Generating substrate bound functional chemokine gradients in vitro

TY - JOUR

T1 - Generating substrate bound functional chemokine gradients in vitro

AU - Hjortø, Gertrud M

AU - Hansen, Morten

AU - Larsen, Niels B

AU - Kledal, Thomas N.

PY - 2009/10

Y1 - 2009/10

N2 - 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.

AB - 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.

KW - Adsorption

KW - Cell Adhesion

KW - Cell Line

KW - Chemokine CX3CL1

KW - Coated Materials, Biocompatible

KW - Humans

KW - Kidney

KW - Materials Testing

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1016/j.biomaterials.2009.06.018

DO - 10.1016/j.biomaterials.2009.06.018

M3 - Journal article

C2 - 19577290

SN - 0142-9612

VL - 30

SP - 5305

EP - 5311

JO - Biomaterials

JF - Biomaterials

IS - 29

ER -