An overview of tissue and whole organ decellularization processes
Open accessAbstract
Biologic scaffold materials composed of extracellular matrix (ECM) are typically derived by processes that involve decellularization of tissues or organs. Preservation of the complex composition and three-dimensional ultrastructure of the ECM is highly desirable but it is recognized that all methods of decellularization result in disruption of the architecture and potential loss of surface structure and composition. Physical methods and chemical and biologic agents are used in combination to lyse cells, followed by rinsing to remove cell remnants. Effective decellularization methodology is dictated by factors such as tissue density and organization, geometric and biologic properties desired for the end product, and the targeted clinical application. Tissue decellularization with preservation of ECM integrity and bioactivity can be optimized by making educated decisions regarding the agents and techniques utilized during processing. An overview of decellularization methods, their effect upon resulting ECM structure and composition, and recently described perfusion techniques for whole organ decellularization techniques are presented herein.
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For thinner tissues, the extent of cellular removal depends on the intensity of agitation or exposure to relatively mild detergent concentrations.
“For thinner tissues, the extent of cellular removal depends on the intensity of agitation or exposure to relatively mild detergent concentrations [23].”
From Effects of Various Decellularization Methods for the Development of Decellularized Extracellular Matrix from Tilapia (<i>Oreochromis niloticus</i>) Viscera (2024) · cites this paper as [23]
Section 4.4 (page unavailable) says that DNA removal from thin tissues depends on agitation speed. Section 4 (page unavailable) describes brief exposure of thin tissues to easily removed detergents or acids, but does not say that cellular removal depends on exposure to relatively mild detergent concentrations. (LLM, conf=0.86)
Experimental — accuracy not yet measuredAll decellularization methods yielded DNA content below the acceptable residual threshold of 0.05 μg/mg.
“with all methods revealing DNA content below the acceptable residual threshold of 0.05 µg/mg [23].”
From Effects of Various Decellularization Methods for the Development of Decellularized Extracellular Matrix from Tilapia (<i>Oreochromis niloticus</i>) Viscera (2024) · cites this paper as [23]
Section 6 (page unavailable) proposes less than 50 ng dsDNA per mg of dry ECM—equivalent to 0.05 µg/mg—as a minimal criterion for decellularization. It does not report that all methods met this criterion. Section 3.1 (page unavailable) instead describes DNA removal as varying by method and tissue. (LLM, conf=0.88)
Experimental — accuracy not yet measuredHypertonic and hypotonic solutions induce cell lysis, dehydration, and apoptosis, thereby removing cellular content.
“Hypertonic and hypotonic solutions are employed in the fabrication of decellularized scaffolds for their osmotic effects, which induce cell lysis, dehydration, and apoptosis, thereby removing cellular content.”
From Bioactive scaffolds for tissue engineering: A review of decellularized extracellular matrix applications and innovations (2025) · cites this paper as [29]
Section 3.1.2 says hypotonic solutions cause cell lysis through osmotic effects and that alternating hypertonic and hypotonic solutions helps rinse cell residue from tissue. It does not attribute dehydration or apoptosis to these solutions; section 3.1.4 instead attributes cellular dehydration to alcohols. Page numbers are unavailable in our copy. (LLM, conf=0.90)
Experimental — accuracy not yet measuredNucleases, including DNase, hydrolyze nucleotide bonds, thereby fragmenting DNA and RNA following cell lysis.
“Nucleases, including DNase, hydrolyze nucleotide bonds, thereby fragmenting DNA and RNA following cell lysis.”
From Bioactive scaffolds for tissue engineering: A review of decellularized extracellular matrix applications and innovations (2025) · cites this paper as [29]
Section 3.2.1 (Enzymes; page unavailable) says DNases and RNases cleave nucleic acid sequences after cell lysis and that endonucleases fragment DNA. It does not specify that this cleavage occurs by hydrolysis of nucleotide bonds, so that mechanistic detail is unsupported by our copy. (LLM, conf=0.81)
Experimental — accuracy not yet measuredDecellularized tissues may experience a lower rejection rate if they are completely depleted of the different sources of immunogens.
“Overall, decellularized tissues may experience a lower rejection rate if they are completely depleted of the different sources of immunogens [7,59].”
From Liver tissue engineering using decellularized scaffolds: Current progress, challenges, and opportunities (2024) · cites this paper as [7]
Section 6, “Evaluation of decellularized ECM” (page unavailable), says residual cellular material may contribute to adverse host responses, supporting the general rationale for removing immunogenic material. It also says decellularization cannot remove 100% of cell material; the paper does not establish a lower rejection rate for tissues completely depleted of all immunogens. (LLM, conf=0.85)
Experimental — accuracy not yet measured
Enzymatic treatments using nucleases, trypsin, alpha-galactosidase, dispase, phospholipase, and collagenase are commonly employed methods for decellularization.
“Enzymatic treatments using nucleases, trypsin, alpha-galactosidase, dispase, phospholipase, and collagenase are commonly employed methods for decellularization [7,146].”
From Liver tissue engineering using decellularized scaffolds: Current progress, challenges, and opportunities (2024) · cites this paper as [7]
Section 3.2.1 (Enzymes; page not provided) lists nucleases, trypsin, collagenase, dispase, and α-galactosidase among enzymes reported in decellularization protocols. It lists lipase, not phospholipase, so the source does not support that item in the claim. (LLM, conf=0.88)
Experimental — accuracy not yet measuredSDS can denature proteins and lyse cells by disrupting protein-protein interactions, while Triton X-100 can disrupt lipid-lipid, lipid-protein, and DNA-protein interactions while preserving protein-protein interaction.
“SDS can denature proteins and lyse cells by disrupting protein-protein interactions, while Triton X-100 can disrupt lipid–lipid, lipid–protein, and DNA-protein interactions while preserving protein-protein interaction [7,59].”
From Liver tissue engineering using decellularized scaffolds: Current progress, challenges, and opportunities (2024) · cites this paper as [7]
Section 3.1.3 (Chemical agents; page not provided) says detergents, including SDS and Triton X-100, solubilize cell membranes and dissociate DNA from proteins. It also describes Triton X-100 as effective for delipidation and SDS as more disruptive to the ECM. The paper does not specify all the interaction types in the claim; moreover, it says detergents can disrupt and dissociate ECM proteins, so it does not support the unqualified assertion that Triton X-100 preserves protein–protein interactions. (LLM, conf=0.84)
Experimental — accuracy not yet measuredSDS-treated ECM scaffolds generally have higher reactivities upon implantation due to disruption of collagen fiber structures and exposure of cryptic antigens.
“Of note, research has shown that SDS-treated ECM scaffolds generally have higher reactivities upon implantation due to the disruption of collagen fiber structures and exposure of cryptic antigens [7,59,94].”
From Liver tissue engineering using decellularized scaffolds: Current progress, challenges, and opportunities (2024) · cites this paper as [7]
Section 6 (page not available) reports that detergents can disrupt collagen in certain tissues, but also says that SDS effects vary and collagen may be unaffected in similar tissues. Section 3.1 (page not available) warns that residual SDS can be cytotoxic. Neither passage establishes that SDS-treated scaffolds generally cause greater reactivity after implantation or that disruption exposes cryptic antigens. (LLM, conf=0.85)
Experimental — accuracy not yet measuredThe efficiency of decellularization via perfusion depends on the route of perfusion, perfusion direction, perfusion conditions, duration, type and concentration of decellularizing agents, and the size of the liver.
“Importantly, the efficiency of decellularization via perfusion depends on various factors, including the route of perfusion (hepatic artery or portal vein or inferior vena cava), perfusion direction (antegrade or retrograde), perfusion conditions (controlled pressure and flow rate), duration of the perfusion, type and concentration of decellularizing agents, and the size of the liver [7,26,170].”
From Liver tissue engineering using decellularized scaffolds: Current progress, challenges, and opportunities (2024) · cites this paper as [7]
Section 4.1 (page unavailable) describes portal-vein perfusion of livers, antegrade and retrograde organ perfusion, and differences in agents and concentrations. It also links pressure and flow to decellularization time in hearts. It does not establish that liver decellularization efficiency depends on every listed factor, particularly hepatic-artery or inferior-vena-cava routes and liver size. (LLM, conf=0.86)
Experimental — accuracy not yet measuredBadylak and his team suggested that a necessary criterion to assure decellularization efficiency is that the DNA fragment length should be less than 200 bp, as determined by gel electrophoresis.
“The third criterion is that the DNA fragment length should be less the 200 bp, as determined by gel electrophoresis.”
From Liver tissue engineering using decellularized scaffolds: Current progress, challenges, and opportunities (2024) · cites this paper as [7]
Section 6 (Evaluation of decellularized ECM) lists DNA fragments shorter than 200 bp among its proposed minimal criteria and says this criterion is evaluated by gel electrophoresis. But it identifies fragment length as the second criterion; the third is the absence of visible nuclear material in stained tissue sections. (LLM, conf=0.90)
Experimental — accuracy not yet measuredBadylak and his team suggested that a necessary criterion to assure decellularization efficiency is the lack of nuclear materials in sections stained with DAPI or H&E.
“The first criterion is the lack of nuclear materials in sections stained with 4’,6-diamidino-2-phenylindole (DAPI) or hematoxylin and eosin (H&E).”
From Liver tissue engineering using decellularized scaffolds: Current progress, challenges, and opportunities (2024) · cites this paper as [7]
Section 6 lists the lack of visible nuclear material in DAPI- or H&E-stained sections among its minimal criteria for decellularization. But it identifies this as the third criterion, not the first; the first concerns the amount of residual dsDNA. (LLM, conf=0.88)
Experimental — accuracy not yet measuredDecellularization is a critical process aimed at eliminating cells and nuclear content while preserving the scaffold's native architecture and functional components.
“Decellularization is a critical process aimed at eliminating cells and nuclear content (e.g., DNA, phospholipids, intracellular proteins) while preserving the scaffold's native architecture and functional components [2,97,98].”
From Immune Response to Extracellular Matrix Bioscaffolds: A Comprehensive Review (2025) · cites this paper as [97]
The effectiveness of detergent penetration in intricate tissue is largely influenced by the method of decellularization used.
“The effectiveness of detergent penetration in this intricate network of tissue is largely influenced by the method of decellularization used [23].”
From Effects of Various Decellularization Methods for the Development of Decellularized Extracellular Matrix from Tilapia (<i>Oreochromis niloticus</i>) Viscera (2024) · cites this paper as [23]
The acceptable residual DNA threshold for decellularized ECM is 0.05 μg/mg.
“with all methods revealing DNA content below the acceptable residual threshold of 0.05 µg/mg [23].”
From Effects of Various Decellularization Methods for the Development of Decellularized Extracellular Matrix from Tilapia (<i>Oreochromis niloticus</i>) Viscera (2024) · cites this paper as [23]
NTIRE's effectiveness is limited by probe size and might not achieve complete decellularization alone.
“Its effectiveness is limited by probe size and might not achieve complete decellularization alone.”
From Bioactive scaffolds for tissue engineering: A review of decellularized extracellular matrix applications and innovations (2025) · cites this paper as [29]
If successful, decellularization would result in developing scaffolds that mimic the mechanical, biological, and anatomical properties of native tissue.
“If successful, decellularization would result in developing scaffolds that mimic the mechanical, biological, and anatomical properties of native tissue [7].”
From Liver tissue engineering using decellularized scaffolds: Current progress, challenges, and opportunities (2024) · cites this paper as [7]
It is very difficult to consider one decellularization agent as an ideal method that efficiently balances depletion of cytoplasmic and nuclear content and preservation of essential structural and bioactive ECM components that promote attachment, growth, and differentiation of reseeded cells.
“Although various methods, including physical, chemical, and enzymatic treatments or a combination of them, have been used for liver decellularization (Fig. 2) [7], it is very difficult to consider one of those agents as an ideal method that efficiently balances the depletion of the cytoplasmic and nuclear content of the tissue and the preservation of essential structural and bioactive ECM”
From Liver tissue engineering using decellularized scaffolds: Current progress, challenges, and opportunities (2024) · cites this paper as [7]
Although chemical agents play a key role in decellularization and removal of immunogenic materials, they may cause ECM damage and affect biocompatibility of yielded decellularized tissues.
“Although chemical agents play a key role in the decellularization and removal of the immunogenic materials from the decellularized tissues, they may cause ECM damage and affect the biocompatibility of the yielded decellularized tissues [5,7,[90], [91], [92]].”
From Liver tissue engineering using decellularized scaffolds: Current progress, challenges, and opportunities (2024) · cites this paper as [7]
Perfusion represents an ideal approach for decellularizing large, thick, compact organs like the liver.
“Perfusion, which involves the infusion of chemical or biological agents through the cannulated intrinsic vascular system, represents an ideal approach for decellularizing large, thick, compact organs like the liver [7,12].”
From Liver tissue engineering using decellularized scaffolds: Current progress, challenges, and opportunities (2024) · cites this paper as [7]
Badylak and his team suggested that a necessary criterion to assure decellularization efficiency is depletion of DNA from the ECM, with the scaffold containing less than 50 ng dsDNA/mg ECM dry weight.
“The second criterion is the depletion of DNA from the ECM, with the scaffold containing less than 50 ng dsDNA/mg ECM dry weight, as quantified using commercially available dsDNA kits.”
From Liver tissue engineering using decellularized scaffolds: Current progress, challenges, and opportunities (2024) · cites this paper as [7]
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- Authors
- Crapo, P. M., Gilbert, T. W., & Badylak, S. F.
- Affiliations
- University of Pittsburgh (US)
- Corresponding author
- Badylak, Stephen F.
- Venue
- Biomaterials
- Publisher
- Elsevier BV
- Published
- 2011-04
- Volume
- 32
- Issue
- 12
- Pages
- 3233-3243
- ISSN
- 0142-9612 (ISSN-L)
- PMC
- PMC3084613
- OpenAlex
- W2059486645
- Cited by
- 3,591
- References
- 127
- Citations by year
- 2026: 246 · 2025: 275 · 2024: 309 · 2023: 308 · 2022: 297 · 2021: 363 · 2020: 301 · 2019: 269 · 2018: 240 · 2017: 265 · 2016: 185 · 2015: 202 · 2014: 145 · 2013: 85 · 2012: 74
- FWCI
- 18.82
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Biomaterials (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR053603)
- Topics
- Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
- Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Medicine › Hydrogels: synthesis, properties, applications
- Keywords
- decellularization, extracellular matrix, biological scaffolds, perfusion decellularization, cell elimination, tissue engineering
- MeSH terms
- Animals, Extracellular Matrix, Organ Specificity, Sterilization, Tissue Engineering, Tissue Scaffolds
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