Abstract
3D cell culture is a helpful approach to study cell-cell interaction in a native-like environment, but is often limited due the challenge of retrieving cells from the material. In this study, we present the use of recombinant lectin B, a sugar-binding protein with four binding cavities, to enable reversible cell integration into a macroporous protein hydrogel matrix. By functionalizing hydrogel precursors with saccharose, lectin B can both bind to sugar moieties on the cellular surface as well as to the modified hydrogel network. Confocal microscopy and flow cytometry analysis revealed cells to be integrated into the network and to adhere and proliferate. Furthermore, the specificity and reversibility was investigated by using a recombinantly produced yellow fluorescent - lectin B fusion protein and a variety of sugars with diverging affinities for lectin B at different concentrations and elution times. Cells could be eluted within minutes by addition of L-fucose to the cell-loaded hydrogels to make cells available for further analysis.
Highlights
Direct cell encapsulation during hydrogel formation leads to an easy handling and a uniform distribution of cells within the materials, but is often challenging due to cell toxicity effects of precursors and the inherent lack of reversibility
We aimed to introduce a new method for the reversible immobilization and adhesion of cells into a macroporous hydrogel system based on reversible binding of cells to sugar residues mediated by a multivalent lectin as a specific molecular adapter
In order to realize binding of yellow fluorescent protein (YFP)-LecB to the macroporous cell culture matrix composed of chemically crosslinked bovine serum albumin (BSA), we used the sugar dimer saccharose to introduce a receptor
Summary
Direct cell encapsulation during hydrogel formation leads to an easy handling and a uniform distribution of cells within the materials, but is often challenging due to cell toxicity effects of precursors and the inherent lack of reversibility. The key challenge is to produce systems from biocompatible precursors, and both gel formation and disintegration for cell release purposes have to be fast, gentle and cell compatible. Tielker et al have recombinantly produced LecB in Escherichia coli and established a purification method through mannose agarose beads to explore the potential of LecB as a tag for single-step protein purification[34]. They constructed a YFP-LecB fusion protein containing LecB as a binding domain and YFP as a reporter for different possible applications. The different sugar affinities of this fusion protein can be used to reversibly immobilize cells into a 3D matrix
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