Skip to main content
Download PDF

Liver microsystems in vitro for drug response

Journal of Biomedical Science volume 26, Article number: 88 (2019) Cite this article

Abstract

Engineering approaches were adopted for liver microsystems to recapitulate cell arrangements and culture microenvironments in vivo for sensitive, high-throughput and biomimetic drug screening. This review introduces liver microsystems in vitro for drug hepatotoxicity, drug-drug interactions, metabolic function and enzyme induction, based on cell micropatterning, hydrogel biofabrication and microfluidic perfusion. The engineered microsystems provide varied microenvironments for cell culture that feature cell coculture with non-parenchymal cells, in a heterogeneous extracellular matrix and under controllable perfusion. The engineering methods described include cell micropatterning with soft lithography and dielectrophoresis, hydrogel biofabrication with photolithography, micromolding and 3D bioprinting, and microfluidic perfusion with endothelial-like structures and gradient generators. We discuss the major challenges and trends of liver microsystems to study drug response in vitro.

Introduction

Drug development and screening is a costly and lengthy process [1, 2]. To decrease the cost and time, researchers have developed various culture systems in vitro to test drug response. With the advances of microengineering, liver microsystems, or so-called liver-on-a-chip techniques, have demonstrated diverse functions and grown vigorously. The liver microsystems in vitro mimic the conditions in vivo for reliable drug response with cells of minimum number, which relieves the demand for animal testing and decreases the duration before human clinical trials [3]. To create a microenvironment as in vivo for cell culture, various engineering tools have been developed, as shown in Fig. 1. To improve the liver cellular function and to recapitulate the cell arrangements in vivo, cell micropatterning techniques, including soft lithography and dielectrophoresis, have been demonstrated. In addition, hydrogel biofabrication techniques, such as photolithography, micromolding and three-dimensional (3D) bioprinting, provide a heterogeneous engineered extracellular matrix (ECM) that offers a 3D liver tissue to study drug response. Moreover, to reproduce the architectures of liver lobule and sinusoidal, the microfluidic perfusion culture systems use endothelial-like structures to mimic flow conditions and gradient generators to reconstruct gradients of oxygen, nutrients and metabolites. In this review, we introduce and compare several representative engineering methods established for diverse cell sources, hydrogels and bioassays to build liver microsystems in vitro to study drug response.

Fig. 1
figure 1

Liver microsystems in vitro for drug responses. Cell micropatterning techniques use soft lithography and dielectrophoresis to arrange precisely the various cells on a micrometer scale. Hydrogel biofabrication techniques apply photolithography, micromolding and 3D bioprinting to reconstruct a 3D heterogeneous extracellular matrix. Microfluidic perfusion culture systems offer endothelial-like structures to mimic flow conditions and gradient generators to reconstruct gradients of oxygen, nutrients and metabolites

Full size image

Cell micropatterning

It is difficult to maintain liver functions of primary hepatocytes in long-term monoculture conditions [4]. To solve this problem, scientists introduced micro-coculture systems with soft lithography adopted from semiconductor fabrication [5,6,7,8,9,10,11]. As shown in Fig. 2a, by soft lithography the hepatocytes, selectively attached on the micropatterned collagen, and the supporting stromal cells (fibroblasts) were further seeded to achieve effective two-dimensional (2D) cell coculture. The coculture condition greatly enhanced the secretion of albumin and urea, markers of protein synthesis and nitrogen metabolism in hepatocytes, relative to hepatocyte 2D monoculture and lasted for several weeks [5]. Moreover, with the soft lithography micropatterning technique, the ratio of fibroblasts to hepatocytes can be optimized with precise control of the area of cell adhesion, e.g., hepatocyte islands of diameter 500 μm with spacing 1200 μm center to center [6, 7]. The system is compatible with bioassays and plate readers on a bench; it has been used in tests of drug hepatotoxicity and drug-drug interactions [5]. Mitochondrial activity was evaluated using tetrazolium-(MTT)-based colorimetric assay to obtain the half-maximal inhibitory concentration (IC50) values. Furthermore, the cell micropatterning technique based on soft lithography has already been commercialized [10] and applied in pathogen studies, including hepatitis B viruses, hepatitis C viruses and plasmodium pathogens [11].

Fig. 2
figure 2

Cell-micropatterning techniques. a. Soft-lithography-based coculture microsystem compatible with bioassays on bench and plate readers [5]. b. DEP driving primary rat hepatocytes toward regions of large electric field to form cell clusters [12]. c. Array of lobule-mimetic-stellate electrodes sequentially constructing a coculture condition with DEP [13]

Full size image

Dielectrophoresis (DEP), another microengineeirng technique for cell sorting in a biocompatible hydrogel matrix or in a DEP buffer solution on applying a non-uniform electric field, has been widely investigated [12,13,14,15]. As shown in Fig. 2b, according to the design of electrode patterns, the DEP force drove hepatocytes towards regions of large electric field to form cell clusters, which facilitates the adjustment of cell organization within the 3D polyethylene-glycol (PEG) hydrogel [12]. As shown in Fig. 2c, with an appropriate operating procedure, hepatoma G2 (HepG2) and human umbilical-vein endothelial cells (HUVEC) were patterned sequentially onto a lobule-mimetic-stellate-electrode array to construct coculture conditions [13], preserving interactions cell to cell that are crucial for further enzyme induction studies [16]. The last, to provide a reusable platform for patterning cells within a 3D hydrogel and a seamless transfer, HepG2 were patterned within an agar hydrogel supported with a paper substrate, which was subsequently positioned into a 96-well plate for culture and analysis [15]. The electric conductivity of the buffer solution or hydrogel matrix must be adjusted for effective DEP actuation without heating and electrolysis [17]. For example, the conductivity of the DEP buffer solution (e.g., 10 mS/m) is much less than that of a normal cell-culture medium DMEM (Dulbecco’s Modified Eagle Medium, conductivity 1800 mS/m) [17]. The frequency of the DEP driving electric signal is another significant parameter that influences the magnitude and direction of the DEP force based on the Clausius–Mossotti factor [18].

Hydrogel biofabrication

From a tissue-engineering point of view, a 3D engineered environment with cells arranged at appropriate positions within an ECM is essential. To obtain such an engineered 3D heterogeneous liver tissue, photolithography, micromolding and 3D bioprinting for a hydrogel, the engineered ECM have been investigated. Inspired by semiconductor fabrication, photolithographic methods have been adopted to transfer the patterns from a mask to the photo-crosslinkable cell-laden hydrogels with UV crosslinking for cell culture [19,20,21,22]. The micrometer resolution is sufficient for construction of the cell environment; serial exposures make heterogeneous microstructures obtainable. The mechanical stiffness of a hydrogel can be adjusted with the exposure dosage and the concentration of the hydrogel prepolymer solution. Using digital light processing (DLP) [21], the gelatin methacryloyl (GelMA, 5%) with human induced pluripotent stem cells (hiPSC) and the GelMA (2.5%) with supporting cells were sequentially crosslinked to form a human hepatic lobule structure (Fig. 3a). Compared with a 2D cell monolayer and a 3D hepatocyte-only monoculture, the engineered liver tissue showed greater albumin, urea secretion and enzyme (cytochrome P450) activities after Rifampicin induction [21], which demonstrated the maturation in vitro of hiPSC-derived hepatic cells with liver-specific gene expressions [26]. However, the photolithographic method might be accompanied by some damage to cells caused by UV radiation and free radicals generated by the photoinitiator [27].

Fig. 3
figure 3

Hydrogel biofabrication of liver tissues. a. Photolithographic method constructing heterogeneous structures for cell coculture [21]. b. Micromolding patterning drug-encapsulated PLGA particles and cell-encapsulated hydrogels to study cancer therapy [23]. c. 3D bioprinting, injecting and curing the biomaterials to form a biomimetic tissue [24]. d. 3D liver tissue printed with a commercial 3D bioprinter [25]

Full size image

Micromolding is another way to pattern hydrogels on a microscale. Unlike photolithographic methods, micromolding is suitable for thermally and chemically crosslinkable hydrogels to avoid UV exposure damage [23, 28,29,30]. The drug-encapsulating poly (lactide-co-glycolide) (PLGA) particles patterned with micromolding were used for cancer studies [23] (Fig. 3b). As shown in the experimental results, the agents for the anti-vascular endothelial growth factor (anti-VEGF) enhanced the efficacy of chemotherapy on inhibiting the growth of endothelial cells, demonstrating a platform in vitro near that of clinical data [31]. By micromolding varied hydrogels embedded with cells and drugs, the method developed a tumor model in vitro for tests of cancer-therapy drug response.

3D printing (additive manufacturing technique) has been applied to biological and medical fields for its great flexibility; various 3D bioprinters are available on the market with diverse tissues printed [32, 33]. 3D bioprinting injects and cures the biomaterials to form a biomimetic tissue [34] and even an organ, including printed liver tissues to assess responses to clinical drugs [24, 25, 35,36,37,38,39,40,41] (Fig. 3c). As shown in Fig. 3d, a 3D printed liver tissue was used to test Trovafloxacin (antibiotic with hepatotoxicity) [25]. The 3D bioprinter can print scaffold-free liver tissue, which is composed of hepatocyte spheroid without any engineered ECM [37]. Another feature of 3D bioprinting is the core-shell structure constructed by a coaxial nozzle [39]. By the coaxial nozzle, the tissue can be printed with a shell for mechanically supporting and a suitable core for liver cell growth [40, 41]. Significant decrease of both albumin secretion and ATP production of the 3D printed liver tissue was observed at doses that induced no hepatotoxicity in standard 2D culture conditions [42], showing that the appropriately printed 3D liver tissues exhibited a greater sensitivity to drug toxicity than the 2D cultured cells [43]. However, the pressure and shear stress at the dispensing nozzle during the printing might cause harm [44]. For example, when the shear stress increased beyond 150 kPa (~ 21.8 psi), the cell viability through a bioprinting nozzle (250 μm) decreased to less than 50%. In general, using 150-μm nozzles, the acceptable dispensing pressure should be less than 10 psi [44, 45]. Although using smaller pressure or a larger nozzle decreases the shear force, the printing speed and resolution are sacrificed. Printing cells with the required resolution with minimum cell damage is hence a critical issue.

Microfluidic perfusion

Although static cell cultures are widely favored in many biological laboratories, a system for microfluidic perfusion culture provides a more biomimetic situation [46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61]. Microfluidic-based microsystems generate flow conditions as in vivo for perfusion cell culture with decreased sample usage and realize a dynamic cell culture with a continuous transfer of nutrition and metabolites. The liver sinusoidal endothelial fenestrations are special differentiations for substance exchange and protection of the hepatocytes from the shear flow of blood [62]. The artificial endothelial-like structures, made of polydimethylsiloxane (PDMS) via micromolding, reproduced the flow rates in vivo (Fig. 4a) [46] (e.g., 10 nL/min in the transport channel and 0.007 nL/min in endothelial-like structures), which retained the phenotypes and functions of primary hepatocytes [46,47,48] and even formed bile canaliculi [49]. The microfluidic system pumped and regulated various drugs of varied concentration on a single chip, which facilitated drug screening. The IC50 values evaluated from the microfluidic chip correlated with the reported median lethal dose (LD50) values in rat experiments [48]. The microfluidic systems also promoted the differentiation efficiency of stem cells to hepatic or hepatocyte-like cells [55, 56].

Fig. 4
figure 4

Microfluidic perfusion culture systems. a. Artificial endothelial-like structures mimicking the microenvironment in vivo to retain phenotypes and functions of primary hepatocytes [46]. b. Complicated model of immune response of neutrophil recruitment [57]. c. Microfluidic gradient generator to study liver zonation [59]

Full size image

Moreover, a microfluidic-based microsystem is suitable for coculture studies. With a porous membrane, microfluidic systems mimicked complicated multiple cell–cell interactions of liver sinusoidal structures [57]. The coculture with non-parenchymal cells of three kinds -- liver sinusoidal endothelial cell, Kupffer cell, hepatic stellate cell -- in a calculated shear flow (shear stress 0.1–0.5 dyn/cm2) environment enhanced albumin secretion and cytochrome P450 (CYP) enzyme activities. Stimulated by the lipopolysaccharide and neutrophil recruitment, the microfluidic system demonstrated an immune response of neutrophil adherence as a prospective drug screening platform (Fig. 4b).

Another advantage of a microfluidic system is the ability to provide a stable gradient for liver zonation as in vivo. The liver zonation is a spatial gradient of oxygen, glucose, albumin, urea and other metabolites caused by the circulation of blood. Zone 1 is rich in oxygen and nutrients, and has higher cell metabolic functions and stronger regenerative capacities, whereas the conditions of hepatocytes in zone 3 are poor and the cell regeneration ability is also weak; the hepatocytes therein are susceptible to drugs and toxic substances. The microfluidic gradient generator [59] established zonation of carbohydrate, glucose, nitrogen and xenobiotic metabolism to build a liver metabolic zonation model for zonal drug toxicity response (Fig. 4c). In this study 3-methylcholanthrene (3-MC) to induce CYP1A enzymes activities [63] was used at varied levels with a gradient generator of concentration (0–2 μM within distance 10 mm) and exposed under acetaminophen (a medicine for pain relief that has hepatotoxicity in an excessive dose [64]) to generate cell toxicity.

The drug metabolism and pharmacokinetics are pivotal points when developing new drugs. For the whole-body drug metabolism and pharmacokinetics analysis, microfluidics is the most suitable platform because it can systematically integrate multiple organs on one single chip [65, 66]. The Gut-liver microfluidic chip is developed for drug metabolism and pharmacokinetics research. The apigenin is used as the model drug and the coculture model has a higher metabolic rate than monoculture model, which is similar to animal experiments [67]. In addition, the small intestine–liver-lung microfluidic chip is used for testing three kinds of anticancer drugs (epirubicine, irinotecan, and cyclophosphamide). The anticancer drugs act on the target cells shows that this platform can replicate the in vivo pharmacokinetic [68]. Besides that, the liver-kidney microfluidic chip is applied to study hepatotoxicity and nephrotoxicity of drug metabolites [69, 70]. The microfluidic provides a drug screening platform for multiple organs.

Comparisons

Table 1 compares the engineering methods, corresponding drug-response studies, advantages and disadvantages to achieve liver microsystems in vitro. As liver is the main detoxifying organ in a human body, the drug hepatotoxicity is important and can be studied with live microsystems in vitro. To evaluate the toxicities at varied drug concentration, cell viability, albumin secretion and IC50 are common factors to quantify hepatotoxicity. For various purposes, such as drug hepatotoxicity, drug-drug interactions, metabolic function and enzyme induction, various drugs were applied.

Table 1 Summary of liver microsystems in vitro
Full size table

In addition, the level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum are also indicators of liver damages and the ratio of AST/ALT is useful in the diagnosis of liver disease [71, 72]. For the microsystem, the AST level in the cell culture medium is measured to evaluate cell injury level [70]. Although the use of ALT or AST as an indicator of liver damage is rare in the field of the liver microsystem, it is still an important way to evaluate hepatitis. As the main organ for drug metabolism, liver plays a crucial role in eliminating many therapeutic drugs. Among the most important drug-metabolizing enzymes is cytochrome 450, a family of enzymes that function as monooxygenases, which are mostly found in the liver [73]. Some of the in vitro live microsystems have demonstrated better enzyme expression or metabolic activities compared to conventional methods [5, 13, 21, 25, 36, 48, 53].

The cells and hydrogels used in the engineering methods are also highlighted. The liver is composed of orderly-aligned hepatocytes and non-parenchymal cells within ECM. Hydrogels, such as collagen [5,6,7,8,9,10,11, 13, 35], agarose [12, 15], PEG [12, 19, 23] and GelMA [19, 21, 24, 28], are widely used in liver microsystems as the engineered ECM [74,75,76] to support the initial growth of cells. In studies of drug response, the source of hepatocytes and the cell types of non-parenchymal cells are crucial [75, 77, 78]. Through the progress of biotechnology, the hepatocytes can be obtained from isolation of human or animal liver cell, stem-cell differentiation and cell line development [2, 3, 79, 80]. For the preclinical research on drugs, the primary cells isolated from a human being or an animal have greater physiological relevance and retain a high level of enzyme activity, phenotype and function [2, 3], but the primary hepatocytes are difficult to obtain and to maintain liver function during long-term culture [2, 3]. Coculture with fibroblasts or other stromal cells is hence widely adopted for long-term culture of primary hepatocytes [5,6,7,8, 10, 11, 20, 30, 35]. Hepatocyte derived from stem cells offers a patient-specific cell source for research on liver drug response in vitro [81, 82], but the differentiation and culture of stem cells is more challenging [83]. Despite a low sensitivity to drugs and loss of some phenotypes, cell lines derived from liver tumors are commonly used in an early stage of microsystem development [12,13,14,15, 19, 22,23,24, 28, 36] for the accessibility and capability of multiple passages [84,85,86].

Conclusions and future trends

We summarize the possibilities and the limitations of liver microsystems in vitro based on engineering methods of cell micropatterning, hydrogel biofabrication and microfluidic perfusion. As mentioned above, the cell-micropatterning techniques focus on patterning cells on a scale of a few micrometers and hydrogel biofabrication focuses on biomaterial patterns on a scale of tens or hundreds of micrometers. Soft lithography is compatible with traditional on-bench bioassays and has been used to test many drugs and even as foreign pathogen models. However, the 2D cell culture has a cell morphology different from conditions in vivo; the usage of fibroblasts is not physiologically identical with non-parenchymal cell types [75]. As for DEP patterning, a non-uniform electric field can pattern cells with a resolution of a few micrometers, but the critical conductivity of the environmental liquid limits its applications. The micropatterned coculture microsystems of hepatocytes are well established, but there are still limitations on forming biomimetic tissues [76].

Hydrogel biofabrication, such as photolithography, micromolding and 3D bioprinting, provides appropriate 3D heterogeneous biomaterial architectures for the corresponding cell types. The 3D cell culture is, in general, more physiologically related to conditions in vivo than 2D cell culture [75]. Photolithography has limitations on material selectivity and UV damage [27], but it can achieve a patterning scale smaller than micromolding and 3D bioprinting [87]. Micromolding can achieve a complicated architecture on stacking the building blocks with diverse geometry [88], but it is less flexible than 3D bioprinting that can directly print a biomaterial in a 3D space. The main challenge of 3D bioprinting liver tissue is that the hepatocyte must bear the pressure and shear stress during the printing [44, 45]. Although a small pressure or big nozzle might be used, the printing speed or the resolution is sacrificed.

The major advantage of microfluidic perfusion culture systems to study liver drug response is the continuous-flow culture environment. To protect the hepatocyte from the flow shear force and to provide a perfused cultured environment as in vivo, the pillar structures and the porous membranes made with polymers are used to mimic the endothelium function, which helps to retain the phenotype and function of the primary hepatocyte and even to form bile canaliculi. With the designed microchannels and automation, a microfluidic system can simultaneously handle drugs of multiple types with varied concentrations, which can realize high-throughput drug screening with a small sample and drug volume. Using primary hepatocytes as the cell source, it can decrease the cell amounts and increase the efficiency of drug screening, which has a great potential to realize personal precision medicine. For the reconstruction of liver zonation in vitro, the gradient generator is facilitated to create a nutrition and metabolic gradient, which is a physiological model that can clarify the zonal drug metabolism.

In sum, we need a powerful tool that can pattern biomaterials and cells on various scales in 3D and can perform drug testing with fluid control on a microscale. With its ability to build complicated tissue and precise fluid control with great flexibility, a multifunctional microsystem might be a solution of next-generation liver microsystems in vitro to study drug response.

Availability of data and materials

Not applicable.

References

  1. Lombardino JG, Lowe JA III. A guide to drug discovery: the role of the medicinal chemist in drug discovery—then and now. Nat Rev Drug Discov. 2004;3:853.

    Article  CAS  PubMed  Google Scholar 

  2. Khetani SR, Berger DR, Ballinger KR, Davidson MD, Lin C, Ware BR. Microengineered liver tissues for drug testing. J Lab Automation. 2015;20:216–50.

    Article  CAS  Google Scholar 

  3. Soldatow VY, LeCluyse EL, Griffith LG, Rusyn I. In vitro models for liver toxicity testing. Toxicol Res. 2013;2:23–39.

    Article  CAS  Google Scholar 

  4. Hewitt NJ, Gómez Lechón MJ, Houston JB, Hallifax D, Brown HS, Maurel P, Kenna JG, Gustavsson L, Lohmann C, Skonberg C, Guillouzo A, Tuschl G, Li AP, LeCluyse E, Groothuis GMM, Hengstler JG. Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab Rev. 2007;39:159–234.

    Article  CAS  PubMed  Google Scholar 

  5. Khetani SR, Bhatia SN. Microscale culture of human liver cells for drug development. Nat Biotechnol. 2008;26:120.

    Article  CAS  PubMed  Google Scholar 

  6. Bhatia SN, Balis UJ, Yarmush ML, Toner M. Microfabrication of hepatocyte/fibroblast co-cultures: role of homotypic cell interactions. Biotechnol Prog. 1998;14:378–87.

    Article  CAS  PubMed  Google Scholar 

  7. Bhatia SN, Balis UJ, Yarmush ML, Toner M. Effect of cell–cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells. FASEB J. 1999;13:1883–900.

    Article  CAS  PubMed  Google Scholar 

  8. Cho CH, Park J, Tilles AW, Berthiaume F, Toner M, Yarmush ML. Layered patterning of hepatocytes in co-culture systems using microfabricated stencils. Biotechniques. 2010;48:47–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wang Y, Su W, Wang L, Jiang L, Liu Y, Hui L, Qin J. Paper supported long-term 3D liver co-culture model for the assessment of hepatotoxic drugs. Toxicol Res. 2018;7:13–21.

    Article  CAS  Google Scholar 

  10. Ramsden D, Zhou J, Tweedie DJ. Determination of a degradation constant for CYP3A4 by direct suppression of mRNA in a novel human hepatocyte model, HepatoPac. Drug Metabol Dispos. 2015;43:1307–15.

    Article  CAS  Google Scholar 

  11. March S, Ramanan V, Trehan K, Ng S, Galstian A, Gural N, Scull MA, Shlomai A, Mota MM, Fleming HE. Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens. Nat Protoc. 2015;10:2027.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Albrecht DR, Underhill GH, Wassermann TB, Sah RL, Bhatia SN. Probing the role of multicellular organization in three-dimensional microenvironments. Nat Methods. 2006;3:369.

    Article  CAS  PubMed  Google Scholar 

  13. Ho C-T, Lin R-Z, Chen R-J, Chin C-K, Gong S-E, Chang H-Y, Peng H-L, Hsu L, Yew T-R, Chang S-F, Liu C-H. Liver-cell patterning lab chip: mimicking the morphology of liver lobule tissue. Lab Chip. 2013;13:3578–87.

    Article  CAS  PubMed  Google Scholar 

  14. Puttaswamy SV, Sivashankar S, Chen R-J, Chin C-K, Chang H-Y, Liu C-H. Enhanced cell viability and cell adhesion using low conductivity medium for negative dielectrophoretic cell patterning. Biotechnol J. 2010;5:1005–15.

    Article  CAS  PubMed  Google Scholar 

  15. Macdonald N, Menachery A, Reboud J, Cooper J. Creating tissue on chip constructs in microtitre plates for drug discovery. RSC Adv. 2018;8:9603–10.

    Article  CAS  Google Scholar 

  16. Kim K, Utoh R, Ohashi K, Kikuchi T, Okano T. Fabrication of functional 3D hepatic tissues with polarized hepatocytes by stacking endothelial cell sheets in vitro. J Tissue Eng Regen Med. 2017;11:2071–80.

    Article  CAS  PubMed  Google Scholar 

  17. Ho C-T, Lin R-Z, Chang W-Y, Chang H-Y, Liu C-H. Rapid heterogeneous liver-cell on-chip patterning via the enhanced field-induced dielectrophoresis trap. Lab Chip. 2006;6:724–34.

    Article  CAS  PubMed  Google Scholar 

  18. Jones TB. Electromechanics of particles; 2005.

    Google Scholar 

  19. Zamanian B, Masaeli M, Nichol JW, Khabiry M, Hancock MJ, Bae H, Khademhosseini A. Interface-directed self-assembly of cell-laden microgels. Small. 2010;6:937–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Liu Y, Zhang L, Wei J, Yan S, Yu J, Li X. Promoting hepatocyte spheroid formation and functions by coculture with fibroblasts on micropatterned electrospun fibrous scaffolds. J Mater Chem B. 2014;2:3029–40.

    Article  CAS  PubMed  Google Scholar 

  21. Ma X, Qu X, Zhu W, Li Y-S, Yuan S, Zhang H, Liu J, Wang P, Lai CSE, Zanella F. Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting. Proc Natl Acad Sci. 2016;113:2206–11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Grix T, Ruppelt A, Thomas A, Amler A-K, Noichl B, Lauster R, Kloke L. Bioprinting perfusion-enabled liver equivalents for advanced organ-on-a-chip applications. Genes. 2018;9:176.

    Article  PubMed Central  CAS  Google Scholar 

  23. Lee W, Park J. The design of a heterocellular 3D architecture and its application to monitoring the behavior of cancer cells in response to the spatial distribution of endothelial cells. Adv Mater. 2012;24:5339–44.

    Article  CAS  PubMed  Google Scholar 

  24. Tamayol A, Najafabadi AH, Aliakbarian B, Arab-Tehrany E, Akbari M, Annabi N, Juncker D, Khademhosseini A. Hydrogel templates for rapid manufacturing of bioactive fibers and 3D constructs. Adv Healthc Mater. 2015;4:2146–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Nguyen DG, Funk J, Robbins JB, Crogan-Grundy C, Presnell SC, Singer T, Roth AB. Bioprinted 3D primary liver tissues allow assessment of organ-level response to clinical drug induced toxicity in vitro. PLoS One. 2016;11:e0158674.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Gieseck RL III, Hannan NR, Bort R, Hanley NA, Drake RA, Cameron GW, Wynn TA, Vallier L. Maturation of induced pluripotent stem cell derived hepatocytes by 3D-culture. PLoS One. 2014;9:e86372.

    Article  PubMed  CAS  Google Scholar 

  27. Billiet T, Vandenhaute M, Schelfhout J, Van Vlierberghe S, Dubruel P. A review of trends and limitations in hydrogel-rapid prototyping for tissue engineering. Biomaterials. 2012;33:6020–41.

    Article  CAS  PubMed  Google Scholar 

  28. Bhise NS, Manoharan V, Massa S, Tamayol A, Ghaderi M, Miscuglio M, Lang Q, Zhang YS, Shin SR, Calzone G. A liver-on-a-chip platform with bioprinted hepatic spheroids. Biofabrication. 2016;8:014101.

    Article  PubMed  CAS  Google Scholar 

  29. Zhang B, Montgomery M, Chamberlain MD, Ogawa S, Korolj A, Pahnke A, Wells LA, Massé S, Kim J, Reis L. Biodegradable scaffold with built-in vasculature for organ-on-a-chip engineering and direct surgical anastomosis. Nat Mater. 2016;15:669.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Stevens KR, Scull MA, Ramanan V, Fortin CL, Chaturvedi RR, Knouse KA, Xiao JW, Fung C, Mirabella T, Chen AX, McCue MG, Yang MT, Fleming HE, Chung K, Jong YP, Chen CS, Rice CM, Bhatia SN. In situ expansion of engineered human liver tissue in a mouse model of chronic liver disease. Sci Transl Med. 2017;9:eaah5505.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Kerbel RS, Kamen BA. The anti-angiogenic basis of metronomic chemotherapy. Nat Rev Cancer. 2004;4:423.

    Article  CAS  PubMed  Google Scholar 

  32. Moroni L, Burdick JA, Highley C, Lee SJ, Morimoto Y, Takeuchi S, Yoo JJ. Biofabrication strategies for 3D in vitro models and regenerative medicine. Nat Rev Mater. 2018;3:21–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Murphy SV, Atala A. 3D bioprinting of tissues and organs. Nat Biotechnol. 2014;32:773.

    Article  CAS  PubMed  Google Scholar 

  34. Pati F, Jang J, Ha D-H, Kim SW, Rhie J-W, Shim J-H, et al. Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink. Nat Commun. 2014;5:3935.

    Article  CAS  PubMed  Google Scholar 

  35. Lee JW, Choi Y-J, Yong W-J, Pati F, Shim J-H, Kang KS, Kang I-H, Park J, Cho D-W. Development of a 3D cell printed construct considering angiogenesis for liver tissue engineering. Biofabrication. 2016;8:015007.

    Article  PubMed  CAS  Google Scholar 

  36. Lewis PL, Green RM, Shah RN. 3D-printed gelatin scaffolds of differing pore geometry modulate hepatocyte function and gene expression. Acta Biomater. 2018;69:63–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kizawa H, Nagao E, Shimamura M, Zhang G, Torii H. Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery. Biochem Biophys Rep. 2017;10:186–91.

    PubMed  PubMed Central  Google Scholar 

  38. Faulkner-Jones A, Fyfe C, Cornelissen D-J, Gardner J, King J, Courtney A, et al. Bioprinting of human pluripotent stem cells and their directed differentiation into hepatocyte-like cells for the generation of mini-livers in 3D. Biofabrication. 2015;7:044102.

    Article  PubMed  Google Scholar 

  39. Ouyang L, Highley CB, Sun W, Burdick JA. A generalizable strategy for the 3D bioprinting of hydrogels from nonviscous photo-crosslinkable inks. Adv Mater. 2017;29:1604983.

    Article  CAS  Google Scholar 

  40. Mistry P, Aied A, Alexander M, Shakesheff K, Bennett A, Yang J. Bioprinting using mechanically robust core–shell cell-laden hydrogel strands. Macromol Biosci. 2017;17:1600472.

    Article  CAS  Google Scholar 

  41. Ma M, Chiu A, Sahay G, Doloff JC, Dholakia N, Thakrar R, et al. Core–shell hydrogel microcapsules for improved islets encapsulation. Adv Healthc Mater. 2013;2:667–72.

    Article  CAS  PubMed  Google Scholar 

  42. Kostadinova R, Boess F, Applegate D, Suter L, Weiser T, Singer T, Naughton B, Roth A. A long-term three dimensional liver co-culture system for improved prediction of clinically relevant drug-induced hepatotoxicity. Toxicol Appl Pharmacol. 2013;268:1–16.

    Article  CAS  PubMed  Google Scholar 

  43. Richert L, Baze A, Parmentier C, Gerets HH, Sison-Young R, Dorau M, Lovatt C, Czich A, Goldring C, Park BK. Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats. Toxicol Lett. 2016;258:207–15.

    Article  CAS  PubMed  Google Scholar 

  44. Nair K, Gandhi M, Khalil S, Yan KC, Marcolongo M, Barbee K, Sun W. Characterization of cell viability during bioprinting processes. Biotechnol J. 2009;4:1168–77.

    Article  CAS  PubMed  Google Scholar 

  45. Chang R, Nam J, Sun W. Effects of dispensing pressure and nozzle diameter on cell survival from solid freeform fabrication–based direct cell writing. Tissue Eng A. 2008;14:41–8.

    Article  CAS  Google Scholar 

  46. Lee PJ, Hung PJ, Lee LP. An artificial liver sinusoid with a microfluidic endothelial-like barrier for primary hepatocyte culture. Biotechnol Bioeng. 2007;97:1340–6.

    Article  CAS  PubMed  Google Scholar 

  47. Toh Y-C, Zhang C, Zhang J, Khong YM, Chang S, Samper VD, van Noort D, Hutmacher DW, Yu H. A novel 3D mammalian cell perfusion-culture system in microfluidic channels. Lab Chip. 2007;7:302–9.

    Article  CAS  PubMed  Google Scholar 

  48. Toh Y-C, Lim TC, Tai D, Xiao G, van Noort D, Yu H. A microfluidic 3D hepatocyte chip for drug toxicity testing. Lab Chip. 2009;9:2026–35.

    Article  CAS  PubMed  Google Scholar 

  49. Wang Y, Toh Y-C, Li Q, Nugraha B, Zheng B, Lu TB, Gao Y, Ng MML, Yu H. Mechanical compaction directly modulates the dynamics of bile canaliculi formation. Integr Biol. 2012;5:390–401.

    Article  Google Scholar 

  50. Shih M-C, Tseng S-H, Weng Y-S, Chu I-M, Liu C-H. A microfluidic device mimicking acinar concentration gradients across the liver acinus. Biomed Microdevices. 2013;15:767–80.

    Article  CAS  PubMed  Google Scholar 

  51. Ma C, Tian C, Zhao L, Wang J. Pneumatic-aided micro-molding for flexible fabrication of homogeneous and heterogeneous cell-laden microgels. Lab Chip. 2016;16:2609–17.

    Article  CAS  PubMed  Google Scholar 

  52. Ma C, Zhao L, Zhou E-M, Xu J, Shen S, Wang J. On-chip construction of liver lobule-like microtissue and its application for adverse drug reaction assay. Anal Chem. 2016;88:1719–27.

    Article  CAS  PubMed  Google Scholar 

  53. Prodanov L, Jindal R, Bale SS, Hegde M, McCarty WJ, Golberg I, Bhushan A, Yarmush ML, Usta OB. Long-term maintenance of a microfluidic 3D human liver sinusoid. Biotechnol Bioeng. 2016;113:241–6.

    Article  CAS  PubMed  Google Scholar 

  54. Schepers A, Li C, Chhabra A, Seney BT, Bhatia S. Engineering a perfusable 3D human liver platform from iPS cells. Lab Chip. 2016;16:2644–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Banaeiyan AA, Theobald J, Paukštyte J, Wölfl S, Adiels CB, Goksör M. Design and fabrication of a scalable liver-lobule-on-a-chip microphysiological platform. Biofabrication. 2017;9:015014.

    Article  PubMed  CAS  Google Scholar 

  56. Ong LJY, Chong LH, Jin L, Singh PK, Lee PS, Yu H, Ananthanarayanan A, Leo HL, Toh YC. A pump-free microfluidic 3D perfusion platform for the efficient differentiation of human hepatocyte-like cells. Biotechnol Bioeng. 2017;114:2360–70.

    Article  CAS  PubMed  Google Scholar 

  57. Du Y, Li N, Yang H, Luo C, Gong Y, Tong C, Gao Y, Lü S, Long M. Mimicking liver sinusoidal structures and functions using a 3D-configured microfluidic chip. Lab Chip. 2017;17:782–94.

    Article  CAS  PubMed  Google Scholar 

  58. Yu F, Deng R, Tong WH, Huan L, Way NC, IslamBadhan A, Iliescu C, Yu H. A perfusion incubator liver chip for 3D cell culture with application on chronic hepatotoxicity testing. Sci Rep. 2017;7:14528.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  59. Kang YBA, Eo J, Mert S, Yarmush ML, Usta OB. Metabolic patterning on a chip: towards in vitro liver zonation of primary rat and human hepatocytes. Sci Rep. 2018;8:8951.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  60. Chong LH, Li H, Wetzel I, Cho H, Toh Y-C. A liver-immune coculture array for predicting systemic drug-induced skin sensitization. Lab Chip. 2018;18:3239–50.

    Article  CAS  PubMed  Google Scholar 

  61. Weng YS, Chang SF, Shih MC, Tseng SH, Lai CH. Scaffold-free liver-on-a-chip with multiscale organotypic cultures. Adv Mater. 2017;29:1701545.

    Article  CAS  Google Scholar 

  62. Braet F, Wisse E. Structural and functional aspects of liver sinusoidal endothelial cell fenestrae: a review. Comp Hepatol. 2002;1:1.

    Article  PubMed  PubMed Central  Google Scholar 

  63. Spatzenegger M, Horsmans Y, Verbeeck RK. Differential activities of CYP1A isozymes in hepatic and intestinal microsomes of control and 3-methylcholanthrene-induced rats. Pharmacol Toxicol. 2000;86:71–7.

    Article  CAS  PubMed  Google Scholar 

  64. Bunchorntavakul C, Reddy KR. Acetaminophen-related hepatotoxicity. Clin Liver Dis. 2013;17:587–607.

    Article  PubMed  Google Scholar 

  65. Lee JB, Sung JH. Organ-on-a-chip technology and microfluidic whole-body models for pharmacokinetic drug toxicity screening. Biotechnol J. 2013;8:1258–66.

    Article  CAS  PubMed  Google Scholar 

  66. Abaci HE, Shuler ML. Human-on-a-chip design strategies and principles for physiologically based pharmacokinetics/pharmacodynamics modeling. Integr Biol. 2015;7:383–91.

    Article  Google Scholar 

  67. Choe A, Ha SK, Choi I, Choi N, Sung JH. Microfluidic gut-liver chip for reproducing the first pass metabolism. Biomed Microdevices. 2017;19:4.

    Article  PubMed  CAS  Google Scholar 

  68. Kimura H, Ikeda T, Nakayama H, Sakai Y, Fujii T. An on-chip small intestine–liver model for pharmacokinetic studies. J Lab Autom. 2015;20:265–73.

    Article  CAS  PubMed  Google Scholar 

  69. Theobald J, Ghanem A, Wallisch P, Banaeiyan AA, Andrade-Navarro MA, Taškova K, et al. Liver-kidney-on-chip to study toxicity of drug metabolites. ACS Biomater Sci Eng. 2017;4:78–89.

    Article  CAS  PubMed  Google Scholar 

  70. Esch MB, Mahler GJ, Stokol T, Shuler ML. Body-on-a-chip simulation with gastrointestinal tract and liver tissues suggests that ingested nanoparticles have the potential to cause liver injury. Lab Chip. 2014;14:3081–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Nyblom H, Berggren U, Balldin J, Olsson R. High AST/ALT ratio may indicate advanced alcoholic liver disease rather than heavy drinking. Alcohol Alcohol. 2004;39:336–9.

    Article  CAS  PubMed  Google Scholar 

  72. Nyblom H, Björnsson E, Simrén M, Aldenborg F, Almer S, Olsson R. The AST/ALT ratio as an indicator of cirrhosis in patients with PBC. Liver Int. 2006;26:840–5.

    Article  CAS  PubMed  Google Scholar 

  73. Spatzenegger M, Jaeger W. Clinical importance of hepatic cytochrome P450 in drug metabolism. Drug Metab Rev. 1995;27:397–417.

    Article  CAS  PubMed  Google Scholar 

  74. Zhang H-B, Xing T-L, Yin R-X, Shi Y, Yang S-M, Zhang W-J. Three-dimensional bioprinting is not only about cell-laden structures. Chin J Traumatol. 2016;19:187–92.

    Article  PubMed  PubMed Central  Google Scholar 

  75. Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Boettger J. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol. 2013;87:1315–530.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Lee K-H, Lee J, Lee S-H. 3D liver models on a microplatform: well-defined culture, engineering of liver tissue and liver-on-a-chip. Lab Chip. 2015;15:3822–37.

    Article  PubMed  CAS  Google Scholar 

  77. Laskin DL. Nonparenchymal cells and hepatotoxicity. Semin Liver Dis. Thieme Medical Publishers. 1990;10:293–304.

    Article  CAS  PubMed  Google Scholar 

  78. Jungermann K, Keitzmann T. Zonation of parenchymal and nonparenchymal metabolism in liver. Annu Rev Nutr. 1996;16:179–203.

    Article  CAS  PubMed  Google Scholar 

  79. Pichard L, Raulet E, Fabre G, Ferrini JB, Ourlin J-C, Maurel P. Human hepatocyte culture. In: Cytochrome P450 Protocols; 2006. p. 283–93.

    Google Scholar 

  80. Beckwitt CH, Clark AM, Wheeler S, Taylor DL, Stolz DB, Griffith L, Wells A. Liver ‘organ on a chip’. Exp Cell Res. 2018;363:15–25.

    Article  CAS  PubMed  Google Scholar 

  81. Alison MR, Poulsom R, Jeffery R, Dhillon AP, Quaglia A, Jacob J, Novelli M, Prentice G, Williamson J, Wright NA. Cell differentiation: hepatocytes from non-hepatic adult stem cells. Nature. 2000;406:257.

    Article  CAS  PubMed  Google Scholar 

  82. Takebe T, Sekine K, Enomura M, Koike H, Kimura M, Ogaeri T, Zhang R-R, Ueno Y, Zheng Y-W, Koike N. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature. 2013;499:481.

    Article  CAS  PubMed  Google Scholar 

  83. Szkolnicka D, Zhou W, Lucendo-Villarin B, Hay DC. Pluripotent stem cell–derived hepatocytes: potential and challenges in pharmacology. Annu Rev Pharmacol Toxicol. 2013;53:147–59.

    Article  CAS  PubMed  Google Scholar 

  84. Castell JV, Jover R, Martnez-Jimnez CP, Gmez-Lechn MJ. Hepatocyte cell lines: their use, scope and limitations in drug metabolism studies. Expert Opin Drug Metab Toxicol. 2006;2:183–212.

    Article  CAS  PubMed  Google Scholar 

  85. Aninat C, Piton A, Glaise D, Le Charpentier T, Langouët S, Morel F, Guguen-Guillouzo C, Guillouzo A. Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab Dispos. 2006;34:75–83.

    Article  CAS  PubMed  Google Scholar 

  86. Guillouzo A, Corlu A, Aninat C, Glaise D, Morel F, Guguen-Guillouzo C. The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chem Biol Interact. 2007;168:66–73.

    Article  CAS  PubMed  Google Scholar 

  87. Yanagawa F, Sugiura S, Kanamori T. Hydrogel microfabrication technology toward three dimensional tissue engineering. Regen Ther. 2016;3:45–57.

    Article  PubMed  PubMed Central  Google Scholar 

  88. Yeh J, Ling Y, Karp JM, Gantz J, Chandawarkar A, Eng G, Blumling Iii J, Langer R, Khademhosseini A. Micromolding of shape-controlled, harvestable cell-laden hydrogels. Biomaterials. 2006;27:5391–8.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

This work was supported by the Ministry of Science and Technology, Taiwan, under grant 107–2221-E-002-122-MY3 and Taiwan Bio-development Foundation.

Author information

Authors and Affiliations

  1. Department of Mechanical Engineering, National Taiwan University, Taipei, Taiwan

    Jyong-Huei Lee & Kuan-Lun Ho

  2. Department of Mechanical and Nuclear Engineering, Kansas State University, Manhattan, KS, 66506, USA

    Shih-Kang Fan

Authors
  1. Jyong-Huei Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kuan-Lun Ho
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shih-Kang Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JHL and KLH collected literature, designed and wrote the manuscript. SKF edited and prepared manuscript for submission. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Shih-Kang Fan.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lee, JH., Ho, KL. & Fan, SK. Liver microsystems in vitro for drug response. J Biomed Sci 26, 88 (2019). https://doi.org/10.1186/s12929-019-0575-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12929-019-0575-0

Keywords

Journal of Biomedical Science

ISSN: 1423-0127