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An efficient protocol for porcine liver decellularization supports subsequent recellularization
Decellularized porcine livers have a wide range of applications in regenerative medicine, however previously existing protocols for organ decellularization still need to be adjusted to a full size porcine liver
Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados
Objective: to establish a protocol that enables effective decellularization of the entire porcine liver in a short period.
Method: Livers (n = 3) were perfused with decellularizing solutions through the portal vein during 3 days at room temperature. To analyze the extracellular matrix (ECM), after decellularization, histology and electron microscopy were performed. The vascular tree integrity was evaluated, by magnetic resonance and computed tomography, injecting gadolinium and iodine, respectively. Toluidine blue dye was also used. The presence of residual cells were analyzed by DAPI and quantification of DNA by spectrophotometry. Collagen IV presence was detected by immunohistochemistry. To recellularize 1E6/ml of HEPG2 cells were cultured over the matrix. Cell presence in the recellularized matrix was visualized by DAPI and secretion of albumin was detected by ELISA.
Results: After 3 days, this new protocol, based on the use of sodium-deoxycholate as detergent, was effective in preserving all ECM structures and the vascular system. DNA quantification indicated that >97% of cells were removed. Electron microscopy showed the presence of collagen in ECM and cells could not be detected. Cell seeding showed that ECM could be recellularized after 15 days.
Considerações Finais/Final considerations/Consideraciones finales
Conclusion: By enabling the achievement of liver scaffolds with preserved tissue integrity in 3 days at room temperature, this novel protocol brings new perspectives to the development of bioartificial livers.
Palavras-chave/Key words/Palabras clave
Decellularization, porcine, recellularization, transplant
MARLON LEMOS DIAS, LANUZA FACCIOLI, Grazielle Suhett, Victor Hoff, Camila Mendez, Marcelo Santiago, Ana Carla Goldberg, Daniella Parente, Antonio Carlos Campos de Carvalho, Regina Goldenberg