Dataset Information

Nanofibrous Scaffolds Support a 3D in vitro Permeability Model of the Human Intestinal Epithelium.

ABSTRACT: Advances in drug research not only depend on high throughput screening to evaluate large numbers of lead compounds but also on the development of in vitro models which can simulate human tissues in terms of drug permeability and functions. Potential failures, such as poor permeability or interaction with efflux drug transporters, can be identified in epithelial Caco-2 monolayer models and can impact a drug candidate's progression onto the next stages of the drug development process. Whilst monolayer models demonstrate reasonably good prediction of in vivo permeability for some compounds, more developed in vitro tools are needed to assess new entities that enable closer in vivo in vitro correlation. In this study, an in vitro model of the human intestinal epithelium was developed by utilizing nanofibers, fabricated using electrospinning, to mimic the structure of the basement membrane. We assessed Caco-2 cell response to these materials and investigated the physiological properties of these cells cultured on the fibrous supports, focusing on barrier integrity and drug-permeability properties. The obtained data illustrate that 2D Caco-2 Transwell® cultures exhibit artificially high trans-epithelial electrical resistance (TEER) compared to cells cultured on the 3D nanofibrous scaffolds which show TEER values similar to ex vivo porcine tissue (also measured in this study). Furthermore, our results demonstrate that the 3D nanofibrous scaffolds influence the barrier integrity of the Caco-2 monolayer to confer drug-absorption properties that more closely mimic native gut tissue particularly for studying passive epithelial transport. We propose that this 3D model is a suitable in vitro model for investigating drug absorption and intestinal metabolism.

SUBMITTER: Patient JD

PROVIDER: S-EPMC6524416 | biostudies-other | 2019

REPOSITORIES: biostudies-other

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Nanofibrous Scaffolds Support a 3D <i>in vitro</i> Permeability Model of the Human Intestinal Epithelium.

Patient Jamie D JD Hajiali Hadi H Harris Kate K Abrahamsson Bertil B Tannergren Christer C White Lisa J LJ Ghaemmaghami Amir M AM Williams Philip M PM Roberts Clive J CJ Rose Felicity R A J FRAJ

Frontiers in pharmacology 20190510

Advances in drug research not only depend on high throughput screening to evaluate large numbers of lead compounds but also on the development of <i>in vitro</i> models which can simulate human tissues in terms of drug permeability and functions. Potential failures, such as poor permeability or interaction with efflux drug transporters, can be identified in epithelial Caco-2 monolayer models and can impact a drug candidate's progression onto the next stages of the drug development process. Whils ...[more]

PMID: 31133850

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Project description:A major challenge of maintaining primary hepatocytes in vitro is progressive loss of hepatocyte-specific functions, such as protein synthesis and cytochrome P450 (CYP450) catalytic activity. We developed a three-dimensional (3D) nanofibrous scaffold made from poly(l-lactide-co-glycolide) (PLGA) polymer using a newly optimized wet electrospinning technique that resulted in a highly porous structure that accommodated inclusion of primary human hepatocytes. Extracellular matrix (ECM) proteins (type I collagen or fibronectin) at varying concentrations were chemically linked to electrospun PLGA using amine coupling to develop an in vitro culture system containing the minimal essential ECM components of the liver micro-environment that preserve hepatocyte function in vitro. Cell-laden nanofiber scaffolds were tested in vitro to maintain hepatocyte function over a two-week period. Incorporation of type I collagen onto PLGA scaffolds (PLGA-Chigh: 100?µg/mL) led to 10-fold greater albumin secretion, 4-fold higher urea synthesis, and elevated transcription of hepatocyte-specific CYP450 genes (CYP3A4, 3.5-fold increase and CYP2C9, 3-fold increase) in primary human hepatocytes compared to the same cells grown within unmodified PLGA scaffolds over two weeks. These indices, measured using collagen-bonded scaffolds, were also higher than scaffolds coupled to fibronectin or an ECM control sandwich culture composed of type I collagen and Matrigel. Induction of CYP2C9 activity was also higher in these same type I collagen PLGA scaffolds compared to other ECM-modified or unmodified PLGA constructs and was equivalent to the ECM control at 7?days. Together, we demonstrate a minimalist ECM-based 3D synthetic scaffold that accommodates primary human hepatocyte inclusion into the matrix, maintains long-term in vitro survival and stimulates function, which can be attributed to coupling of type I collagen. STATEMENT OF SIGNIFICANCE:Culturing primary hepatocytes within a three-dimensional (3D) structure that mimics the natural liver environment is a promising strategy for extending the function and viability of hepatocytes in vitro. In the present study we generate porous PLGA nanofibers, that are chemically modified with extracellular matrix proteins, to serve as 3D scaffolds for the in vitro culture of primary human hepatocytes. Our findings demonstrate that the use of ECM proteins, especially type I collagen, in a porous 3D environment helps to improve the synthetic function of primary hepatocytes over time. We believe the work presented within will provide insights to readers for drug toxicity and tissue engineering applications.

Project description:The small intestine is the primary site of drug absorption following oral administration, making paramount the proper monitoring of the absorption process. In vitro tools to predict intestinal absorption are particularly important in preclinical drug development since they are less laborious and cost-intensive and raise less ethical considerations compared to in vivo studies. The Caco-2 model is considered the gold standard of in vitro intestinal models regarding the prediction of absorption of orally delivered compounds. However, this model presents several drawbacks, such as the expression of tighter tight junctions, not being suitable to perform permeability of paracellular compounds. Besides, cells are representative of only one intestinal cell type, without considering the role of non-absorptive cells on the absorption pathway of drugs. In the present study, we developed a new three-dimensional (3D) intestinal model that aims to bridge the gap between in vitro tools and animal studies. Our 3D model comprises a collagen layer with human intestinal fibroblasts (HIFs) embedded, mimicking the intestinal lamina propria and providing 3D support for the epithelium, composed of Caco-2 cells and mucus-producing HT29-MTX cells, creating a model that can better resemble, both in terms of composition and regarding the outcomes of drug permeability when testing paracellular compounds, the human small intestine. The optimization of the collagen layer with HIFs was performed, testing different collagen concentrations and HIF seeding densities in order to avoid collagen contraction before day 14, maintaining HIF metabolically active inside the collagen disks during time in culture. HIF morphology and extracellular matrix (ECM) deposition were assessed, confirming that fibroblasts presented a normal and healthy elongated shape and secreted fibronectin and laminin, remodeling the collagen matrix. Regarding the epithelial layer, transepithelial electrical resistance (TEER) values decreased when cells were in the 3D configuration, comparing with the 2D analogs (Caco-2 and coculture of Caco-2+HT29-MTX models), becoming more similar with in vivo values. The permeability assay with fluorescein isothiocyanate (FITC)-Dextran 4 kDa showed that absorption in the 3D models is significantly higher than that in the 2D models, confirming the importance of using a more biorelevant model when testing the paracellular permeability of compounds.

Dataset Information

Nanofibrous Scaffolds Support a 3D in vitro Permeability Model of the Human Intestinal Epithelium.

Publications

Nanofibrous Scaffolds Support a 3D <i>in vitro</i> Permeability Model of the Human Intestinal Epithelium.

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