A new method for incorporating functional heparin onto the surface of islets of Langerhans

Abstract

A novel technique is described to conjugate macromolecular heparin complexes to cell surfaces. The method is based on the dual properties of avidin-expressing binding sites for both biotin and a macromolecular complex of heparin. A quartz crystal microbalance with dissipation monitoring (QCM-D) revealed sequential binding of biotin, avidin, and heparin complexes. Large particle flow cytometry confirmed functional integrity. Confocal microscopy of the heparinized islets showed evenly distributed fluorescence. An in vitro Chandler loop model demonstrated that the biocompatibility of the new method is comparable to the previous method used on artificial materials with regard to coagulation and antithrombin uptake. The technique presented allows human islets of Langerhans to successfully be covered with functional heparin as a means to reduce instant blood-mediated inflammatory reactions induced by the innate immune system.

FIG. 1.

Use of QCM-D to monitor the stepwise build-up of a heparin coat with biotin/avidin. The polystyrene surface was initially coated with albumin in order to create a matrix to which biotin could bind. For each step in which an increased mass is added to the sensor, a corresponding dampening of the frequency is obtained (f, blue line). The corresponding dissipation change, ΔD, is indicated by the pink line. This binding was followed by exposure to avidin and finally to the heparin conjugate. The biological function of the coat was then tested by allowing AT to bind to the surface-linked heparin.

FIG. 2.

Large particle flow cytometry. Islets were analyzed in a BioSorter 1000 large particle flow cytometer. Particles were distinguished by size, optical density, and intensity of fluorescence markers. Background fluorescence of control and heparinized islets (A, B). Heparinized islets detected using fluorochrome-labeled AT or avidin (C, D). Heparinized islets labeled with AT (E) or avidin (F) after incubation with FXa. Color images available online at www.liebertpub.com/ten.

FIG. 3.

Binding of heparin to human islets as visualized by confocal microscopy. Human islets were heparinized and analyzed by confocal microcopy. Bound heparin was visualized using fluorochrome-labeled avidin (red). The upper two panels show representative untreated (A) and heparinized (B) human islets. The lower two panels show representative cross sections of untreated (C) and heparinized (D) human islets. DAPI (blue) was used to detect cell nuclei in the islets. Scale bars: 200 μm.