Use of Alginate Hydrogels as Non-Immunogenic Reactive Substrates for Neutrophil Adhesion in in Vitro Flow Adhesion Assays

In vitro cell-free adhesion assays have previously been useful in elucidating neutrophil receptor-ligand interactions and cell signaling. However, these cell-free assays often involve the use of plastic or glass substrate that can generate a significant amount of non-specific adhesion. Moreover, neutrophils are highly active cells that can undergo significant non-specific activation on plastic and glass substrates – particularly when experiments are conducted at body temperature. Alginate hydrogels are non-immunogenic in nature and thus support cell adhesion only when chemically grafted with active biological molecules. As such alginate hydrogels are attractive for in vitro cell adhesion studies. We show that alginate hydrogels modified with leukocyte adhesion ligands such as the selectins and ICAM-1 can support rolling and firm adhesion of neutrophils in a parallel plate flow chamber. Specifically, neutrophils interacting with P-selectin coupled gels displayed slow rolling in the flow chamber while maintaining their spherical shape at 37oC – suggesting that cells remain unactivated in the absence of an external activating stimulus. When ICAM-1 was coupled to the gel surface, neutrophil firm adhesion was observed. Compared to the traditional glass cover slip, alginate hydro-gels also present a better structural model of the cell wall. Finally, we show that alginate gels were able to withstand vacuum pressure during experimentation for extended periods of time. Overall, the described alginate hydrogel for use in parallel plate flow chamber shear adhesion assays can allow further study of neutrophil rolling and firm adhesion dynamics at more physiological conditions.