Thymus Extracellular Matrix-Derived Scaffolds Support Graft-Resident Thymopoiesis and Long-Term In Vitro Culture of Adult Thymic Epithelial Cells.

The thymus provides the physiological microenvironment critical for the development of T lymphocytes, the cells that orchestrate the adaptive immune system to generate an antigen-specific response. A diverse population of stroma cells provides surface-bound and soluble molecules that orchestrate the intrathymic maturation and selection of developing T cells. Forming an intricate 3D architecture, thymic epithelial cells (TEC) represent the most abundant and important constituent of the thymic stroma. Effective models for in and ex vivo use of adult TEC are still wanting, limiting the engineering of functional thymic organoids and the understanding of the development of a competent immune system. Here a 3D scaffold is developed based on decellularized thymic tissue capable of supporting in vitro and in vivo thymopoiesis by both fetal and adult TEC. For the first time, direct evidences of feasibility for sustained graft-resident T-cell development using adult TEC as input are provided. Moreover, the scaffold supports prolonged in vitro culture of adult TEC, with a retained expression of the master regulator Foxn1. The success of engineering a thymic scaffold that sustains adult TEC function provides unprecedented opportunities to investigate thymus development and physiology and to design and implement novel strategies for thymus replacement therapies.

Development and Validation of a New Storage Procedure to Extend the In-Use Stability of Azacitidine in Pharmaceutical Formulations.

Stability studies performed by the pharmaceutical industry are principally designed to fulfill licensing requirements. Thus, post-dilution or post-reconstitution stability data are frequently limited to 24 h only for bacteriological reasons, regardless of the true physicochemical stability which could, in many cases, be longer. In practice, the pharmacy-based centralized preparation may require preparation in advance for administration, for example, on weekends, holidays, or in general when pharmacies may be closed. We report an innovative strategy for storing resuspended solutions of azacitidine, a well-known chemotherapic agent, for which the manufacturer lists maximum stability of 22 h. By placing the syringe with the azacitidine reconstituted suspension between two refrigerant gel packs and storing it at 4 °C, we found that the concentration of azacitidine remained above 98% of the initial concentration for 48 h, and no change in color nor the physicochemical properties of the suspension were observed throughout the study period. The physicochemical and microbiological properties were evaluated by HPLC-UV and UHPLC-HRMS analysis, FTIR spectroscopy, pH determination, visual and subvisual examination, and sterility assay. The HPLC-UV method used for evaluating the chemical stability of azacitidine was validated according to ICH. Precise control of storage temperature was obtained by a digital data logger. Our study indicates that by changing the storage procedure of azacitidine reconstituted suspension, the usage window of the drug can be significantly extended to a time frame that better copes with its use in the clinical environment.

Human Mesenchymal Stem Cell Combined with a New Strontium-Enriched Bioactive Glass: An ex-vivo Model for Bone Regeneration.

A 3D cellular model that mimics the potential clinical application of a biomaterial is here applied for the first time to a bioactive glass, in order to assess its biological potential. A recently developed bioactive glass (BGMS10), whose composition contained strontium and magnesium, was produced in the form of granules and fully investigated in terms of biocompatibility in vitro. Apart from standard biological characterization (Simulated Body Fluid (SBF) testing and biocompatibility as per ISO10993), human bone marrow mesenchymal stromal/stem cells (BM-MSCs) were used to investigate the performance of the bioactive glass granules in an innovative 3D cellular model. The results showed that BGMS10 supported human BM-MSCs adhesion, colonization, and bone differentiation. Thus, bioactive glass granules seem to drive osteogenic differentiation and thus look particularly promising for orthopedic applications, bone tissue engineering and regenerative medicine.

Label-free toxicology screening of primary human mesenchymal cells and iPS-derived neurons.

The high-throughput, label-free Corning Epic assay has applications in drug discovery, pharmacogenomics, cell receptor signaling, cell migration, and viral titration. The utility of Epic technology for biocompatibility testing has not been well established. In manufacturing of medical devices, in vitro and in vivo biocompatibility assessments are mandatory, according to ISO 10993. The new medical device regulation MDR 745/2017 specifies that ex vivo assays that can closely recapitulate in vivo scenarios are needed to better evaluate biomedical devices. We propose herein that Epic technology-which enables detection of variations in cell mass distribution-is suitable for biocompatibility screening of compounds. In this study, we challenged primary human osteoblasts, endothelial cells, and neurons derived from induced pluripotent stem cells with specific concentrations of methyl methacrylate (MMA). Polymeric MMA has long been applied in cranioplasty, where it makes contact with multiple cell types. Application of Epic technology yielded real-time cytotoxicity profiles for all considered cell types. The results were compared with those from microscopic observation of the same culture plate used in the Epic analyses. The Epic assay should be further examined for its utility for cell biology, genomics, and proteomics companion assays. Our results suggest that Epic technology can be applied to biocompatibility evaluation of human cells in medical device development.

An Alternative Approach to Investigate Biofilm in Medical Devices: A Feasibility Study.

Biofilms are assemblages of bacterial cells irreversibly associated with a surface where moisture is present. In particular, they retain a relevant impact on public health since through biofilms bacteria are able to survive and populate biomedical devices causing severe nosocomial infections that are generally resistant to antimicrobial agents. Therefore, controlling biofilm formation is a mandatory feature during medical device manufacturing and during their use. In this study, combining a crystal violet staining together with advanced stereomicroscopy, we report an alternative rapid protocol for both qualitative and semi-quantitative biofilm determination having high specificity, high repeatability, and low variability. The suggested approach represents a reliable and versatile method to detect, monitor, and measure biofilm colonization by an easy, more affordable, and reproducible method.

Ex situ bioengineering of bioartificial endocrine glands: a new frontier in regenerative medicine of soft tissue organs.

Ex situ bioengineering is one of the most promising perspectives in the field of regenerative medicine allowing for organ reconstruction outside the living body; i.e. on the laboratory bench. A number of hollow viscera of the cardiovascular, respiratory, genitourinary, and digestive systems have been successfully bioengineered ex situ, exploiting biocompatible scaffolds with a 3D morphology that recapitulates that of the native organ (organomorphic scaffold). In contrast, bioengineering of entire soft tissue organs and, in particular endocrine glands still remains a substantial challenge. Primary reasons are that no organomorphic scaffolding for endocrine viscera have as yet been entirely assembled using biocompatible materials, nor is there a bioreactor performance capable of supporting growth within the thickness range of the regenerating cell mass which has proven to be reliable enough to ensure formation of a complete macroscopic gland ex situ. Current technical options for reconstruction of endocrine viscera include either biocompatible 3D reticular scaffolds lacking any organomorphic geometry, or allogenic/xenogenic acellular 3D matrices derived from a gland similar to that to be bioengineered, eventually recellularized by autologous/heterologous cells. In 2007, our group designed, using biocompatible material, an organomorphic scaffold-bioreactor unit for bioengineering ex situ the human thyroid gland, chosen as a model for its simple anatomical organization (repetitive follicular cavities). This unit reproduces both the 3D native geometry of the human thyroid stromal/vascular scaffold, and the natural thyrocyte/vascular interface. It is now under intense investigation as an experimental tool to test cellular 3D auto-assembly of thyroid tissue and its related vascular system up to the ex situ generation of a 3D macroscopic thyroid gland. We believe that these studies will lay the groundwork for a new concept in regenerative medicine of soft tissue and endocrine organs; i.e. that the organomorphism of a biocompatible scaffold-bioreactor complex is essential to both the 3D organization of seeded stem cells/precursor cells and their phenotypic fate as glandular/parenchymal/vascular elements, eventually leading to a physiologically competent and immuno-tolerant bioconstruct, macroscopically suitable for transplantation and clinical applications.