3D Bioprinted Liver-on-a-Chip for Drug Cytotoxicity Screening

Abstract

Tissues on a chip are sophisticated three-dimensional (3D) in vitro microphysiological systems designed to replicate human tissue conditions within dynamic physicochemical environments. However, the current fabrication methods for tissue spheroids on a chip require multiple parts and manual processing steps, including the deposition of spheroids onto prefabricated “chips.” These challenges also lead to limitations regarding scalability and reproducibility. To overcome these challenges, we employed 3D printing techniques to automate the fabrication process of tissue spheroids on a chip. This allowed the simultaneous high-throughput printing of human liver spheroids and their surrounding polymeric flow chamber “chips” containing inner channels in a single step. The fabricated liver tissue spheroids on a liver-on-a-chip (LOC) were subsequently subjected to dynamic culturing by a peristaltic pump, enabling assessment of cell viability and metabolic activities. The 3D printed liver spheroids within the printed chips demonstrated high cell viability (>80%), increased spheroid size, and consistent adenosine triphosphate (ATP) activity and albumin production for up to 14 days. Furthermore, we conducted a study on the effects of acetaminophen (APAP), a nonsteroidal anti-inflammatory drug, on the LOC. Comparative analysis revealed a substantial decline in cell viability (<40%), diminished ATP activity, and reduced spheroid size after 7 days of culture within the APAP-treated LOC group, compared to the nontreated groups. These results underscore the potential of 3D bioprinted tissue chips as an advanced in vitro model that holds promise for accurately studying in vivo biological processes, including the assessment of tissue response to administered drugs, in a high-throughput manner.

Impact statement

Tissues on a chip provide sophisticated three-dimensional (3D) in vitro systems that replicate human physiological conditions within dynamic environments. However, current fabrication methods involve intricate manual steps, which hinder scalability and reproducibility. To overcome this, we employed 3D printing to automate the creation of tissue spheroids on a chip, enhancing both reproducibility and scalability. This innovative approach underscores the potential of 3D bioprinted tissue chips, offering opportunities for drug discovery, toxicity testing modeling, and personalized medicine applications.

Introduction

The use of animal models for preclinical drug tests often leads to unexpected side effects during human clinical trials due to interspecies differences.^1–3 Moreover, animal models for drug testing are expensive and raise ethical concerns. To overcome these limitations, there has been a growing interest in the development of in vitro human tissue models. Although two-dimensional (2D) cultures of cells, such as human hepatocytes, are commonly used, they fail to fully replicate the native tissue microenvironment.^4,5 In contrast, three-dimensional (3D) models, such as spheroids, although not entirely a human replicate, can mimic cell-cell and cell-matrix interactions and exhibit improved metabolic competency compared to 2D cultures.^6,7 One of the major challenges for new drug development of personalized medicine is avoiding liver toxicity. Therefore, enhanced accuracy in predicting drug hepatotoxicity makes the development of 3D liver models a promising approach for preclinical drug toxicity screening.

To replicate the liver microenvironment more accurately, researchers have developed in vitro microphysiological systems, such as liver-on-a-chip (LOC).^5,8,9 The LOC is an in vitro 3D hepatic model that mimics the dynamic physicochemical hepatic microenvironment of the liver on a microscopic scale.^10,11 Compared to the static microenvironment, the dynamic cultures in the LOC have demonstrated an enhanced ability of hepatocytes to metabolize substrates for specific cytochrome P450 enzymes and transport metabolites into the canaliculi.¹² This advancement holds promise in reducing reliance on animal studies for preclinical drug assessment.

Many studies have shown the feasibility of these systems. For example, a microfluidic 3D hepatocyte chip, known as the 3D HepaTox Chip, has been previously introduced.¹³ More sophisticated systems that replicate a hepatobiliary system were developed.^14,15 Also, systems that allow the interaction of the liver with other tissues have been introduced.^5,16,17 These chips have the capability to detect the IC₅₀ values of drugs and monitor the dose-dependent drug response in hepatocytes, thereby providing valuable insights into the potential toxicity of drugs during preclinical evaluation.

Despite the advantages offered by LOC systems, their fabrication typically involves multiple assembly steps and manual labor, leading to potential variability between samples.^5,9 This inconsistency among individual samples can undermine the reliability of drug screening results, which goes against the primary goal of using in vitro liver models as alternatives to animal testing. In addition, as the assembly process becomes more complex and the number of prefabricated chip parts increases, there is an elevated risk of contamination of cells and spheroids as these are introduced within the LOC system. Consequently, there is a pressing need for an automated LOC fabrication technique capable of simultaneously constructing the chip platform with its tissue spheroid components. Such a technique would ensure higher reproducibility and consistency across samples with better throughput.

This study employed the utilization of 3D bioprinting technology to fabricate an LOC system, leveraging its precise deposition capabilities for polymeric materials and cell-laden hydrogel in a controlled manner with simultaneous automated printing and delivery of liver spheroids onto the chip (Fig. 1). We optimized the printing settings to ensure consistent and reliable chip-to-chip printing during the fabrication process. Following the successful fabrication, the printed LOC was subjected to dynamic culturing, and the metabolic activities of liver spheroids were thoroughly evaluated. Furthermore, to determine the feasibility of the 3D printed LOC as an in vitro model for toxicity testing, the system was treated with a hepatotoxic drug, and the resulting outcomes were compared with those of a nontreated control group.

FIG. 1.

Schematic illustration of 3D bioprinted LOC. 3D bioprinting technology was utilized to fabricate an LOC system, leveraging its precise deposition of polymeric materials for a chip structure and liver spheroid-laden hydrogel. 3D, three dimensional; LOC, liver-on-a-chip.

Materials and Methods

Liver spheroid culture

The human hepatoma cell line HepG2 cells (HB-8065; ATCC, Manassas, VA) were cultured and expanded in Dulbecco's modified Eagle's medium (DMEM, high glucose) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic (AA) solution. The cells were maintained in a humidified incubator at 37°C with 5% CO₂, and the medium was changed every 3 days. To prepare the 3D spheroids, HepG2 cells at passages 3–5 were detached and transferred to a six-well round bottom microcavity plate (Cat. No. 4440; Corning, Corning, NY) at a seeding density of 2.16 × 10⁶ cells per well (750 cells per microcavity). The HepG2 liver spheroids were formed after 4 days of culture in the incubator with the medium supplemented with rat tail collagen Type I (10 μg/mL, Cat No. 354236; Corning). Unless otherwise mentioned, all the cell culture and analysis reagents were purchased from Thermo Fisher Scientific (Waltham, MA).

Bioink preparation

Gelatin methacryloyl (GelMA, 80% degree of substitution) was prepared through the methacrylate of porcine skin-derived gelatin (Type A, 300 bloom; Millipore Sigma, St. Louis, MO), following the method described by Shirahama et al.¹⁸ A mixture of glycerol (Millipore Sigma) and DMEM (high glucose, no phenol red) in a 1:9 (v/v) ratio was prepared. The following materials were dissolved in the glycerol/DMEM solution: 2.5 w/v% GelMA, 0.3 w/v% hyaluronic acid (MW 100 k; Lifecore Biomedical, Chaska, MN), 1.0 w/v% gelatin, and 0.2 w/v% lithium phenyl(2,4,6-trimethyl benzoyl) phosphinate (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan).

The materials were completely dissolved at 37°C, and the bioink was subsequently sterile filtered using a 0.45 μm syringe filter. Simultaneously, the liver spheroids cultured for 4 days were carefully collected, and the supernatant medium was aspirated. The number of harvested spheroids was calculated by examining over 95% confluency in microcavities of the six-well plate (2886 microcavities/well) through a microscope. Subsequently, a volume of 1 mL of sterile-filtered bioink was carefully blended with the entirety of the harvested liver spheroids.

Bioprinting of LOC

The G-code for the LOC was generated by utilizing in-house G-code generation software. The LOC consisted of a polymeric chip structure and spheroid-laden bioink. Poly (ɛ-caprolactone) (PCL, MW 50k; Polysciences, Inc., Warrington, PA) to construct the polymeric chip structure and the liver spheroid-laden bioink were loaded into printing syringes, respectively, in a 3D integrated tissue and organ printing (ITOP) system, which contains an X-, Y-, and Z-axis stage/controller and multiple dispensing modules.¹⁹

The metal nozzle for PCL had an inner diameter (ID) of 300 μm, while the tapered nozzle for the bioink had an inner diameter of 610 μm. The LOC was printed on a poly(ethylene terephthalate) film adhered to a glass microscope slide under sterile conditions. The spheroid-laden bioink was solidified using an integrated ultraviolet (UV) light source (200 mW/cm² for 10 s) within the ITOP system. Before use, all syringes and nozzles underwent ethylene oxide (EO) sterilization, and the ITOP system was sterilized with 70% ethanol. The UV intensity was regularly calibrated using a radiometer (Accu-Cal 50L; Dymax, Torrington, CT) before every printing session.

LOC setup

To prepare the LOC platform, computer-aided design models of tubing supports and conical tube holders were created using Fusion 360 (Autodesk, San Rafael, CA) and were 3D printed using the Ender 3 3D printer (Creality, Shenzhen, China). The 3D printed LOC platform, pump tubing (MPP-038-F-PVC; Meinhard, Golden, CO), and extension tubing (No. MFLX06417-21; VWR, Radnor, PA) were presterilized in EO gas and assembled in the biosafety cabinet, followed by set up in the incubator.

The printed LOC was transferred to an incubator (5% CO₂, 37°C) and connected to a peristaltic pump (MP^2–6; Elemental Scientific, Omaha, NE) for medium perfusion at a flow rate of 10 μL/min. The medium reservoir, a 50 mL conical tube (Corning), was filled with 5 mL of DMEM (high glucose) supplemented with 10% FBS and 1% AA. The culture medium was refreshed on a daily basis, and the spent medium was gathered and preserved at −80°C for further analysis.

Cell viability assay

The complete spheroid-laden hydrogel construct was extracted from the printed LOCs, and the cell viability of spheroids in the hydrogel construct was evaluated using the Live/Dead™ Staining Assay kit (n = 3 per group, Invitrogen, Waltham, MA). The spheroids were stained with 0.15 mM calcein-AM (live) and 2 mM ethidium homodimer-1 (dead) for 1 h in the incubator. A confocal microscope (Fluoview FV10i; Olympus, Tokyo, Japan) was used to capture 3D z-stacked fluorescent images, which were analyzed using ImageJ (National Institutes of Health, Bethesda, MD) and Imaris software (Oxford Instruments, Abingdon, United Kingdom). Cell viability was calculated using equation (1),

The spheroid aspect ratio was determined using the following equation (2): $A s p e c t r a t i o = \frac{h e i g h t o f o r g a n o i d}{w i d t h o f o r g a n o i d} (w i d t h > h e i g h t)$ (2)

To evaluate the metabolic activity of spheroids, a CellTiter-Glo^® 3D assay was performed according to the manufacturer's instructions (n = 3 per group, Promega, Madison, WI). Briefly, the spheroid-laden samples were immersed in a 1:1 v/v mixture of media and CellTiter-Glo solution in a 48-well plate with gentle shaking at room temperature for 30 min. The supernatant was transferred to a white 96-well plate, and the luminescence was measured using a Veritas™ microplate luminometer (Turner BioSystems, Sunnyvale, CA). The number of spheroids in the hydrogel sample was determined in the Live/Dead step to calculate the adenosine triphosphate (ATP) value per spheroid.

Functional analysis

The collected medium samples were thawed and analyzed for their albumin contents using human albumin ELISA kits (n = 3 per group). The albumin ELISA kit was obtained from Abcam (Cambridge, United Kingdom). The ELISA was performed according to the manufacturer's protocol, and the absorbance was measured using a microplate reader (SpectraMax M5; Molecular Devices, San Jose, CA).

Drug cytotoxicity test

The printed line LOCs were prepared for two experimental groups: a control group without any drug treatment and a drug-treated group for conducting a drug cytotoxicity test. In both the control and drug-treated groups of LOCs, the culture medium was replaced and collected daily over a period of 7 days. Specifically, the control LOC group was continuously cultured in the culture medium for the entire 7-day duration. In contrast, the drug-treated group was initially cultured in the standard medium, but starting from day 2 and continuing until day 7, the medium was replaced with a sterile filtered medium containing 20 mM acetaminophen (APAP; Millipore Sigma). Both groups were then analyzed following the aforementioned procedures.

Statistical analysis

All data were collected with n = 3 or greater and analyzed using Prism 9.0 software (GraphPad Software, San Diego, CA) with one-way analysis of variance followed by pairwise post hoc testing using Tukey's multiple comparison test. A p-value <0.05 was considered statistically significant and indicated with asterisks in the graph. The significance levels were represented by the following symbols: *, **, ***, and **** for p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively.

Results

LOC design and fabrication

The hepG2 cells were cultured in a six-well round bottom microcavity plate to form spheroids (Supplementary Fig. S1A). After 4 days, the liver spheroids were mixed with bioink and loaded into a 1 mL dispensing syringe. The syringe was equipped with a pink tapered nozzle (ID = 610 μm) and inserted into the ITOP printer as the second syringe. The outer shell of the LOC was 3D printed using the first PCL syringe (green in G-code), and the liver spheroid-laden bioink was printed inside with the second syringe (red in G-code cross-section) (Supplementary Fig. S1B).

The LOC was designed with a unidirectional medium flow path from the inlet port to the zig-zag channel (width = 1.7 mm) and the outlet port. The LOC walls were printed with a double PCL layer to prevent medium leakage, and the inlet and outlet ports were designed to fit the medium-flowing tubing properly. The step design was incorporated at the outlet port to facilitate the thorough filling of the LOC with the medium before initiating aspiration through the outlet tubing. This modification, as opposed to using a conventional LOC without the step, effectively mitigates the accumulation of air bubbles within the LOC system.

In fabricating the LOC, the first syringe with a metal nozzle dispensed PCL and printed the base layer, including the channel walls (Supplementary Fig. S1B, C). Then, the dispensing nozzle was switched to the second syringe, which accurately deposited the liver spheroid bioink into the inner medium channels.

The printing stage was moved to a UV source integrated into the ITOP system that automatically projected UV light for 10 s at an intensity of 200 mW/cm², crosslinking the GelMA-based bioink. The printing syringe was changed back to the first syringe, and PCL was deposited to stack the channel walls, creating additional space for medium flow on top of the spheroids. Finally, the PCL was printed on top to cover the LOC and create inlet and outlet ports. The printed chip's dimensions were ∼2 × 1 × 1 cm³. The total fabrication time for a single LOC was 30 min. Supplementary Movie S1 shows the complete printing process used in the fabrication of LOC.

The LOC platform, tubing supports, and a conical tube holder were designed and 3D printed (Supplementary Fig. S2). The platform was designed to accommodate a maximum of three LOC slots. The tubing support was intended to facilitate the tubing connection into the inlet and outlet of the LOC, and it could easily slide into the moving rails of the LOC platform. The conical tube holder could hold up to three 50 mL conical tubes, serving as a medium reservoir. The peristaltic pump and tubing were sterilized and preset inside the incubator. Once the LOC was printed, it was placed in the platform, and the tubing support was slid through the moving rail to adjust the location for proper tubing connection.

Liver spheroid printing optimization

The printing conditions were optimized to improve the liver spheroid viability, proliferation, and morphology. First, the effect of the UV crosslinking time on spheroid viability was evaluated (Fig. 2A). Spheroids embedded in the hydrogel were crosslinked with UV light for 0, 10, or 120 s at an intensity of 200 mW/cm². The hydrogel did not crosslink without UV exposure, and the spheroids were cultured statically for 4 days. On day 1, the average viability of the spheroids in the 0- and 10-s groups was 21% and 14% higher compared with the 120-s group, respectively (Fig. 2B). On day 4, the 0- and 10-s groups exhibited 10% and 13% higher viability, respectively, compared to the 120-s group. All groups showed similar sizes and aspect ratios of liver spheroids on days 1 and 4 (Fig. 2C, D).

FIG. 2.

Optimization of liver spheroid bioprinting. (A) Live/Dead™ images of liver spheroids in hydrogel after different UV exposure times (0, 10, and 120 s) and cultured for 1 and 4 days in the incubator. The effect of UV exposure on (B) cell viability, (C) size, and (D) morphology (n = 3). (E) Live/Dead images of liver spheroids in hydrogel after dispensing through nozzles with different inner diameters (1000, 610, and 330 μm) and cultured for 1 and 4 days. The effect of nozzle size on (F) cell viability, (G) size, and (H) morphology (n = 3). “ns” indicates non-significance, and asterisks (*), (**), (***), and (****) indicate p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. UV, ultraviolet.

Second, the effect of the syringe nozzle size on the liver spheroid was evaluated (Fig. 2E). Nozzles with three different IDs were prepared, including a regular 1 mL pipette tip (ID = 1000 μm), a pink tapered plastic nozzle (ID = 610 μm), and an orange metal nozzle (ID = 330 μm).

On day 1, the 1000 and 610 μm groups showed 8% higher viability than the 330 μm group (Fig. 2F). On day 4, the 1000 μm group had the highest viability (93%) compared to the 610 μm (86%) and 330 μm (84%) groups. All groups showed similar sizes of liver spheroids on days 1 and 4 (Fig. 2G). The aspect ratio was significantly lower in the 330 μm group than in the 610 and 1000 μm groups on day 1 (Fig. 2H). With the data collected above, we used 10 s for the bioink crosslinking and 610 μm tapered nozzle for further LOC bioprinting.

Cellular activities and albumin production

The LOC was bioprinted and dynamically cultured in an incubator for up to 14 days. Live/Dead images showed that the printed liver spheroids proliferated actively, leading to an increase in spheroid size over time (Fig. 3A). The viability of printed liver spheroids remained consistently over 85% through day 14 (Fig. 3B). The size of liver spheroids steadily increased, with an average size of 400 μm on day 14 (Fig. 3C). Furthermore, the ATP production per spheroid increased progressively over the 14-day culture period compared to day 0 (Fig. 3D).

FIG. 3.

Dynamic culture of LOC for 14 days. (A) Live/Dead images of liver spheroids in LOC on days 7 and 14, (B) cell viability, (C) spheroid size, and (D) ATP levels (n = 6). (E) Albumin levels in media were collected every 2 days from the LOC (n = 3). “ns” indicates non-significance, and asterisks (*), (**), and (***) indicate P < 0.05, P < 0.01, and P < 0.001, respectively. ATP, adenosine triphosphate.

The medium was changed and collected every 2 days during LOC culture to assess albumin secretion. The results showed that the albumin production from the LOC steadily increased over time and reached its highest level on day 14 (Fig. 3E). The results indicate that the optimized bioprinting conditions allowed the successful culture of spheroids on the LOC for an extended period, while maintaining high viability and function.

Drug cytotoxicity test

The feasibility of using a 3D printed LOC for evaluating drug cytotoxicity was assessed by administering APAP, a nonsteroidal anti-inflammatory drug known to be the leading cause of drug-induced acute liver failure.²⁰ On day 2 of the experiment, the drug-treated group received a dose of 20 mM APAP, while both groups were cultured with the same culture media for the initial 2 days.

On day 7, Live/Dead images were captured, revealing a higher number of dead cells and smaller spheroid sizes in the drug-treated group compared to the control group (Fig. 4A). The viability of spheroids in the drug-treated group was found to be 45% lower compared with the control group (Fig. 4B). Furthermore, the average diameter of liver spheroids in the drug-treated group (132 μm) was significantly smaller compared with the control group (404 μm) (Fig. 4C). In addition, minimal ATP production was detected in the drug-treated group (Fig. 4D). In the control group, albumin secretion displayed a slight increase over time, while in the drug-treated group, there was a dramatic decrease in albumin secretion following APAP administration (Fig. 4E).

FIG. 4.

Assessment of the response of liver spheroids to 20 mM APAP administered on day 2 in the LOC. (A) Live/Dead images of control and 20 mM APAP group on day 7. Corresponding (B) cell viability, (C) spheroid size, and (D) ATP activity on day 7 (n = 3). (E) Albumin production of LOC in collected medium sample from day 1 to 7 (n = 3). Asterisks (*), (**), (***), and (****) indicate p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. APAP, acetaminophen.

Discussion

3D bioprinting technologies were implemented to develop the LOC system containing liver spheroids, aiming to enhance the reproducibility of chip fabrication and reduce sample-to-sample variability. Both the creation of chips and the deposition of liver spheroids were done simultaneously using a 3D printer, allowing a high-throughput uniform and self-contained sterile unit to be reliably manufactured. Through the optimization of liver spheroid bioprinting, we achieved high cell viability and maintained spheroid morphology. The dynamic culture within the LOC system facilitated continuous monitoring of cell viability and metabolic activity, while also enabling the assessment of hepatotoxicity associated with administered drugs. This comprehensive approach allowed us to evaluate the functionality and performance of the 3D printed LOC system effectively.

One of the key advantages of the printed LOC system is its automated fabrication process, which greatly enhances the consistency between samples. In contrast, previously reported LOC systems relied on manual assembly of pre-fabricated chip parts, such as poly(methyl methacrylate) and polydimethylsiloxane, along with the incorporation of cells. This manual assembly approach often led to variations in the final product.^5,9

The implementation of 3D printing technology allowed the precise deposition of spheroid-laden bioink volumes in each chip, leading to a remarkable enhancement in sample-to-sample consistency. In this study, the spheroid-laden bioink was dispensed at a known volume of 35 μL, achieving a dispensing rate of 2.76 μL/s. This deposition strategy resulted in an approximate yield of 120 spheroids per chip. In addition, the automated fabrication process significantly reduced the number of manual assembly steps needed, effectively minimizing the potential risk of contamination. In addition, the preprogrammed printer capabilities give us the option to 3D print hundreds of these chips in short periods of time.

HepG2 cell lines cultured as 2D monolayers exhibit reduced functionality and altered phenotype compared to human hepatocytes in vivo.^21,22 Therefore, the LOC system was printed, incorporating HepG2 liver spheroids with bioink to promote improved cell-cell and cell-matrix interactions. To maintain the quality of liver spheroids during the bioprinting process, two printing conditions were optimized. First, the impact of UV crosslinking on GelMA-based bioink and liver spheroids was assessed.

The results demonstrated that 10-s UV exposure had no significant effect on spheroid viability, size, or morphology, while 120 s of exposure resulted in decreased viability on days 1 and 4. Considering the low hydrogel volume (35 μL), a 10-s UV projection was sufficient for crosslinking the GelMA hydrogel in the LOC system. Furthermore, the GelMA concentration in our bioink was predetermined at 2.5%. This specific concentration was chosen as it was found to be the minimum amount capable of maintaining the mechanical stability of the construct inside the chip under dynamic medium flow conditions (data not shown).

Second, the effect of nozzle size on printed liver spheroids was evaluated. Typically, liver spheroids cultured for 4 days possess an average diameter of ∼150 μm. The findings revealed that the 330 μm ID nozzle generated shear stress during extrusion, leading to changes in their spherical morphology. However, the 610 μm ID nozzle exhibited similar viability and morphology to a typical 1 mL micropipette tip with a 1000 μm ID. The optimized LOC system demonstrated successful cultivation for up to 14 days without medium leakage or air bubble formation. The printed LOCs exhibited high cell viability (>80%), an increase in spheroid size, consistent ATP production, and elevated albumin production for the duration of 14 days.

The printed LOC system was utilized to investigate the cytotoxic effects. APAP, which is a well-known hepatotoxic drug, was selected as a model compound to evaluate the efficacy of the developed LOC system in mimicking acute liver failure resulting from APAP overdose. The LOC group treated with 20 mM APAP demonstrated a significant decrease in cell viability (<40%), reduced ATP production, and smaller spheroid size compared to the nontreated group after 7 days of culture. These findings align with previously reported studies, indicating a decline in liver spheroid viability, metabolic activity, size, and albumin production under APAP conditions. Thus, the developed LOC system effectively replicates the detrimental effects of APAP and demonstrates its potential as a valuable tool for studying drug-induced hepatotoxicity.^9,23

The findings of this study demonstrate the potential of bioprinted human tissue chip systems as a valuable tool for assessing the toxicity of drugs and chemicals. Utilizing advanced 3D printing techniques, we hold a significant advantage in integrating diverse cell types into the chip. We envision the expansion of the bioprinted strategy into a more comprehensive body-on-a-chip system by incorporating multiple tissue types, such as lung and heart spheroids, through the utilization of diverse bioinks. In our upcoming studies, we will investigate the hepatotoxic effects of unidentified compounds. In addition, the integration of biosensors, such as aptamers, along with environmental sensors monitoring factors like temperature, oxygen levels, and pH, could enable real-time monitoring of cellular function within the chip. This integration would enhance the functionality and versatility of the chip, opening up new possibilities for advanced in vitro modeling and toxicological studies.

Conclusion

We developed a one-step printing method for the fabrication of human tissue on a chip system, utilizing human liver spheroids embedded within a GelMA-based bioink. The findings demonstrated that the optimized bioprinting conditions and the utilization of spheroids, as opposed to single cells, resulted in high cell viability, consistent ATP production, and albumin secretion over a 14-day period. Moreover, the LOC system effectively identified the cytotoxic effects of APAP, successfully mimicking acute liver failure. The automated bioprinting approach enhanced chip-to-chip consistency, enabling more precise assessments of liver spheroid viability and metabolic activity in hepatotoxic microenvironments. Ultimately, the one-step 3D printed LOC system has the potential to serve as a high-throughput screening in vitro model, facilitating accurate investigations of in vivo biological processes and monitoring tissue responses to administered drugs.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

The project or effort depicted was or is sponsored by the Department of the Defense, Defense Threat Reduction Agency (HDTRA11910013). The content of the information does not necessarily reflect the position or the policy of the federal government, and no official endorsement should be inferred. J.P.R.L.L.P. was supported by Brazilian Sao Paulo State Foundation—FAPESP (#2021/11291-2).

Supplementary Material

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Supplementary Material

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