RESUMEN
Hepatocytes, the main epithelial cell type in the liver, perform most of the biochemical functions of the liver. Thus, maintenance of a primary hepatocyte phenotype is crucial for investigations of in vitro drug metabolism, toxicity, and development of bioartificial liver constructs. Here, we report the impact of topographic cues alone and in combination with soluble signals provided by encapsulated feeder cells on maintenance of the primary hepatocyte phenotype. Topographic features were 300 nm deep with pitches of either 400, 1400, or 4000 nm. Hepatocyte cell attachment, morphology and function were markedly better on 400 nm pitch patterns compared with larger scale topographies or planar substrates. Interestingly, topographic features having biomimetic size scale dramatically increased cell adhesion whether or not substrates had been precoated with collagen I. Albumin production in primary hepatocytes cultured on 400 nm pitch substrates without collagen I was maintained over 10 days and was considerably higher compared to albumin synthesis on collagen-coated flat substrates. In order to investigate the potential interaction of soluble cytoactive factors supplied by feeder cells with topographic cues in determining cell phenotype, bioactive heparin-containing hydrogel microstructures were molded (100 µm spacing, 100 µm width) over the surface of the topographically patterned substrates. These hydrogel microstructures either carried encapsulated fibroblasts or were free of cells. Hepatocytes cultured on nanopatterned substrates next to fibroblast carrying hydrogel microstructures were significantly more functional than hepatocytes cultured on nanopatterned surfaces without hydrogels or stromal cells significantly elevated albumin expression and cell junction formation compared to cells provided with topographic cues only. The simultaneous presentation of topographic biomechanical cues along with soluble signaling molecules provided by encapsulated fibroblasts cells resulted in optimal functionality of cultured hepatocytes. The provision of both topographic and soluble signaling cues could enhance our ability to create liver surrogates and inform the development of engineered liver constructs.
Asunto(s)
Materiales Biocompatibles/farmacología , Heparina/química , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Nanoestructuras/química , Animales , Materiales Biocompatibles/química , Línea Celular , Células Cultivadas , Técnicas de Cocultivo , Fibroblastos/química , Fibroblastos/citología , Heparina/farmacología , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Ratones , Fenotipo , Ratas , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
In this article, porous poly(D,L-lactide-co-glycolide) (PLGA) microsphere scaffolds with a size of â¼ 400 µm and pores of â¼ 20 µm were prepared for constructing injectable three-dimensional hepatocyte spheroids. The porous sites of PLGA microspheres provided a spatial space for hepatocyte distribution. Hepatocytes spheroids were cocultured with human umbilical vein endothelial cell, bone marrow mesenchymal stem cell, or NIH/3T3 cells by combining the porous PLGA microspheres with the relatively hydrophobic culture strategy. The combination of open porous microspheres, hepatocytes, and nonparenchymal cells was demonstrated for application in functional hepatic tissue reconstruction. Hepatocellular-specific functions can sustained up to 2 weeks in the support of coculturing with nonparenchymal cells. The spheroidal hepatocyte coculture system had the advantages of an injectable delivery, higher cell seeding density, protection from exerted shear stress, better exchange of nutrients, oxygen and metabolites, and heterotypic cell-cell contact within and between microspheres.