BOVINE DIAPHRAGM FOR MUSCLE 3D ORGANOID

A primary cell 3D organoid technology represents a physiologically very interesting model for basic research and drug screening, in fact this system can demonstrate effect and toxicity of drugs reducing the costs. Bioengineered scaffolds derived from the decellularized extracellular matrix (ECM) obtained from discarded animal organs and tissues are attractive candidates for 3D organoid production. The use of a natural decellularized scaffold preserves the physiological and mechanical properties and ECM proteins for attachment, migration, and proliferation of cells, reduces immunogenicity and preserves their histological structures. Bovine diaphragm seems to be an adequate candidate to obtain a muscle 3D organoid. Morphologically, the diaphragm is a flat muscle in which the peripheral muscle fibers are separated from the deeper ones by the interposition of a large aponeurotic lamina which constitutes the tendon centre. We have recently observed that bovine diaphragm is constituted by a multilayer in which muscle fibres layers are separated by connective tissue. Starting from diaphragm muscles obtained from animals euthanized at slaughterhouse, we evaluated three different points, costal caudal (12th rib), costal intermediate (9th rib), sternal. The caudal dorsal portion of diaphragm was used to produce the 3D muscle scaffold, being this part histologically the most appropriate for our purpose. A protocol to decellularize the diaphragm scaffold was tuned, evaluating the complete elimination of nuclei and the integrity of collage fibres by histological staining. To obtain preliminary data about biocompatibility of diaphragm scaffold, the decellularized samples were recellularized using primary cells from a fresh biopsy of bovine cutaneous trunci muscle, used as a representative bovine fast-twitch skeletal muscle, immediately after slaughter. In conclusion, we demonstrated that decellularized caudal dorsal portion of bovine diaphragm can be a promise muscle 3D organoid, which maintain its structure after decellularization cycles and does not loss its biocompatibility.