Abstract
In a typical cell culture system, growth factors immobilized on the cell culture surfaces can serve as a reservoir of bio-signaling molecules, without the need to supplement them additionally into the culture medium. In this paper, we report on the fabrication of albumin/heparin (Alb/Hep) assemblies for controlled binding of basic fibroblast growth factor (FGF-2). The surfaces were constructed by layer-by-layer adsorption of polyelectrolytes albumin and heparin and were subsequently stabilized by covalent crosslinking with glutaraldehyde. An analysis of the surface morphology by atomic force microscopy showed that two Alb/Hep bilayers are required to cover the surface of substrate. The formation of the Alb/Hep assemblies was monitored by the surface plasmon resonance (SPR), the infrared multiinternal reflection spectroscopy (FTIR MIRS) and UV/VIS spectroscopy. The adsorption of FGF-2 on the cross-linked Alb/Hep was followed by SPR. The results revealed that FGF-2 binds to the Alb/Hep assembly in a dose and time-dependent manner up to the surface concentration of 120 ng/cm2. The bioactivity of the adsorbed FGF-2 was assessed in experiments in vitro, using calf pulmonary arterial endothelial cells (CPAE). CPAE cells could attach and proliferate on Alb/Hep surfaces. The adsorbed FGF-2 was bioactive and stimulated both the proliferation and the differentiation of CPAE cells. The improvement was more pronounced at a lower FGF-2 surface concentration (30 ng/cm2) than on surfaces with a higher concentration of FGF-2 (120 ng/cm2).
Highlights
In a typical cell culture system, systematic administration of growth factors in the culture medium is required to stimulate cell growth
A decrease in the measured signal is observed during the rinsing step, when the unbound part of the albumin or heparin is removed from the surface
After replacement of the albumin solution by the citrate buffer (CB), the albumin layer was subjected to the heparin solution and after 20 minutes about 20 ng/cm2 of heparin was adsorbed
Summary
In a typical cell culture system, systematic administration of growth factors in the culture medium is required to stimulate cell growth. It is desirable to attach the protein molecule to the surface by making use of its natural affinity to the specific biological substances This strategy is commonly applied to adsorb basic fibroblast growth factor (FGF-2) on heparinized surfaces. [16,17,18]), a control over the surface composition; the type of the terminal deposited layer of the coating can be tuned depending on the application This technical aspect is used in growth factor delivery from eLbL films. Albumin, which serves as a primary anchoring layer to the substrate, can adsorb and strongly on a wide range of surfaces This is in contrast to other types of polyelectrolytes used in eLbL technique that require a presence of a stable charge on the surfaces to initiate the eLbL self-assembly process. The bioactivity of the adsorbed FGF-2 and the overall cellular responses to the Alb/Hep assembly loaded with FGF-2 were verified and studied in in vitro experiments, using calf pulmonary arterial endothelial cells (CPAE) as a model cell line
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