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Layered patterning of hepatocytes in co-culture systems using microfabricated stencils.

ABSTRACT: Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling microenvironments in which cell behavior can be observed. Here we present a novel approach to generate layered patterning of hepatocytes on micropatterned fibroblast feeder layers using microfabricated polydimethylsiloxane (PDMS) stencils. We fabricated PDMS stencils to pattern circular holes with diameters of 500 microm. Hepatocytes were co-cultured with 3T3-J2 fibroblasts in two types of patterns to evaluate and characterize the cellular interactions in the co-culture systems. Results of this study demonstrated uniform intracellular albumin staining and E-cadherin expression, increased liver-specific functions, and active glycogen synthesis in the hepatocytes when the heterotypic interface between hepatocytes and fibroblasts was increased by the layered patterning technique. This patterning technique can be a useful experimental tool for applications in basic science, drug screening, and tissue engineering, as well as in the design of bioartificial liver devices.

SUBMITTER: Cho CH

PROVIDER: S-EPMC3147300 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Layered patterning of hepatocytes in co-culture systems using microfabricated stencils.

Cho Cheul H CH Park Jaesung J Tilles Arno W AW Berthiaume François F Toner Mehmet M Yarmush Martin L ML

BioTechniques 20100101 1

Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling microenvironments in which cell behavior can be observed. Here we present a novel approach to generate layered patterning of hepatocytes on micropatterned fibroblast feeder layers using microfabricated polydimethylsiloxane (PDMS) stencils. We fabricated PDMS stencils to pattern circular holes with diameters of 500 microm. Hepatocytes were co-cultured with 3T3-J2 fibroblasts in ...[more]

PMID: 20078427

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Project description:Polymeric particles are ideal drug delivery systems due to their cellular uptake-relevant size. Microparticles could be developed for direct injection of drug formulations into a diseased site, such as a tumor, allowing for drug retention and slow drug exposure over time through sustained release mechanisms. Bombyx mori silk fibroin has shown promise as a biocompatible biomaterial both in research and the clinic. Silk has been previously used to make particles using an emulsion-based method with poly(vinyl alcohol) (PVA). In this study, polydimethylsiloxane-based microfluidic devices were designed, fabricated, and characterized to produce silk particles through self-association of silk when exposed to PVA. Three main variables resulted in differences in particle size and size distribution, or polydispersity index (PDI). Utilizing a co-flow microfluidic device decreased the PDI of the silk particles as compared to an emulsion-based method (0.13 versus 0.65, respectively). With a flow-focusing microfluidics device, lowering the silk flow rate from 0.80 to 0.06 mL/h resulted in a decrease in the median particle size from 6.8 to 3.0 μm and the PDI from 0.12 to 0.05, respectively. Lastly, decreasing the silk concentration from 12% to 2% resulted in a decrease in the median particle size from 5.6 to 2.8 μm and the PDI from 0.81 to 0.25, respectively. Binding and release of doxorubicin, a cytotoxic drug commonly used for cancer treatment, with the fabricated silk particles was evaluated. Doxorubicin loading in the silk particles was approximately 41 µg/mg; sustained doxorubicin release occurred over 23 days. When the cytotoxicity of the released doxorubicin was tested on KELLY neuroblastoma cells, significant cell death was observed. To demonstrate the potential for internalization of the silk particles, both KELLY and THP-1-derived macrophages were exposed to fluorescently labelled silk particles for up to 24 h. With the macrophages, internalization of the silk particles was observed. Additionally, THP-1 derived macrophages exposure to silk particles increased TNF-α secretion. Overall, this microfluidics-based approach for fabricating silk particles utilizing PVA as a means to induce phase separation and silk self-assembly is a promising approach to control particle size and size distribution. These silk particles may be utilized for a variety of biomedical applications including drug delivery to multiple cell types within a tumor microenvironment.

Dataset Information

Layered patterning of hepatocytes in co-culture systems using microfabricated stencils.

Publications

Layered patterning of hepatocytes in co-culture systems using microfabricated stencils.

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