Processing decellularized tissues into nanoscale extracellular matrix (ECM) components for scaffold fabrication involves a series of carefully controlled steps to preserve the native architecture and bioactive cues essential for tissue regeneration. The approach leverages the natural ECM’s ability to provide structural support, biochemical signaling, and mechanical cues necessary for cell attachment, proliferation, and differentiation. This method is particularly relevant for complex organs such as the liver, heart, and lung, where the ECM’s composition and organization are critical for function.

The first step in the process is the decellularization of the source tissue, which involves the removal of cellular material while retaining the ECM’s structural and functional proteins. For liver, heart, and lung tissues, this is typically achieved through a combination of physical, chemical, and enzymatic treatments. Physical methods include freeze-thaw cycles and mechanical agitation, which disrupt cell membranes. Chemical agents such as detergents (e.g., sodium dodecyl sulfate or Triton X-100) solubilize cellular components, while enzymatic treatments (e.g., DNase and RNase) degrade nucleic acids. The choice of decellularization protocol must balance thorough cell removal with minimal ECM disruption. For example, liver ECM requires gentle detergent concentrations to preserve its delicate vascular and lobular architecture, whereas heart tissue may tolerate more aggressive treatments due to its denser collagen network.

Following decellularization, the ECM is processed into nanoscale components. This is achieved through mechanical disruption (e.g., milling or homogenization) followed by solubilization in acidic or enzymatic solutions. The resulting ECM suspension can be further processed into hydrogels, electrospun nanofibers, or 3D-printed scaffolds. Liver-derived ECM hydrogels, for instance, retain growth factors such as hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), which are crucial for hepatocyte function and angiogenesis. Similarly, heart ECM processed into nanofibers preserves laminin and fibronectin, promoting cardiomyocyte adhesion and alignment. Lung ECM, when decellularized and processed into a thin film or porous scaffold, maintains elastin and collagen IV, supporting alveolar epithelial cell growth and gas exchange.

Crosslinking is a critical step to enhance the mechanical stability and degradation resistance of the scaffold. Natural crosslinkers such as genipin, a biocompatible alternative to glutaraldehyde, are often used due to their low cytotoxicity. Genipin forms stable bonds between ECM collagen fibers, improving tensile strength while maintaining biocompatibility. For liver scaffolds, crosslinking must be optimized to avoid excessive stiffness, which can impair hepatocyte function. In heart tissue engineering, balanced crosslinking ensures sufficient elasticity to mimic the native myocardium’s contractile properties. Lung scaffolds require mild crosslinking to preserve the ECM’s porous structure for efficient oxygen diffusion.

Preservation of bioactive cues is paramount for scaffold functionality. The ECM’s native composition includes glycosaminoglycans (GAGs), growth factors, and matricellular proteins that guide cell behavior. To retain these components, decellularization must avoid harsh conditions that denature proteins. For example, liver ECM processed with low-concentration detergents retains over 80% of its original GAG content, which is critical for hepatocyte maturation. In heart scaffolds, preserving transforming growth factor-beta (TGF-β) and fibroblast growth factor (FGF) enhances cardiomyocyte proliferation and reduces fibrosis. Lung ECM scaffolds with intact elastin and surfactant proteins improve alveolar epithelial cell differentiation and barrier function.

Recellularization strategies depend on the target organ and cell type. For liver scaffolds, primary hepatocytes or stem cell-derived hepatocyte-like cells are seeded via perfusion through the preserved vascular network. Studies show that dynamic culture conditions, such as bioreactors with flow perfusion, enhance cell viability and function by improving nutrient delivery and waste removal. Heart scaffolds require cardiomyocytes or cardiac progenitor cells, often seeded in combination with endothelial cells to promote vascularization. Electrical stimulation during culture can further mature engineered cardiac tissue. Lung scaffolds are repopulated with alveolar epithelial cells and pulmonary endothelial cells, with ventilation-mimicking bioreactors used to simulate mechanical stretch and airflow.

Clinical translation faces several challenges. Scalability is a major hurdle, as producing decellularized ECM scaffolds in large quantities while maintaining consistency is difficult. Batch-to-batch variability in ECM composition can affect scaffold performance. Immunogenicity, though reduced by decellularization, remains a concern if residual cellular antigens persist. Regulatory approval requires rigorous demonstration of safety and efficacy, particularly for organs like the liver, where scaffold failure could have severe consequences. Vascularization is another critical issue; while ECM scaffolds retain their microvascular architecture, achieving full perfusion post-implantation remains challenging. In heart scaffolds, integration with host vasculature is necessary to prevent ischemia, whereas lung scaffolds must ensure gas exchange without thrombosis or inflammation.

Long-term stability and functionality of recellularized scaffolds in vivo are still under investigation. Preclinical studies in animal models show promising results, such as improved liver function in rats with partial hepatectomy after implantation of ECM-derived scaffolds. However, human trials are limited, and durability beyond a few months is not well-documented. For heart applications, electrical coupling between engineered and host tissue is essential to prevent arrhythmias, a risk not yet fully mitigated. Lung scaffolds must demonstrate resistance to infection and fibrosis over time.

In summary, processing decellularized tissues into nanoscale ECM components offers a biologically relevant platform for liver, heart, and lung regeneration. Crosslinking methods must balance mechanical reinforcement with biocompatibility, while preservation of bioactive cues ensures proper cell guidance. Recellularization strategies are organ-specific and benefit from dynamic culture systems. Despite progress, clinical translation requires addressing scalability, immunogenicity, vascularization, and long-term functional integration. Advances in these areas could position ECM-based scaffolds as a viable therapeutic option for organ repair and replacement.