as drug responses [78]. The advent of coaxial printing and sacrificial printing allowed the fabrication of vascularized liver-on-chip platforms, enabling the study and real-time analysis of liver-specific phenomena (e.g., zonation) [77; 86]. Blood flow carries nutrients and oxygen to liver tissue, during which the partial pressure of oxygen drops as a result of progressing across the liver sinusoid (i.e., the functional unit of the liver from periportal to perivenous hepatocytes) [87]. The difference between oxygen levels in the liver tissue (known as zonation) plays a role in regulating functionality and the response of the liver to metabolic and toxic stimuli by facilitating differential metabolism. Non-perfused cell cultures suffer from oxygen scarcity since proximate cells consume a considerable portion of available oxygen, reducing the adequate presence of oxygen in cell populations away from the oxygen source [87]. Nonetheless, perfused on-chip platforms not only can distribute oxygen to different culture sites on the chip to sustain cell proliferation, but also can enable the creation of oxygen zones with different saturation levels by intentional manipulation of flow rate and oxygen levels to study the oxygen sensitivity of liver tissue [53]. In addition, liver fibrosis is a pathophysiological process caused by different insults (e.g., viral infection, alcoholism, metabolic disease, drugs, and chemical exposure), resulting in complicated HC-NPC interactions that can bring about liver stiffening, cirrhosis, and failure. However, it is difficult to reproduce a liver fibrosis model in vitro following standard approaches due to the complex factors involved in liver fibrosis.