Method for perfusion decellularization of porcine whole liver and kidney for use as a scaffold for clinical‐scale bioengineering engrafts
Abstract
Background: Whole‐organ engineering provides a new alternative source of donor organs for xenotransplantation. Utilization of decellularized whole‐organ scaffolds, which can be created by detergent perfusion, is a strategy for tissue engineering. In this article, our aim is to scale up the decellularization process to human‐sized liver and kidney to generate a decellularized matrix with optimal and stable characteristics on a clinically relevant scale. Methods: Whole porcine liver and kidney were decellularized by perfusion using different detergents (1% SDS, 1% Triton X‐100, 1% peracetic acid (PAA), and 1% Na DOC) via the portal vein and renal artery of the liver and kidney, respectively. After rinsing with PBS to remove the detergents, the obtained liver and kidney extracellular matrix (ECM) were processed for histology, residual cellular content analysis, and ECM components evaluation to investigate decellularization efficiency, xenoantigens removal, and ECM preservation. Results: The resulting liver and kidney scaffolds in the SDS‐treated group showed the most efficient clearance of cellular components and xenoantigens, including DNA and protein, and preservation of the extracellular matrix composition. In comparison, cell debris was observed in the other decellularized groups that were generated using Triton X‐100, PAA, and Na DOC. Special staining and immunochemistry of the porcine liver and kidney ECM s further confirmed the disrupted three‐dimension ultrastructure of the ECM in the Triton X‐100 and Na DOC groups. Additionally, Triton X‐100 effectively eliminated the residual SDS in the SDS‐treated group, which ensured the scaffolds were not cytotoxic to cells. Thus, we have developed an optimal method that can be scaled up for use with other solid whole organs. Conclusions: Our SDS‐perfusion protocol can be used for porcine liver and kidney decellularization to obtain organ scaffolds cleared of cellular material, xenoimmunogens, and preserved vital ECM components.
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Triton X-100 tends to break DNA into larger fragments, which might induce an adverse tissue remodeling response, and while it preserves ECM protein structures better than SDS, it may still pose immunogenic risks due to remnant DNA.
“However, one study reported that Triton X-100 tends to break DNA into larger fragments, which might induce an adverse tissue remodeling response. This finding indicates that while Triton X-100 preserves ECM protein structures better than SDS, it may still pose some immunogenic risks due to remnant DNA [95].”
From Liver tissue engineering using decellularized scaffolds: Current progress, challenges, and opportunities (2024) · cites this paper as [95]
Quantification of residual DNA as a potent immunogen and indirect index of remaining cell materials is a practical tool to estimate remaining immunogen amount throughout dECM.
“The quantification of residual DNA as a potent immunogen and indirect index of remaining cell materials is a practical tool to estimate the remaining immunogen amount throughout the dECM [54–56].”
From Immunogenicity of decellularized extracellular matrix scaffolds: a bottleneck in tissue engineering and regenerative medicine (2023) · cites this paper as [55]
PAA's adverse effects on ECM structure and composition and later repopulation seem negligible; however, it is not a strong agent in removing immunogen cell materials and is mainly used for sterilization.
“PAA’s adverse effects on ECM structure and composition and later repopulation seem negligible; however, it is not a strong agent in removing immunogen cell materials and is mainly used for sterilization [48, 55].”
From Immunogenicity of decellularized extracellular matrix scaffolds: a bottleneck in tissue engineering and regenerative medicine (2023) · cites this paper as [55]
TX100 could be applied to eliminate residual SDS from decellularized tissues, making TX100/SDS a good combination for better cell removal and minimization of cytotoxicity synchronously.
“TX100 could also be applied to eliminate residual SDS from decellularized tissues, making TX100/SDS a good combination for better cell removal and minimization of cytotoxicity synchronously [55].”
From Immunogenicity of decellularized extracellular matrix scaffolds: a bottleneck in tissue engineering and regenerative medicine (2023) · cites this paper as [55]
TX100 breaks DNA down into larger fragments than ionic detergents, which is disadvantageous in terms of immunogenicity of dECM scaffolds.
“Moreover, TX100 breaks DNA down into larger fragments than ionic detergents, which is disadvantageous in terms of the immunogenicity of dECM scaffolds [55].”
From Immunogenicity of decellularized extracellular matrix scaffolds: a bottleneck in tissue engineering and regenerative medicine (2023) · cites this paper as [55]
SDC is a potent agent for extracting cellular proteins from dense tissues.
“SDC, another ionic detergent, is a potent agent for extracting cellular proteins from dense tissues [55].”
From Immunogenicity of decellularized extracellular matrix scaffolds: a bottleneck in tissue engineering and regenerative medicine (2023) · cites this paper as [55]
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Decellularization of tissues and organs
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“This finding indicates that while Triton X-100 preserves ECM protein structures better than SDS, it may still pose some immunogenic risks due to remnant DNA [95].”
BackgroundImmune Response to Extracellular Matrix Bioscaffolds: A Comprehensive Review
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“Implementation of scalable systems of perfusion decellularization is also an alternative, especially in highly vascularized tissues [188–190].”
Strategies for decellularization, re-cellularization and crosslinking in liver bioengineering
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- Authors
- Wang, Y., Bao, J., Wu, Q., Zhou, Y., Li, Y., Wu, X., Shi, Y., Li, L., & Bu, H.
- Affiliations
- Sichuan University (CN)
- Southwest Medical University (CN)
- Corresponding author
- Bu, Hong
- Venue
- Xenotransplantation
- Publisher
- Wiley
- Published
- 2014-10-07
- Volume
- 22
- Issue
- 1
- Pages
- 48-61
- ISSN
- 0908-665X (ISSN-L)
- 1399-3089
- OpenAlex
- W1988411148
- Cited by
- 125
- References
- 5
- Citations by year
- 2026: 2 · 2025: 6 · 2024: 8 · 2023: 19 · 2022: 7 · 2021: 17 · 2020: 8 · 2019: 10 · 2018: 6 · 2017: 18 · 2016: 12 · 2015: 10 · 2014: 1
- FWCI
- 13.56
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Xenotransplantation (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Natural Science Foundation of China (81200315)
- Topics
- Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
- Health Sciences › Medicine › Surgery › Xenotransplantation and immune response
- Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
- Keywords
- perfusion decellularization, porcine liver, porcine kidney, sodium deoxycholate, Triton X-100, peracetic acid, extracellular matrix preservation
- MeSH terms
- Animals, Basement Membrane, Deoxycholic Acid, Detergents, DNA, Extracellular Matrix, Kidney, Liver, Male, Membrane Proteins, Microscopy, Electron, Scanning, Peracetic Acid, Perfusion, Portal Vein, Renal Artery, Sodium Dodecyl Sulfate, Swine, Swine, Miniature, Octoxynol, Tissue Scaffolds, Bioengineering
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