The ‘cardiosphere’ is a 3D cluster of cardiac progenitor cells recapitulating a stem cell niche-like microenvironment with a potential for disease and regeneration modelling of the failing human myocardium. In this multicellular 3D context, it is extremely important to decrypt the spatial distribution of cell markers for dissecting the evolution of cellular phenotypes by direct quantification of fluorescent signals in confocal microscopy. In this study, we present a fully automated method, named CARE (‘CARdiosphere Evaluation’), for the segmentation of membranes and cell nuclei in human-derived cardiospheres. The proposed method is tested on twenty 3D-stacks of cardiospheres, for a total of 1160 images. Automatic results are compared with manual annotations and two open-source software designed for fluorescence microscopy. CARE performance was excellent in cardiospheres membrane segmentation and, in cell nuclei detection, the algorithm achieved the same performance as two expert operators. To the best of our knowledge, CARE is the first fully automated algorithm for segmentation inside in vitro 3D cell spheroids, including cardiospheres. The proposed approach will provide, in the future, automated quantitative analysis of markers distribution within the cardiac niche-like environment, enabling predictive associations between cell mechanical stresses and dynamic phenotypic changes.

Co‐culture of mesenchymal stromal cells (MSCs) with articular chondrocytes (ACs) has been reported to improve the efficiency of utilization of a small number of ACs for the engineering of implantable cartilaginous tissues. However, the use of cells of animal origin and the generation of small‐scale micromass tissues limit the clinical relevance of previous studies. Here we investigated the in vitro and in vivo chondrogenic capacities of scaffold‐based constructs generated by combining primary human ACs with human bone marrow MSCs (BM‐MSCs). The two cell types were cultured in collagen sponges (2 × 6 mm disks) at the BM‐MSCs:ACs ratios: 100:0, 95:5, 75:25 and 0:100 for 3 weeks. Scaffolds freshly seeded or further precultured in vitro for 2 weeks were also implanted subcutaneously in nude mice and harvested after 8 or 6 weeks, respectively. Static co‐culture of ACs (25%) with BM‐MSCs (75%) in scaffolds resulted in up to 1.4‐fold higher glycosaminoglycan (GAG) content than what would be expected based on the relative percentages of the different cell types. In vivo GAG induction was drastically enhanced by the in vitro preculture and maximal at the ratio 95:5 (3.8‐fold higher). Immunostaining analyses revealed enhanced accumulation of type II collagen and reduced accumulation of type X collagen with increasing ACs percentage. Constructs generated in the perfusion bioreactor system were homogeneously cellularized. In summary, human cartilage grafts were successfully generated, culturing BM‐MSCs with a relatively low fraction of non‐expanded ACs in porous scaffolds. The proposed co‐culture strategy is directly relevant towards a single‐stage surgical procedure for cartilage repair. Copyright © 2012 John Wiley & Sons, Ltd.

The pericardial tissue is commonly used to produce bio-prosthetic cardiac valves and patches in cardiac surgery. The procedures adopted to prepare this tissue consist in treatment with aldehydes, which do not prevent post-graft tissue calcification due to incomplete xeno-antigens removal. The adoption of fixative-free decellularization protocols has been therefore suggested to overcome this limitation. Although promising, the decellularized pericardium has not yet used in clinics, due to the absence of proofs indicating that the decellularization and cryopreservation procedures can effectively preserve the mechanical properties and the immunologic compatibility of the tissue. The aim of the present work was to validate a procedure to prepare decellularized/cryopreserved human pericardium which may be implemented into cardiovascular homograft tissue Banks. The method employed to decellularize the tissue completely removed the cells without affecting ECM structure; furthermore, uniaxial tensile loading tests revealed an equivalent resistance of the decellularized tissue to strain, before and after the cryopreservation, in comparison with the fresh tissue. Finally, immunological compatibility, showed a minimized host immune cells invasion and low levels of systemic inflammation, as assessed by tissue transplantation into immune-competent mice. Our results indicate, for the first time, that fixative-free decellularized pericardium from cadaveric tissue donors can be banked according to Tissue Repository-approved procedures without compromising its mechanical properties and immunological tolerance. This tissue can be therefore treated as a safe homograft for cardiac surgery.