Construction of Thymus Organoids from Decellularized Thymus Scaffolds

Abstract

Summary One of the hallmarks of modern medicine is the development of therapeutics that can modulate immune responses, especially the adaptive arm of immunity, for disease intervention and prevention. While tremendous progresses have been made in the past decades, manipulating the thymus, the primary lymphoid organ responsible for the development and education of T lymphocytes, remains a challenge. One of the major obstacles is the difficulty to reproduce its unique extracellular matrix (ECM) microenvironment that is essential for maintaining the function and survival of thymic epithelial cells (TECs), the predominant population of cells in the thymic stroma. Here, we described the construction of functional thymus organoids from decellularized thymus scaffolds repopulated with isolated TECs. Thymus decellularization was achieved by freeze/thaw cycles to induce intracellular ice crystal formation, followed by detergent-induced cell lysis. Cellular debris was removed with extensive wash. The decellularized thymus scaffolds can largely retain the 3-D extracellular matrix (ECM) microenvironment that can support the re-colonization of TECs. When transplanted into athymic nude mice, the reconstructed thymus organoids can effectively promote the homing of bone marrow-derived lymphocyte progenitors and support the development of a complex T cell repertoire. Bioengineering of thymus organoids can be a promising approach to rejuvenate/modulate the function of T-cell mediated adaptive immunity in regenerative medicine.

Tissue-Specific Decellularization Methods: Rationale and Strategies to Achieve Regenerative Compounds

“…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].…”

Current and Future Therapeutic Approaches for Thymic Stromal Cell Defects

“…Early attempts using natural ECM from decellularized mouse thymi following repopulation with freshly isolated stromal cells, lacked TEC organization and did not support ex vivo thymopoiesis either ( 162 , 163 ). Thymus decellularization was achieved by freeze/thaw cycles and detergent-induced cell lysis ( 164 ). This method cannot be used to obtain human natural scaffolds, as it cannot be applied to the much larger human thymi.…”

When the Damage Is Done: Injury and Repair in Thymus Function

“…Finally, there are also several approaches that do not rely on the endogenous thymus at all, but rather concentrate on de novo formation of whole organs ex vivo that can be transplanted into patients as required ( 186 , 187 ). Although in vivo evidence of their efficacy is still only limited, several approaches have been used to generate artificial thymuses ex vivo , including decellularizing the tissue, which has been performed in several tissues including the thymus, as well as generating synthetic matrices to support T cell development ( 188 – 190 ). Both of these approaches would require some cellular input to generate a functional thymus; namely the thymic epithelial microenvironment would need to be recapitulated with specific factors or, more likely, cells such as TECs derived from multipotent progenitors or reprogrammed, as above.…”