The dorsal portion of the bovine diaphragm as a useful tissue for producing a 3D muscle scaffold

Three-dimensional (3D) organoids are an innovative approach to obtain an in vitro model for ex vivo studies to overcome the limitations of monolayer cell culture and reduce the use of animal models. An organoid of skeletal muscle requires the presence of the extracellular matrix to represent a functional muscle in vitro, which is why decellularized tissue is an optimal choice. Various muscles have been considered to produce a muscle organoid, most from rodents or small animals, and only recently some studies have been reported on the muscles of large animals. This work presents a muscular organoid produced from the bovine diaphragm, which has a peculiar multilayered structure with different fibre orientations depending on the considered area. This paper analyses the anatomical structure of the bovine diaphragm, selects the most appropriate portion, and presents a decellularization protocol for a multilayered muscle. In addition, a preliminary test of recellularization with primary bovine myocytes was presented with the future aim of obtaining a 3D muscle allogenic organoid, completely bovine-derived. The results demonstrate that the dorsal portion of bovine diaphragm presents a regular alternation of muscular and fibrous layers and that the complete decellularization does not affect the biocompatibility. These results provide a strong foundation for the potential application of this portion of tissue as a scaffold for in vitro studies of muscle organoids.