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Decellularization and recellularization of liver in a perfusion bioreactor with mesenchymal stem cells
Introduction: Organ transplantation is the preferred therapy for end-stage organ failure, but the demand is still higher than the number of donations. Recently, the technique for whole organ decellularization for posterior recellularization appeared as a promising option. With the appropriate cells, this solution could render organs inadequate for transplantation to become suitable free rejection surrogates. Research is still needed for what should be the best decellularization protocols and the type of cells to utilize. Perfusion is a method that can better distribute the solution and nutrients in tissue, mimicking the conditions of the tissue in vivo, and stem cells are a good option for recellularization as they can differentiate into the desired cell type and can be obtained from the patients themselves.
Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados
Objectives: Utilize a perfusion bioreactor manufactured in the laboratory to decellularize and recellularize a rat liver with mesenchymal stem cells. Methods and Results: To strengthen the partnership of national industries with the academia, a partnership with the start-up Eva Scientific was established to construct a perfusion bioreactor. The bioreactor consists of a peristaltic pump connected to a glass chamber where the organs were placed. The decellularization was performed using distilled water for 24h, a 0.5% solution of sodium dodecyl sulfate for 24h followed by phosphate buffered saline with 1% penicillin, streptomycin and gentamicin for removal of residual SDS for 24h. Confirmation of decellularization was made by macroscopic observation, showing the discoloration of the organ. Histological analysis, using hematoxylin and eosin staining, showed absence of cellular nucleus. DNA quantification analysis showed less than 100ng/mg of wet tissue in the decellularized liver. Glycosaminoglycans were measured by the dimethylmethylene blue method as an indicator of matrix preservation, maintaining 50% of their total in the decellularized liver. Peracetic acid (PA) 0.1% with ethanol (EtOH) 4% and PSG 1% were utilized for 3h for sterilization. To verify the sterilization, supplemented culture media DMEM with 10% fetal bovine FBS and 1% PSG was perfused through the organs for 24h at 37°C and with 5% CO2. The nonexistence of turbidity in the media indicated the absence of microorganism growth. Liver recellularization was realized in a multistep seeding process with the total of 109 cells utilized, obtaining an efficiency > 97%. All the processes were carried out in a laminar flow hood. After 7 days, the live cells were shown marked with fluorescein diacetate and the dead cells marked with propidium iodide. Discussion: The perfusion method in the bioreactor guarantees a homogeneous distribution of the solutions, saving time and quantity of reagent. The organs rapidly acquired a white translucent color with the SDS solution, indicating loss of cellular mass. The histology confirmed the decellularized organs by the absence of nucleus colored with hematoxylin. According to data in the literature, a quantity of DNA lower than 100ng/mg of wet tissue indicates the absence of cells in the tissue. The association of PA, EtOH with antibiotics was efficient to sterilize the decellularized organ. The multistep seeding was highly efficient, guaranteeing that the cells stayed inside the tissue. The presence of live cells after 7 days shows that the cells can survive the process.
Considerações Finais/Final considerations/Consideraciones finales
Acknowledgments: MCTIC, FINEP, CNPq and Stem Cell Research Institute(IPCT)
Palavras-chave/Key words/Palabras clave
Tissue engineering, stem cells, decellularizaton, recellularization, bioreactor, liver
MAURICIO FELISBERTO BORGES, Natasha Natasha Maurmann, Patricia Pranke