*4.12. Thymus*

The thymus is an innervated organ part of the lymphatic and endocrine systems. The function of the thymus is to allow the development and maturation of the T-cell repertoire, and therefore, it has a main role in the immune response. Specifically, T-cell precursors are generated in the bone marrow and migrate to the thymus to become thymocytes, ultimately maturing immunocompetent T-cells. Endothelial and epithelial cells are the main cellular components of the thymus and, along with thymocytes, contribute to creating specific ECMs and microenvironment. The complex interaction network in the thymus includes cytokines, chemokines, matrix metalloproteases, laminin, collagen type IV, and multiple isoforms of fibronectin and glycoproteins, among others, with specific roles and precisely tuned toward the T-cell development process.

Thymus organ cultures are achieved via serial disaggregation and reaggregation of the tissue, and they are useful for ex vivo study of thymus function and complex cell interactions [187]. The rationale of thymus decellularization is mainly related to modeling thymus development, as well as the generation of potential regenerative or therapeutic approaches for in vivo immune response modulation [188]. To this aim, decellularization should be soft enough to keep intact the key ECM components, and should allow further proper recellularization with thymic epithelial cells and endothelial cells. Specifically, the thymus's dECM-derived bioengineered structure has to be able to reproduce T-cell differentiation and maturation processes. Freeze–thawing, followed by SDS and Triton X-100 detergent treatments, is a common decellularization technique [189]. Thymic epithelial cell-seeded dECM scaffolds, also called thymic reconstructed organoids, have been implanted in immunocompromised mice, yielding the development of populations of mature T-cells overwise absent in these animals [190].

#### **5. The Clinical Outcome and Market of the dECM**

Translational research is already a reality for some dECM-derived approaches, including several ongoing clinical trials and products on the market (see Table 2). The most common products are decellularized tissue pieces, serving as implantable materials for tissue formation, with proprietary- or patented-specific decellularization procedures [191–193].

Decellularized products on the market are generally issued with the ISO standard for biological medical devices (ISO10993-1, the standard for biological evaluation of medical devices), while recently, a specific standard for the evaluation of decellularized products has become available (ASTM F3354-19, Standard Guide for Evaluating Extracellular Matrix Decellularization Processes) [8]. Characterization includes in vitro and in vivo studies to provide data related to the removal of donor DNA and to the safety of implantable commercial products [194,195].

For some specific tissues, there are multiple decellularized products, competing for the same application market and claiming different properties due to differences in decellularization treatments. Comparative clinical case studies are common, and they provide useful information related to clinical success and outcomes of the different commercial dECMs available for each specific application [196– 199]. In this sense, there is a lack of standardized tissue-specific decellularization methods, which would serve as the standard control for comparative purposes [5,200]. Such standardized controls would be useful not only for the assessment of products already on the market, but also to perform more efficient, comparable, and reliable experimental research studies [201].


