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Impact of Nanotopography, Heparin Hydrogel Microstructures, and Encapsulated Fibroblasts on Phenotype of Primary Hepatocytes.

ABSTRACT: 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.

SUBMITTER: You J

PROVIDER: S-EPMC4372509 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Multiplexed, sensitive, and on-chip molecular diagnostic assays are essential in both clinical and research settings. In past work, running reactions in nanoliter- to femtoliter-sized volumes such as microwells or droplets has led to significant increases in detection sensitivities. At the same time, hydrogels have emerged as attractive scaffolds for bioassays due to their nonfouling, flexible, and aqueous properties. In this paper, we combine these concepts and develop a novel platform in which hydrogel compartments are used as individually confined reaction volumes within a fluorinated oil phase. We fabricate functional and versatile hydrogel microstructures in microfluidic channels that are physically isolated from each other using a surfactant-free fluorinated oil phase, generating picoliter- to nanoliter-sized immobilized aqueous reaction compartments that are readily functionalized with biomolecules. In doing so, we achieve monodisperse reaction volumes with an aqueous interior while exploiting the unique chemistry of a hydrogel, which provides a solid and porous binding scaffold for biomolecules and is impenetrable to oil. Furthermore, our lithographically defined reaction volumes are readily customized with respect to geometry and chemistry within the same channel, allowing rational tuning of the confined reaction volume on a post-to-post basis without needing to use surfactants to maintain stability. We design and implement a multiplexed signal amplification assay in which gel-bound enzymes turn over small molecule substrate into fluorescent product in the oil-confined gel compartment, providing significant signal enhancement. Using short (20 min) amplification times, the encapsulation scheme provides up to 2 orders of magnitude boost of signal in nucleic acid detection assays relative to direct labeling and does not suffer from any cross-talk between the posts. We ultimately demonstrate up to 57-fold increase in nucleic acid detection sensitivity compared to a direct labeling scheme.

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Impact of Nanotopography, Heparin Hydrogel Microstructures, and Encapsulated Fibroblasts on Phenotype of Primary Hepatocytes.

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