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ORIGINAL RESEARCH ARTICLE
Protocol for a liver–bone organoid platform to study senescence-driven interorgan crosstalk
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1 Department of Diagnostic Radiology, Yong Loo Lin School of Medicine, National University of Singapore,
Singapore
2 Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Department of Pathophysiology, Shanghai Jiao Tong University School of Medicine, Shanghai,
China
3 Department of Orthopaedics, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai,
China
OR, 026170024 https://doi.org/10.36922/OR026170024
Received: 23 April 2026 | Revised: 2 June 2026 | Accepted: 9 June 2026 | Published online: 26 June 2026
© 2026 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
Abstract
Liver–bone crosstalk is increasingly recognized as an important regulatory axis in aging-associated diseases, such as metabolic liver disease and osteoporosis. However, conventional two-dimensional co-culture systems and animal models do not adequately capture the multicellular architecture, extracellular matrix context, and bidirectional secretory communication that underlie interorgan senescence propagation. Here, we describe a protocol for constructing a senescent liver– bone organoid platform by integrating a bone matrix-inspired three-dimensional (3D) bioprinted bone organoid system with a mouse duct-derived liver organoid culture system, followed by engineered senescence induction and conditioned-medium exchange. In this protocol, bone marrow-derived mesenchymal stem cells are incorporated into a gelatin/alginate/hydroxyapatite hybrid bioink and printed into porous 3D constructs, whereas liver ductal structures are embedded in Matrigel and expanded into liver organoids before directed differentiation. Senescence is induced in both organoid types using doxorubicin, and systemic aging-related signals are modeled by treating organoids with serum from older mice. Bidirectional communication is then interrogated through reciprocal conditioned-medium transfer assays. This platform enables investigation of interorgan senescence propagation, liver-bone axis dysfunction, and candidate mediators such as 27-hydroxycholesterol in a physiologically relevant 3D setting.
Keywords
Liver–bone organoid
Liver–bone crosstalk
Senescence
3D bioprinting
Funding
This study was funded by Noncommunicable Chronic Diseases-National Science and Technology Major Project (2024ZD0530900).
Conflict of interest
Ming He serves as an Editorial Board Member of this journal, but was not in any way involved in the editorial and peer-review process conducted for this paper, directly or indirectly. The authors declare that they have no competing interests.
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