(616g) Applications of Proteolipobeads in 3D Ligand Display and Cellular Interactions

Fried, E., City College of New York
Gilchrist, M. L., City College of New York
Gupta, M., City College of New York of CUNY
Nicoll, S., City College of New York

Biomaterials and biomedical research are often focused on the re-creation of complexities found in cells and cellular microenvironments. We hypothesize that biomembrane-microsphere assemblies are a viable biomimetic means to present ligands/bound factors to stem cells and mimic cellular communication in the stem cell niche. Our platform, termed proteolipobeads (PLBs), features laterally-mobile proteins and peptides positioned at the interface of a synthetic biomembrane. The presentation of ligands/bound factors involved in cell-cell interactions (e.g. cadherin) has begun in 2D culture systems by surface patterning but has not yet been well established in 3D stem cell culture systems.

We present results on three aspects of this study: 1) the fabrication and characterization of functionalized microspheres that contain supported lipid bilayers; 2) the construction and characterization of biocompatible hydrogel/microsphere hybrid scaffolds that integrate lipid bilayer/microsphere assemblies and 3) preliminary experiments of human mesenchymal stem cells (hMSCs) microencapsulated into matrix/PLB hybrid scaffolds. Special attention was given to the viability of hMSCs within this platform, as well as the frequency and extent of hMSC-PLB interactions. A primary feature of the PLB construct is a membrane-embedded synthetic peptide, which serves as a conjugation point for both ligands displayed at the PLB exterior and for biomembrane-microsphere tethering at the PLB interior. Moreover, the phyics of lipid-peptide interactions permits spatial control of the peptide, and consequently, ligand display.

Confocal microscopy studies were conducted to assess the diffusivity of laterally mobile PLB constituents (via FRAP) and to visualize N-cadherin-mediated PLB interactions with hMSCs in situ. Immunohistochemical staining was used to detect N-Cadherin within the hybrid matrices relative to counterstained supported lipid bilayers. Finally, we used flow cytometry to detect the level and homogeneity of specific fluorescently labeled proteins, peptides, and lipid bilayers included in the PLB assemblies. This work constitutes a new method for displaying a wide range of complex membrane proteins and signaling molecules to live cells within 3D matrices.