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

GUSTAVO A NARI; MARIANA P CID; ROMINA COMÍN; LAURA REYNA; GUSTAVO JURI; RICARDO TABORDA; NANCY A SALVATIERRA

REVISTA ESPANOLA DE ENFERMEDADES DIGESTIVAS

ARAN EDICIONES

Lugar: Comunidad de Madrid, España; Año: 2013 p. 138 - 143

1130-0108

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