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Long-segment airway stenosis as well as their neoplastic transformation is life-threatening and still currently represent unsolved clinical problems. Indeed, despite several attempts, definitive surgical procedures are not presently available, and a suitable tracheal reconstruction or replacement remains an urgent clinical need. A possible innovative strategic solution to restore upper airway function may be represented by the creation of a bioprosthetic trachea, obtained through the combination of tissue engineering and regenerative medicine.Here we describe a two-step protocol for the ex vivo generation of tracheal segments. The first step involves the application of a decellularization technique that allows for the production of a naturally derived extracellular matrix (ECM)-based bio-scaffold, that maintains the macro- and micro-architecture as well as 9 the matrix-related signals distinctive of the original tissue. In the second step chondrocytes are seeded onto decellularized trachea, using a rotating bioreactor to ensure a correct scaffold repopulation.This multi-step approach represents a powerful tool for in vitro reconstruction of a bioengineered trachea that may constitute a promising solution to restore upper airway function. In addition, the procedures here described allow for the creation of a suitable 3D platform that may find useful applications, both for toxicological studies as well as organ transplantation strategies., (© 2021. Springer Science+Business Media, LLC.)

Ovarian failure is the most common cause of infertility. Although numerous strategies have been proposed, a definitive solution for recovering ovarian functions and restoring fertility is currently unavailable. One innovative alternative may be represented by the development of an "artificial ovary" that could be transplanted in patients for re-establishing reproductive activities. Here, we describe a novel approach for successful repopulation of decellularized ovarian bioscaffolds in vitro. Porcine whole ovaries were subjected to a decellularization protocol that removed the cell compartment, while maintaining the macrostructure and microstructure of the original tissue. The obtained bioscaffolds were then repopulated with porcine ovarian cells or with epigenetically erased porcine and human dermal fibroblasts. The results obtained demonstrated that the decellularized extracellular matrix (ECM)-based scaffold may constitute a suitable niche for ex vivo culture of ovarian cells. Furthermore, it was able to properly drive epigenetically erased cell differentiation, fate, and viability. Overall, the method described represents a powerful tool for the in vitro creation of a bioengineered ovary that may constitute a promising solution for hormone and fertility restoration. In addition, it allows for the creation of a suitable 3D platform with useful applications both in toxicological and transplantation studies.

Bi-dimensional culture systems have represented the most used method to study cell biology outside the body for over a century. Although they convey useful information, such systems may lose tissue-specific architecture, biomechanical effectors, and biochemical cues deriving from the native extracellular matrix, with significant alterations in several cellular functions and processes. Notably, the introduction of three-dimensional (3D) platforms that are able to re-create in vitro the structures of the native tissue, have overcome some of these issues, since they better mimic the in vivo milieu and reduce the gap between the cell culture ambient and the tissue environment. 3D culture systems are currently used in a broad range of studies, from cancer and stem cell biology, to drug testing and discovery. Here, we describe the mechanisms used by cells to perceive and respond to biomechanical cues and the main signaling pathways involved. We provide an overall perspective of the most recent 3D technologies. Given the breadth of the subject, we concentrate on the use of hydrogels, bioreactors, 3D printing and bioprinting, nanofiber-based scaffolds, and preparation of a decellularized bio-matrix. In addition, we report the possibility to combine the use of 3D cultures with functionalized nanoparticles to obtain highly predictive in vitro models for use in the nanomedicine field.

Ovarian failure is the most common cause of infertility and affects about 1% of young women. One innovative strategy to restore ovarian function may be represented by the development of a bioprosthetic ovary, obtained through the combination of tissue engineering and regenerative medicine.We here describe the two main steps required for bioengineering the ovary and for its ex vivo functional reassembling. The first step aims at producing a 3D bioscaffold, which mimics the natural ovarian milieu in vitro. This is obtained with a whole organ decellularization technique that allows the maintenance of microarchitecture and biological signals of the original tissue. The second step involves the use of magnetic activated cell sorting (MACS) to isolate purified female germline stem cells (FGSCs). These cells are able to differentiate in ovarian adult mature cells, when subjected to specific stimuli, and can be used them to repopulate ovarian decellularized bioscaffolds. The combination of the two techniques represents a powerful tool for in vitro recreation of a bioengineered ovary that may constitute a promising solution for hormone and fertility function restoring. In addition, the procedures here described allow for the creation of a suitable 3D platform with useful applications both in toxicological and transplantation studies.

Tissue engineering provides unique opportunities for disease modeling, drug testing, and regenerative medicine applications. The use of cell-seeded scaffolds to promote tissue development is the hallmark of the tissue engineering. Among the different types of scaffolds (derived from either natural or synthetic polymers) used in the field, the use of decellularized tissues/organs is specifically attractive. The decellularization process involves the removal of native cells from the original tissue, allowing for the preservation of the three-dimensional (3D) macroscopic and microscopic structures of the tissue and extracellular matrix (ECM) composition. Following recellularization, the resulting scaffold provides the seeded cells with the appropriate biological signals and mechanical properties of the original tissue. Here, we describe different methods to create viable scaffolds from decellularized heart and liver as useful tools to study and exploit ECM biological key factors for the generation of engineered tissues with enhanced regenerative properties.

The development of new approaches for organ transplantation has become crucial in the last years. In particular, organ engineering, involving the preparation of acellular matrices that provide a natural habitat for reseeding with an appropriate population of cells, is an attractive although technically demanding approach. We here describe a method that allows for the derivation of functional in vitro hepatic organoids and that does not require a previous selection of all the parenchymal hepatocytes and non-parenchymal cells, namely, Kupffer cells, liver endothelial cells, and hepatic stellate cells. The procedure also replaces the costly standard collagenase perfusion step with a trypsin-based enzymatic digestion that results in high-yield decellularization. A combination of physical and chemical treatments through deep immersion and intraluminal infusion of two different consecutive solutions is used: (1) deionized water (DI) and (2) DI + Triton X 1% + ammonium hydroxide (NH 4 OH) 0.1%. This ensures the isolation of the hepatic constructs that reliably maintain original architecture and ECM components while completely removing cellular DNA and RNA. The procedure is fast, simple, and cheap and warrants an optimal organoid functionality that may find applications in both toxicological and transplantation studies.