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2D and 3D liver models

  • Sarah H. Saxton
    Affiliations
    Department of Bioengineering, University of Washington, Seattle, WA 98105, USA

    Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98105, USA
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  • Kelly R. Stevens
    Correspondence
    Corresponding author. Address: 850 Republican St., Seattle, WA 98109, USA; Tel.: (206) 897-1519.
    Affiliations
    Department of Bioengineering, University of Washington, Seattle, WA 98105, USA

    Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98105, USA

    Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98105, USA

    Brotman Baty Institute, University of Washington, Seattle, WA 98105, USA
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Published:August 26, 2022DOI:https://doi.org/10.1016/j.jhep.2022.06.022

      Keywords

      The liver performs hundreds of essential functions for the human body, including protein synthesis, nitrogen metabolism, detoxification, and drug metabolism. Yet hepatocytes, the primary functional cell of the liver, have been historically difficult to culture ex vivo. To overcome this hurdle, a variety of complementary in vitro liver models have been introduced. These models, which can be broadly categorized into 2D vs. 3D models, now offer a sizable toolkit with which to study human hepatic biology and disease. Culture format restrictions and hepatic functionality (e.g., albumin secretion, cytochrome induction, and urea secretion) stratify each model, though none of them are both simple to use and effective for predicting all hepatic functions. Future work must focus on model affordability/accessibility, throughput, and long-term maintenance of a broad range of hepatic functions.

      2D adherent cultures

      Cells from various hepatic cell sources are plated onto a cell culture dish as a monolayer. The simplicity of 2D approaches makes them particularly amenable for high-throughput studies, such as pharmaceutical screening.
      Hepatic cell lines have been developed from liver tumors and immortalized hepatocytes, including HepaRG, HepG2, HuH-7, and others. These cell lines are expandable and affordable, making them especially useful for studies that require large numbers of cells. However, most cell lines exhibit abnormal liver-specific functions (e.g., drug metabolism), are genetically abnormal, and imperfectly replicate all aspects of hepatocyte biology.
      • Serras A.S.
      • Rodrigues J.S.
      • Cipriano M.
      • Rodrigues A.v.
      • Oliveira N.G.
      • Miranda J.P.
      A critical perspective on 3D liver models for drug metabolism and toxicology studies.
      Primary human hepatocytes can be isolated from human or other mammalian livers. These cells retain their hepatic phenotype and functions for a short period of time (days) in a 2D monolayer but then progressively dedifferentiate.
      • Khetani S.R.
      • Bhatia S.N.
      Microscale culture of human liver cells for drug development.
      Primary hepatocyte morphology and functional stability can be prolonged in 2D by incorporating elements that mimic the hepatic microenvironment, such as culturing hepatocytes at high-density to increase cellular interactions, and/or growing cells on a collagen-coated surface or between 2 layers of extracellular matrix, known as sandwich culture.
      • Serras A.S.
      • Rodrigues J.S.
      • Cipriano M.
      • Rodrigues A.v.
      • Oliveira N.G.
      • Miranda J.P.
      A critical perspective on 3D liver models for drug metabolism and toxicology studies.
      However, even in these formats hepatic function declines within a few days in culture, limiting use to short-term studies of function and acute toxicity. Furthermore, this model continues to be limited by cell sourcing, as long-term expansion of functional adult primary human hepatocytes in vitro has not yet been achieved.
      iPSC-derived hepatocytes: Induced pluripotent stem cells (iPSC) can be differentiated into hepatocyte-like cells by recapitulating developmental signaling networks in vitro. While iPSC-derived hepatocytes offer a potentially unlimited cell source, current differentiation protocols attain only a fetal-like hepatocyte state, which precludes studies of adult hepatic biology.
      • Serras A.S.
      • Rodrigues J.S.
      • Cipriano M.
      • Rodrigues A.v.
      • Oliveira N.G.
      • Miranda J.P.
      A critical perspective on 3D liver models for drug metabolism and toxicology studies.
      ,
      • Corbett J.L.
      • Duncan S.A.
      iPSC-derived hepatocytes as a platform for disease modeling and drug discovery.
      Addition of other cell populations in co-culture: Addition of a variety of stromal or non-parenchymal liver cell populations to hepatic cultures prolongs hepatic function of most of the hepatic cell types above. Additionally, further spatially patterning these different cell populations in 2D culture (e.g., micropatterned co-culture
      • Khetani S.R.
      • Bhatia S.N.
      Microscale culture of human liver cells for drug development.
      ,
      • Underhill G.H.
      • Khetani S.R.
      Bioengineered liver models for drug testing and cell differentiation studies.
      ) stabilizes hepatic morphology and function for weeks, enabling chronic toxicity and metabolism studies not possible with other 2D models. However, this method requires expertise and equipment not easily accessible to all laboratories.
      • Serras A.S.
      • Rodrigues J.S.
      • Cipriano M.
      • Rodrigues A.v.
      • Oliveira N.G.
      • Miranda J.P.
      A critical perspective on 3D liver models for drug metabolism and toxicology studies.

      3D culture

      3D culture offers functional and morphological advantages by recapitulating cell-cell and cell-extracellular matrix interactions that are absent in 2D culture, though these methods are often more difficult to translate into high-throughput approaches.
      Spheroids: Primary hepatocytes can form spheroids through culture in hanging drops, microwells, non-adherent plates, or other aggregation strategies. Spheroid culture prolongs maintenance of hepatic phenotype and function, which is further improved by co-aggregation with stromal or non-parenchymal cells.
      • Underhill G.H.
      • Khetani S.R.
      Bioengineered liver models for drug testing and cell differentiation studies.
      • Stevens K.R.
      • Ungrin M.D.
      • Schwartz R.E.
      • Ng S.
      • Carvalho B.
      • Christine K.S.
      • et al.
      InVERT molding for scalable control of tissue microarchitecture.
      • Proctor W.R.
      • Foster A.J.
      • Vogt J.
      • Summers C.
      • Middleton B.
      • Pilling M.A.
      • et al.
      Utility of spherical human liver microtissues for prediction of clinical drug-induced liver injury.
      Liver organoids: Hepatic cells can transiently proliferate and self-organize to form liver organoids that mimic the structure and function of human liver when cultured under specific media and matrix conditions. Various organoid models have been developed from numerous hepatic cell types that vary in their similarity to human hepatocytes, including iPSC-derived hepatoblasts,
      • Takebe T.
      • Sekine K.
      • Enomura M.
      • Koike H.
      • Kimura M.
      • Ogaeri T.
      • et al.
      Vascularized and functional human liver from an iPSC-derived organ bud transplant.
      hepatic stem/biliary cells,
      • Cordero-Espinoza L.
      • Dowbaj A.M.
      • Kohler T.N.
      • Strauss B.
      • Sarlidou O.
      • Belenguer G.
      • et al.
      Dynamic cell contacts between periportal mesenchyme and ductal epithelium act as a rheostat for liver cell proliferation.
      and primary fetal and pediatric hepatocytes.
      • Hu H.
      • Gehart H.
      • Artegiani B.
      • Löpez-Iglesias C.
      • Dekkers F.
      • Basak O.
      • et al.
      Long-term expansion of functional mouse and human hepatocytes as 3D organoids.
      Though hepatic organoid culture shows potential to generate long-term models that conserve liver structure and function, current organoids remain functionally immature or biliary in phenotype.
      Liver slice culture: Resected liver biopsies can be sliced into thin tissue sections and cultured ex vivo for up to 2 weeks. Liver slices retain the cellular diversity of the liver and maintain in vivo cellular architecture but are subject to major patient-to-patient variability and cellular death after resection and culture.
      • Palma E.
      • Doornebal E.J.
      • Chokshi S.
      Precision-cut liver slices: a versatile tool to advance liver research.
      Organ on a chip and microfluidic models: Using fabrication methods such as soft lithography, hepatocytes with or without non-parenchymal cells can be spatially patterned in 3D. Fluid flow can be introduced to recapitulate important mechanical stimuli that are missing from most other in vitro models. While such models offer physiologic improvements, they can be labor-intensive to make and maintain, limiting their throughput.
      • Serras A.S.
      • Rodrigues J.S.
      • Cipriano M.
      • Rodrigues A.v.
      • Oliveira N.G.
      • Miranda J.P.
      A critical perspective on 3D liver models for drug metabolism and toxicology studies.
      ,
      • Underhill G.H.
      • Khetani S.R.
      Bioengineered liver models for drug testing and cell differentiation studies.
      Bioprinted liver: By marrying 3D printing technologies with cytocompatible biological “inks”, engineers can bioprint tissue models that incorporate parenchymal and support cells in spatially patterned arrangements. For example, advances in stereolithography
      • Grigoryan B.
      • Paulsen S.J.
      • Corbett D.C.
      • Sazer D.W.
      • Fortin C.L.
      • Zaita A.J.
      • et al.
      Multivascular networks and functional intravascular topologies within biocompatible hydrogels.
      and sacrificial scaffolds
      • Kinstlinger I.S.
      • Saxton S.H.
      • Calderon G.A.
      • Vasquez Ruiz K.
      • Yalacki D.R.
      • Deme P.R.
      • et al.
      Generation of model tissues with dendritic vascular networks via sacrificial laser-sintered carbohydrate templates.
      have enabled early models of the liver, though future improvements to printing materials and resolution are still needed to accurately replicate liver architecture.

      Financial support

      NIH R01DK128551, The Wellcome Leap Fund, and the W. M. Keck Foundation.

      Authors’ contributions

      SHS and KS: conceptualization, writing, and figure design.

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following are the supplementary data to this article:

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