3D printing functional liver discoids

The fields of tissue engineering and regenerative medicine have been evolving at a rapid rate as cell therapies are being translated into the clinic and humanized tissues models become an increasingly popular alternative to animal testing. For example, promising results have been reported about the use of dopaminergic neurons derived from human embryonic stem cells (hESCs) for treating Parkinson’s disease in terms of safety and efficacy (1). Additionally, the recent trend from funding bodies, like the National Institutes of Health, indicate a shift away from the use of animal model and toward humanized systems, including three-dimensional (3D) bioprinted tissues (2). 3D bioprinting serves as a highly promising technology for advancing research and technology development in regenerative medicine and tissue engineering. It represents an important and growing subset of additive manufacturing that can be used to produce tissues to replace diseased or damaged organs as well as serve as a tool for drug screening (3). The capability of 3D printing to generate complex structures based on computer aided design files has huge implications for engineering functional tissues. For example, 3D bioprinting can overcome the limitations of existing 2D monolayer cultures, which tend to de-differentiate and rapidly lose tissue-specific functions—an important consideration when using them for applications in screening drugs for toxicity and efficacy. A recent Cell Stem Cell paper focused on how patient derived induced pluripotent stem cell lines can be used to generate complex neural constructs that replicate disease phenotypes (4,5). Also, work by my own group published in Bioelectric Medicine used 3D bioprinting to model Alzheimer’s disease—indicating the power of this technology for studying complicated human biology (5,6). Such in vitro human models have become increasingly important as the National Institutes of Health recently decided to no longer fund grants that focused exclusively on animal research. The generation of larger tissues and organs produced by bioprinting represents a potential way to address organ shortages (7).

Despite the tremendous potential of 3D bioprinting to generate complex living structures, many challenges must be addressed before this technology can be adopted widely, including scaling up the production of cells to make these bioprinted tissues, printing these constructs in a fashion amenable to high throughput screening while retaining physiologically relevant biology, and vascularizing these tissues to generate larger tissues and organs suitable for transplantation. Some of the major challenges when producing vasculature is how to design and implement these complex structures inside of a construct while ensuring that perfusion can occur and be maintained, which have been discussed and addressed in two recent publications in Science (8) and Science Advances (9). Additional challenges include producing the large quantities of cells needed for generating such constructs as the printing process often requires millions of cells per milliliter of bioink used when printing (10,11). These challenges must be addressed to fully realize the potential of 3D bioprinting for generating transplantable organs.

In terms of more immediate clinical applications, functional 3D bioprinted constructs can be produced on a smaller scale that lends themselves to high throughput drug screening, which is the focus of the paper entitled “A functional human liver tissue model: 3D bioprinted co-culture discoids” being discussed here (12). These human tissue models can serve as a way to quickly perform high throughput screening potential drugs for efficacy and toxicity due to their ability to replicate features of healthy and diseased tissues (13). Figure 1 shows a comparison of traditional animal models for drug screening with the use of bioprinted tissues for high throughput screening for evaluating liver toxicity.

Figure 1 Comparison of the traditional methods for screening potential hepatotoxicity using rodent models compared to the high throughput screening of human bioprinted liver tissues. Created in Biorender. 3D, three dimensional.

One of the major challenges when developing new pharmaceutical drugs is predicting if these formulations will induce unwanted side effects like liver toxicity (14). The phenomenon is known as drug induced liver injury (DILI) cannot be accurately predicted by animal models or 2D cell cultures (14,15). Thus, the use of humanized 3D tissue models has been investigated as an alternative method to assess the potential of therapeutics to induce this effect, including this recent paper published in Biomaterials Advances. Here Dr. Thomas Angelini and his colleagues bioprinted a functional liver model that could serve a way to perform high throughput screening of drugs for toxicity (12). This novel approach used a custom 3D bioprinter to produce disc-shaped liver tissue constructs that they call “discoids” composed of different ratios of hepatocytes, cholangiocytes, and human umbilical vascular endothelial cells (HUVECs) mixed in bioinks composed of collagen-1 into a highly permeable support medium made from packed polyethylene glycol (PEG) microgels, enabling them to maintain cell function and viability. The use of support baths has become a popular strategy for printing structures using delicate bioinks. This concept was initially pioneered by the Feinberg lab at Carnegie Mellon University who termed the process freeform reversible embedding of soft hydrogels (FRESH) as it enabled them to print soft polymeric materials like collagen and fibrin into stable structures (16).

Here the authors have chosen to print into a support bath consisting of PEG microgels with an average size of 9.46±4.25 µm that were produced through emulsification followed by cross-linking to ensure consistency. They chose to use PEG due to its non-adhesive properties. Accordingly, the bioink-cell mixture retains its shape post printing as the structure would be encapsulated inside on the inert support bath. They also characterized the rheology of their collagen based bioink containing cells and fond that it had a viscosity of 12.5±0.8 mPa·s. In terms of the cells themselves, they printed human hepatocytes both alone and in combination with HUVECs and human cholangiocytes—the specialized epithelial cells that line the bile ducts that enable its transport in the liver. Different cell mixtures were extruded in these “discoid” shapes using a custom bioprinter that extruded in a spiral path when generating the desired structure. The combination of the printer, support bath, and structure size enabled the authors to print these structures into 96 well plates—which would enable high throughput screening of drugs. Additionally, they were able to achieve this rate of fabrication in under an hour—making this a relatively rapid process.

This work also showed that their novel bioprinted “discoid” geometric structure enhanced the cohesion between cells while enhancing metabolic activity when compared to the same cultures printed into spheroids. The printed spheroids also exhibited instability in comparison to the “discoids”, emphasizing the important role of geometry in tissue engineering. They also found that adding HUVECs to these constructs increased production of albumin and urea, which indicated the functionality of the tissues. Interestingly, the incorporation of cholangiocytes leads to decreases in the secretion of albumin and urea both in the presence and absence of HUVECs in the construct. This functionality was further confirmed by mRNA analysis of the gene expression patterns associated with adsorption, distribution, metabolism and excretion that showed comparable levels of expression in these tissues compared to human liver tissue. They also demonstrated these tissues exhibited cytochrome P450 (CYP) enzymatic activity associated with liver tissues through substrate-based assays. They demonstrated that their uniquely shaped bioprinted “discoids” maintained their structure and function for 21 days—showing their stability and indicating the potential for screening drugs for chronic toxicity effects.

The study highlights the importance of cellular composition, tissue size, and geometry in optimizing liver tissue model function and stability, providing valuable insights for future 3D printing research in for applications in high throughput tissue engineering. Additionally, the research demonstrates that the printing method is crucial as it enables proper formation of the tissue architecture with appropriate cellular composition when producing functional constructs. This study also emphasizes the need to do a thorough characterization of the biological and functional properties of such tissue constructs and benchmark them against their human tissue counterparts. This breakthrough in 3D bioprinting technology represents a significant step toward more reliable and accessible in vitro liver models that could transform drug development and toxicity testing processes, ultimately leading to safer and more effective pharmaceutical products in humans. Advances like ones detailed in this paper will lead to more widespread uptake of 3D bioprinting for clinical applications, including printing complete organs.

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-25-107/prf

Funding: This work was supported by funding from the NSERC Discovery Grant program, the NSERC Alliance Grant program, the B.C. Ministry of Forests, the CIHR Project Grant program, and the NSERC Idea to Innovation Grant program. S.M.W. is the C.E.O. of Axolotl Biosciences and she holds shares in this company.

Conflicts of Interest: The author has completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-25-107/coif). S.M.W. reports that this work was supported by funding from the NSERC Discovery Grant program, the NSERC Alliance Grant program, the B.C. Ministry of Forests, the CIHR Project Grant program, and the NSERC Idea to Innovation Grant program. S.M.W. is the C.E.O. of Axolotl Biosciences and she holds shares in this company. The author has no other conflicts of interest to declare.

Ethical Statement: The author is 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.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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