Microengineered discoid liver platforms: bridging geometry, functions, and scalability

In the article entitled “A functional human liver tissue model: 3D bioprinted co-culture discoids,” published in Biomater Adv, the authors present a human liver tissue bioprinting strategy capable of fabricating disc-shaped (discoid) liver constructs with a thickness of approximately 200 µm and a diameter of up to 3 mm (1). The authors’ approach addresses several key limitations in liver tissue engineering. First, the discoid geometry better reflects the in vivo conditions than typical spheroid or monolayer cultures. Second, the bioprinting strategy enables spatial arrangement, promoting intercellular crosstalk that is critical for liver-specific functions. Importantly, the model demonstrates stable functional output over extended culture periods, making it a potentially useful platform for pharmacological applications. Initially, spherical polyethylene glycol (PEG) microgels were fabricated and used as support media during bioprinting. Hepatocytes, epithelial cells, and type I collagen were mixed to prepare a solution, which was extruded into PEG microgels. Among the epithelial cells, hepatocytes were primarily co-cultured with human umbilical vein endothelial cells (HUVECs) to enhance hepatic functions, such as albumin and urea synthesis. In addition to demonstrating hepatocyte function, the study further characterized gene expressions related to absorption, distribution, metabolism, and excretion (ADME), as well as hepatic enzymatic activities within the bioprinted discoid liver model. Therefore, this article highlights the potential of the three-dimensional (3D) bioprinted discoid liver model as an encouraging but preliminary platform for pharmacological applications.

Various biological liver models, including those derived from induced pluripotent stem cells (iPSCs), have been widely utilized in liver-related studies (2-4). While two-dimensional (2D) monolayer cultures remain commonly used, 3D liver models have gained increasing attention due to their ability to better recapitulate in vivo-like functions through enhanced cell-cell and cell-matrix interactions (5). To further improve the functional performance of 3D liver tissue models and facilitate their large-scale production, incorporation of perfusion is considered essential (6). However, the complexity associated with current fabrication techniques and perfusable system integration poses significant challenges to their widespread adoption, particularly in pharmaceutical applications (7).

The authors present a scalable 3D liver tissue fabrication platform utilizing cytocompatible PEG microgels as the support media. The resulting discoid liver constructs, with a uniform thickness and <5% variability in effective diameter, were printed at a rate exceeding one tissue per minute, enabling the production of 96 tissues within approximately an hour. The constructs maintained stable albumin and urea production, expressed key genes associated with ADME, and demonstrated metabolic activity toward test compounds for over 21 days. This approach overcomes limitations related to non-Newtonian bioink flow, offering a robust strategy for high-throughput liver tissue modeling.

The proposed liver model, developed through PEG microgel synthesis and characterization with theoretical evaluation of printing quality, can be fabricated rapidly and at a scale comparable to previously reported models (8,9). While PEG microgels served effectively as a supporting medium for bioprinting, the synthesized microgels displayed a relatively broad size distribution. This heterogeneity in particle size could lead to localized variability within bioprinted constructs, thereby reducing the precision of structural control in certain regions of spheroids, as reflected by the uneven filling observed in the hexagonal array presented by the authors. Although the authors did not explicitly mention a successive (iterative) bioprinting approach, which is commonly employed in extrusion-based bioprinting, their strategy appears capable of generating bulk liver constructs with substantial volumetric height. The authors’ concept of producing approximately 96 tissue constructs per hour appears technically feasible and reliable. However, the absence of supporting experimental data or quantitative validation raises concerns regarding the reproducibility of this throughput. This is also related to the lack of detailed discussion on potential extrusion-based bioprinting issues, such as shear stress-induced cell damage or nozzle clogging, which commonly affect printing fidelity and cell viability. Despite their advantages, including high-speed, large-scale printing capability and the use of porous microgels to promote structural maturation, this model requires a higher cell density to more accurately replicate hepatic function, particularly when compared with the cell density found in mammalian liver tissue (10). Increasing the cell density within the cell-collagen solution would require careful consideration of its non-Newtonian behavior and strategies to address it. One possible approach is to incorporate hepatic organoids or spheroids, which could help overcome cell density limitations when integrated with their printing strategy (11,12). In such cases, whether by increasing cell density or incorporating hepatic organoids, the microgel synthesis parameters, including PEG concentration and particle size, should be optimized to provide sufficient mechanical support for bioprinted constructs and to ensure accurate printability that reflects the overall weight determined by cell density as well as the size and morphology of the organoids.

In addition, the authors demonstrated hepatic functional activity by co-culturing hepatocytes with HUVECs and cholangiocytes within their liver models. Previous studies have reported that HUVECs can enhance hepatocyte functions (13,14). In the discoid models, HUVECs not only provided structural support but also enhanced albumin and urea synthesis in hepatocytes, accompanied by improved cell-cell cohesion. Beyond HUVECs, the authors also investigated a cholangiocyte co-culture model. The cholangiocyte co-culture led to decreased hepatocyte functions, reducing albumin and urea production compared to HUVEC incorporation. This suppression likely arises from cholangiocyte overgrowth and altered microenvironmental cues, including polarity and paracrine signaling (15). These results underscore the need to regulate cholangiocyte phenotype and cell ratios for accurate reconstruction of the native liver function in 3D bioprinted models. In the native liver, in addition to hepatocytes, there are hepatic stellate cells and liver sinusoidal endothelial cells (2). Considering the native tissue microenvironment, as the authors have already noted, various cell types should be incorporated into the model, including hepatic stellate cells, liver sinusoidal endothelial cells, and Kupffer cells, to recapitulate better the cellular complexity and functional heterogeneity of the liver tissue. Therefore, future studies should perform functional characterization with numerous cell types, including assessment of cell type-dependent deformation of the printed discoids, such as shrinkage after a few days of maturation. Such information could provide valuable insights for the field of hepatology.

In this article, 3D bioprinted liver discoids, particularly hepatocyte-HUVEC co-cultures, were shown to maintain transcriptional and functional characteristics closely resembling those of native human liver tissue. Their comprehensive mRNA profiling of ADME-associated genes revealed stable expression of key phase I and II metabolizing enzymes up to 21 days in culture. The authors assessed the enzymatic activity of critical CYP450 isoforms (CYP1A2, CYP2C9, CYP2D6, CYP3A4) and UGT1A1 in printed co-cultures. Metabolite formation rates in the discoids after two weeks matched or exceeded those observed in freshly thawed hepatocyte suspensions. The enhancement of UGT1A1 and CYP1A2 activities upon supplementation with the ROCK inhibitor Y-27632 and hepatocyte growth factor further demonstrated the tunability of this system and suggested a path toward long-term maintenance of hepatic function. The authors demonstrated meaningful maintenance of liver-specific functions, yet the bioprinted model, despite its relatively large size, does not reflect the microarchitectural organization of the native liver. This structural limitation suggests a functional-structural gap that reduces physiological relevance. Although the co-culture with HUVECs increased albumin and urea production, the study did not provide evidence of vascular niche formation. Despite the use of CD31 immunolabeling, organized endothelial structures linking the observed enhancement to functional improvement were not presented. This reflects a common issue in engineered tissues where biochemical gains occur without corresponding architectural maturation.

The technique and findings presented in the work of Subramaniam et al. are noteworthy and demonstrate considerable potential, although further validation is warranted. Still, in our view, aspects such as achieving physiologically relevant cell densities and incorporating a broader range of liver-specific cell types warrant further investigation in studies with larger sample sizes and more comprehensive functional analyses. Finally, one important aspect not addressed in this study is the potential for 3D tissue models to be applied in transplantation, which is increasingly critical due to the shortage of donor organs (9,16). Since this approach requires a microgel supporting bed, intraoperative bioprinting cannot be applied at present, and demonstrating transplantation-related data would represent a more meaningful advancement of this platform. The discoid 3D liver models presented here warrant further investigation in this context. Evaluating the in vivo viability, integration, and functional performance of these constructs after implantation would be the next essential step. Such advancements would not only enhance the physiological relevance of the model for hepatology research but also pave the way for its use in fabricating transplantable liver tissues.

In addition, the development of clear operational protocols, particularly to address potential structural instability or functional decline under physiologically challenging conditions, will be essential before this high-throughput printing strategy can be widely adopted. Nonetheless, the described approach for producing uniform and metabolically active 3D liver constructs represents a compelling and potentially scalable alternative to conventional liver tissue engineering techniques.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Annals of Translational Medicine. The article has undergone external peer review.

Peer Review File: Available at https://atm.amegroups.com/article/view/10.21037/atm-25-123/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-25-123/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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References

1. Subramaniam V, Abrahan C, Higgins BR, et al. A functional human liver tissue model: 3D bioprinted co-culture discoids. Biomater Adv 2025;173:214288. [Crossref] [PubMed]
2. Ma L, Wu Y, Li Y, et al. Current Advances on 3D-Bioprinted Liver Tissue Models. Adv Healthc Mater 2020;9:e2001517. [Crossref] [PubMed]
3. Lu J, Einhorn S, Venkatarangan L, et al. Morphological and Functional Characterization and Assessment of iPSC-Derived Hepatocytes for In Vitro Toxicity Testing. Toxicol Sci 2015;147:39-54. [Crossref] [PubMed]
4. Khetani SR, Bhatia SN. Microscale culture of human liver cells for drug development. Nat Biotechnol 2008;26:120-6. [Crossref] [PubMed]
5. Jin M, Yi X, Liao W, et al. Advancements in stem cell-derived hepatocyte-like cell models for hepatotoxicity testing. Stem Cell Res Ther 2021;12:84. [Crossref] [PubMed]
6. Rennert K, Steinborn S, Gröger M, et al. A microfluidically perfused three dimensional human liver model. Biomaterials 2015;71:119-31. [Crossref] [PubMed]
7. Kolesky DB, Homan KA, Skylar-Scott MA, et al. Three-dimensional bioprinting of thick vascularized tissues. Proc Natl Acad Sci U S A 2016;113:3179-84. [Crossref] [PubMed]
8. Ma X, Qu X, Zhu W, et al. Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting. Proc Natl Acad Sci U S A 2016;113:2206-11. [Crossref] [PubMed]
9. Yanagi Y, Nakayama K, Taguchi T, et al. In vivo and ex vivo methods of growing a liver bud through tissue connection. Sci Rep 2017;7:14085. [Crossref] [PubMed]
10. Yamasaki C, Ishida Y, Yanagi A, et al. Culture density contributes to hepatic functions of fresh human hepatocytes isolated from chimeric mice with humanized livers: Novel, long-term, functional two-dimensional in vitro tool for developing new drugs. PLoS One 2020;15:e0237809. [Crossref] [PubMed]
11. Takebe T, Zhang RR, Koike H, et al. Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant. Nat Protoc 2014;9:396-409. [Crossref] [PubMed]
12. Sato K, Zhang W, Safarikia S, et al. Organoids and Spheroids as Models for Studying Cholestatic Liver Injury and Cholangiocarcinoma. Hepatology 2021;74:491-502. [Crossref] [PubMed]
13. Salerno S, Campana C, Morelli S, et al. Human hepatocytes and endothelial cells in organotypic membrane systems. Biomaterials 2011;32:8848-59. [Crossref] [PubMed]
14. Enomoto Y, Enomura M, Takebe T, et al. Self-formation of vascularized hepatic tissue from human adult hepatocyte. Transplant Proc 2014;46:1243-6. [Crossref] [PubMed]
15. Ardalani H, Sengupta S, Harms V, et al. 3-D culture and endothelial cells improve maturity of human pluripotent stem cell-derived hepatocytes. Acta Biomater 2019;95:371-81. [Crossref] [PubMed]
16. Forbes SJ, Gupta S, Dhawan A. Cell therapy for liver disease: From liver transplantation to cell factory. J Hepatol 2015;62:S157-69. [Crossref] [PubMed]