Decellularization in Regenerative Medicine: State-of-the-Art and Emerging Frontiers

Abstract

Abstract There is a huge gap between the number of donors available and the number of patients in need of organ transplants. Decellularisation of organs and tissues has emerged as a pivotal approach in regenerative medicine, enabling the creation of natural extracellular matrix scaffolds for human organ regeneration. The process involves removing cellular components from native organs/tissues and preserving the structural and biochemical integrity of the extracellular matrix (ECM). It decreases immunogenicity and provides a framework conducive to recellularisation. The principle of this methodology features complete cell removal, retention of extracellular proteins, and maintenance of biomechanical properties such as tensile strength and elasticity. The decellularization methods range from detergent-based and enzymatic protocols to physical techniques, each balancing efficiency with preservation of scaffold architecture. In our lab, we established a detergent-based decellularization protocol for goat valves and tested for recellularization using a culture method. We evaluated the decellularized heart valves using histological, histochemical, immunohistochemical, and ultrastructural methods, along with DNA quantification using PCR methods. Applications of these techniques range from experimental models to translational use in cardiac surgery, vascular grafts, and whole-organ regeneration, with growing potential in personalized medicine through patient-derived stem cell recellularization. Despite these advances, challenges remain in achieving complete decellularization without ECM damage, ensuring effective sterilization, scaling protocols for clinical use, and navigating regulatory and ethical frameworks. Future directions focus on refining recellularization strategies, biomechanical validation, and long-term implantation studies, ultimately positioning decellularized scaffolds as platforms for multi-organ regeneration. Together, these attempts emphasize the transformative capability of decellularization in filling current gaps in transplantation and pushing the limits of regenerative therapies. Fig. 1. Graphical Abstract Introduction The aim of regenerative medicine is to restore or replace injured tissues and organs by harnessing biological systems and bioengineered products. One of the recent approaches is decelllariazation of organs and tissues. In this process, the cellular components are removed from the native organs/tissues and preserving the extracellular matrix (ECM). The resulting three-dimensional scaffold can be recellularized with autologous or allogeneic cells to create functional tissue grafts. Over the last two decades, researchers have established protocols for decellularising a variety of tissues and whole organs. The tissues include the skin, blood vessels, heart valves, heart, lung, liver, kidney, pancreas, and intestine.(Tabatabai et al., 2024). The research in this field has established that ECM scaffolds can be produced, maintained structurally intact, and can be partially recellularised in vitro and in vivo. Thus, it supports their potential role as biologically derived scaffolds for tissue engineering and as alternatives to transplantation (Hillebrandt et al., 2019). This paper discusses the basic principles, methods, and applications of decellularization in regenerative medicine, highlights current translational achievements, the challenges and future directions in the area. Biological Rationale and Design Principles The extracellular matrix (ECM) is a complex network of structural proteins (collagen and elastin), adhesive glycoproteins (fibronectin and laminins), proteoglycans, glycosaminoglycans (GAGs), and bound signalling molecules, such as growth factors and chemokines. It is responsible for providing structural support and mechanical integrity, and biochemical cues that regulate cell adhesion, proliferation, migration, and differentiation. It provides tissue-specific architecture, including vascular and ductal networks and compartmentalization (e.g., myocardium vs. valves, cortex vs. medulla in kidney). The decellularized ECM (dECM) scaffolds attempt to preserve these features while eliminating cells and nuclear material that can provoke inflammation or immune rejection.(Liu et al., 2025). A perfect decellularization process should remove immunogenic cellular components and preserve ECM composition. Removal of immunogenic cellular components includes cells and cell fragments, nuclear material and major histocompatibility complex (MHC) antigens and species‑specific epitopes (e.g., α‑Gal in porcine tissues) (Hillebrandt et al., 2019). It should preserve key structural proteins, GAGs, and bioactive molecules, maintain ECM architecture at macro-, micro-, and nano‑scales, and retain appropriate mechanical properties. It should not contain cytotoxic residues, detergents, enzymes, or sterilants and should be reproducible and scalable, adaptable to human-sized tissues. In practice, decellularization protocols represent a compromise: more aggressive treatments improve cell removal but often damage the ECM, whereas gentler protocols preserve ECM but may leave residual DNA and antigens.(Liu et al., 2025). Decellularization Methods Decellularization strategies can be grouped into chemical, enzymatic, and physical approaches, which are typically combined in multi-step protocols tailored to tissue type, density, and thickness.(Neishabouri et al., 2022). Fig. 2. Various decellularization strategies Chemical methods Detergents are the mainstay of chemical decellularization. The chemicals for decellularization include ionic detergents (e.g., sodium dodecyl sulfate, SDS), Nonionic detergents (e.g., Triton X-100) and Zwitterionic detergents (e.g., CHAPS, sulfobetaines). The ionic detergents effectively solubilize cell membranes and nuclear envelopes but can denature proteins; however, they alter ECM mechanics and bioactivity (Scarritt et al., 2015). The nonionic detergents are milder, but better preserve protein–protein interactions and often result in improved ECM preservation (Tabatabai et al., 2024). The Zwitterionic detergents combine features of ionic and nonionic agents and may allow more selective decellularisation with less ECM damage in some tissues (Scarritt et al., 2015). The other chemicals reported for this purpose are acids and bases (peracetic acid, ammonium hydroxide); hyper- and hypotonic solutions (e.g., hypertonic saline, deionised water) and alcohols and organic solvents. Enzymatic methods Enzymatic treatments are often used in combination with detergents. The enzymes used for the purpose are nucleases (Neishabouri et al., 2022), Proteases (e.g., trypsin) and collagenases and elastases (mainly used to generate soluble dECM fractions or hydrogels.(Liu et al., 2025). Enzymatic steps typically follow initial detergent-based lysis to clear residual material and improve scaffold biocompatibility. Physical methods Physical approaches can enhance chemical and enzymatic decellularization or serve as alternatives in specific contexts. These include freeze–thaw cycles, mechanical agitation or sonication (Neishabouri et al., 2022) pressure‑ and flow‑based strategies.(Scarritt et al., 2015) and supercritical CO₂ and other advanced techniques have been explored for decellularisation and sterilisation with reduced chemical residue. Perfusion vs. immersion decellularization For thick tissues and whole organs, perfusion decellularization —routing solutions through the native vasculature or ductal system—provides more homogeneous exposure and efficient removal of cellular components while maintaining vascular patency (Hillebrandt et al., 2019) In contrast, immersion decellularization (static or agitated) is more suitable for thin tissues (e.g., membranes, small biopsies) but can lead to diffusion gradients and uneven decellularization. Organ‑ and Tissue‑Specific Considerations The optimal decellularization protocol depends on the composition, density, vascularity, and function of the target tissue.(Neishabouri et al., 2022). Soft tissues and membranes (small intestine submucosa, dermis, pericardium, and amniotic membrane) are relatively thin and collagen rich. They can be efficiently decellularized using mild detergents and enzymatic treatments and have already been translated into several commercial products used clinically for hernia repair, wound healing, and reconstructive surgery.(Liu et al., 2025). Heart valves, blood vessels, and the myocardium decellularization has been achieved using perfusion with SDS, Triton X‑100, or combinations thereof, followed by nuclease treatment.(Scarritt et al., 2015). In our laboratory, we decellularized heart valves using detergent methods. The lung decellularization has been achieved through the perfusion‑based method using pulmonary artery and trachea and the detergent method (milder detergents or amphoteric agents). This technique preserves the branching architecture and at least part of the basement membrane while removing cellular components (Scarritt et al., 2015). Highly vascularized and dense organs, such as the liver and kidney, require protocols that clear cells from the parenchyma while maintaining sinusoids, glomeruli, and collecting systems. SDS‑based perfusion combined with nucleases has been shown to have better results (Scarritt et al., 2015). Characterisation and Quality Control of Decellularised Scaffolds Proper characterization and quality control are crucial for both research and clinical translation. Commonly assessed parameters include residual cellular content and immunogenicity, ECM composition and structure, mechanical and functional properties and sterility and safety. Residual cellular content and immunogenicity can be assessed by DNA quantification, histological staining, and immunohistochemistry and ultrastructure. In vitro immune assays and in vivo implantation studies to assess host