# Three-Dimensional-Bioprinted Liver Chips and Challenges
## 1 Introduction  
Approximately 10 years and $2.6 billion are required to bring a drug to market [1], while nearly 90% of proposed drugs fail to acquire clinical approval [2, 3]. Even after attaining clinical approval, adverse drug reactions/events (ADRs/ADEs) are prevalent. ADR/ADE refers to allergic reactions, medication errors, and overdoses. The National Medical Products Administration (NMPA) of China received 1.514 million reports of ADRs/ADEs in 2019 alone [4]. In the United States, ADEs account for virtually one-third of hospital adverse events (1 million emergency department visits per year) and prolongs hospital stays by 1.7 to 4.6 days [5]. Animal testing raises moral concerns and is not accurate in predicting the pathophysiology of human disease [6, 7]--roughly half of the preclinically approved drugs turn out to be toxic to humans [8], highlighting the importance of conducting drug testing directly on human cells [9]. Despite being widely accepted and useful, two-dimensional (2D) cell cultures face challenges in emulating in vivo-like cell bioactivity (e.g., cell morphology and cell-cell and cell-matrix interactions) [10, 11]. Sandwich culture and micro-patterning are two common 2D cell culture methods [12]. On the other hand, compared to 2D cell cultures, three-dimensional (3D) cell cultures recapitulate in vivo function, structure, and the reaction of human tissue to drugs more effectively [13]. Moreover, the 3D extracellular matrix (ECM) enhances mechano-responses, proliferation, survival, and differentiation of cells. Spheroid culture, biopolymer and prefabricated scaffolds, hydrogels, and cell sheets are the most commonly used 3D cell culture techniques [12, 14, 15]. Microfluidics allows for multiplexing biotechnological techniques and enabling applications ranging from single-cell analysis [16; 17; 18; 19; 20] to on-chip applications [21; 22]. Polymeric microfluidic chips (particularly polydimethylsiloxane (PDMS) as the most widely used material for microfluidic chip fabrication) can serve as bioreactors for 3D cell culture due to biocompatibility as well as the inertness of PDMS to aqueous solutions, temperature, and pressure encountered in cell culture [12]. Organ-on-chip (OOC) technology, the intersection of microfluidics and tissue engineering, refers to the extraction and culture of specific organ cells on a continuously perfused microfluidic chip to create cellular microenvironments in vitro with desired structural, mechanical, and fluidic properties [7; 23; 24]. Many of the OOC designs enable real-time imaging and analysis of metabolic responses of living cells in a functional organ context by mimicking (i) systematic organ interaction (e.g., drug distribution and absorption) [25; 26], (ii) tissue-level organization of parenchymal cells [27; 28], and (iii) epithelial and multicellular vascular interfaces of organs (e.g., a network of blood vessels) [6; 29; 30]. In 2004, the first report on the study of human disease or physiology at the organ level using a microfluidic chip was published [31]. This was a system for capturing the systemic interaction between the liver and lungs on a very small (one square inch) silicon chip known as a cell culture analogue (CCA) [6]. The concept of OOC was introduced in 2010 as a means to study lung function [32]. Culturing on microfluidic chips offers the possibility of programmed circulation of fluids and reagents directly on the chip to nourish the cells (e.g., oxygen) and remove waste material, which promotes the proliferation of cultured cells during long culture periods [6]. Integration of OOC platforms with sensors allows real-time monitoring of fluid pressure, cell migration, and tissue barrier integrity, as well as glucose, lactate, oxygen, and pH levels [33; 34]. OOC methods can also mimic the interaction of multiple organs on a single chip by culturing different cell types in adjacent channels separated by a porous membrane (e.g., the blood-brain barrier) [35].