Generating substrate bound functional chemokine gradients in vitro

Gertrud Malene Hjortø, Morten Hansen, Niels Bent Larsen, Thomas N Kledal

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    Microcontact printing (mCP) is employed to generate discontinuous microscale gradients of active fractalkine, a chemokine expressed by endothelial cells near sites of inflammation where it is believed to form concentration gradients descending away from the inflamed area. In vivo, fractalkine is a transmembrane molecule extending its chemokine domain into the vascular lumen. Substrate bound in vitro gradients may thus closely resemble in vivo conditions. Direct mCP of sensitive proteins like fractalkine may cause partial protein denaturation and will not ensure correct orientation of the biologically active part of the molecules. Here, indirect mCP of a capture antibody recognizing a molecular tag on the target protein is successfully used to pattern tagged fractalkine in microscale gradient patterns. Fractalkine functions as an adhesion molecule for leukocytes. Cells expressing the fractalkine receptor are found to attach to the gradient structure at a density correlated with the fractional area covered by fractalkine. This indicates that the patterned fractalkine maintains its biological function. The method can be applied to in vitro studies of cell responses to the wide range of naturally surface-bound chemokines (haptotactic gradients). The use of a capture antibody facilitates control of the orientation of tagged molecules, thereby ensuring a high degree of bio-functionality through correct presentation and reduced protein denaturation.
    Original languageEnglish
    JournalBiomaterials
    Volume30
    Issue number29
    Pages (from-to)5305-5311
    ISSN0142-9612
    DOIs
    Publication statusPublished - 2009

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