Preparation of a three-dimensional extracellular matrix by decellularization of rabbit livers

Abstract

Introduction: the availability of transplantable livers is not sufficient to fulfill the current demand for grafts, with the search for therapeutic alternatives having generated different lines of research, one of which is the use of decellularized three-dimensional biological matrices and subsequent cell seeding to obtain a functional organ. Objective: to produce a decellularization protocol from rabbit liver to generate a three-dimensional matrix. Methods: a combination of physical, chemical (Triton X-100 and SDS) and enzymatic agents to decellularize rabbit livers was used. After 68 h of retrograde perfusion, a decellularized translucent matrix was generated. To evaluate if the decellularization protocol was successful, with the extracellular matrix being preserved, we carried out histological (light microscopy and scanning electron microscopy) and biochemical (DNA quantification) studies. Results: the decellularization process was verified by macroscopic observation of the organ using macroscopic staining, which revealed a correct conservation of bile and vascular trees. A microscopic observation corroborated these macroscopic results, with the hematoxylin-eosin staining showing no cells or nuclear material and the presence of a portal triad. Wilde s staining demonstrated the conservation of reticulin fibers in the decellularized matrix. In addition, scanning electron microscopy revealed a preserved Glisson s capsule and a decellularized matrix, with the DNA quantification being less than 10 % in the decellularized liver compared to control. Finally, the time taken to develop the decellularization protocol was less than 96 hours. Conclusion: the proposed decellularization protocol was correct, and was verified by an absence of cells. The hepatic matrix had preserved vascular and bile ducts with a suitable three-dimensional architecture permitting further cell seeding.