Comprehensive Characterization of an “Off/On” Rhodol-Based Lysosomal Tracker for Orthogonal Cellular Analysis by Confocal Imaging

Two florescent xanthene-cyanamide lysosomal trackers emitting strongly at ∼525 nm were prepared from fluorescein and rhodol methyl esters in microwave-assisted reactions. Both forms named “off” (nonfluorescent lactam) and “on” (strongly fluorescent ring-opened amide) have been comprehensively characterized out by using a combination of NMR spectroscopy, X-ray analysis, fluorimetry and confocal microscopy. Known rhodamines bearing electron-withdrawing groups (EWGs) exhibit an equilibrium between non-fluorescent (off) and fluorescent (on) depending on the dielectric constant of the medium. Here, cyanamide was introduced as EWG amine into the fluorescein and rhodol framework. Unlike rhodamine-type dyes, the ring-opened forms of fluorescein- and rhodol-cyanamides are stable in protic solvents under circumneutral and basic pH conditions. The osteoblastic cell line MC3T3-E1 from C57BL/6 mouse calvaria was used for confocal imaging where the different organelles and nuclei were distinguished by using an orthogonal combination of fluorescent dyes.     

Multi-Material 3D Microprinting of Magnetically Deformable Biocompatible Structures

Two-photon polymerization (2PP) allows precise 3D printing at the micrometer scale, and by associating it with magnetic materials, the creation of remotely actuatable micro-structures. Such structures attract a growing interest for biomedical applications, thanks to their size and to the biocompatibility of some photoresist materials. Gelatin methacryloyl (Gel-MA) is one such material, and can be used to create physiological scaffolds for cell culture. Here, the physico-chemical properties of two resins are exploited, the first being a silica-based hybrid polymer, the OrmoComp, and the second a Gel-MA-based hydrogel. A 2PP manufacturing protocol is defined and designed to print both materials in succession as a single structure, which is then linked to a neodymium-iron-boron (NdFeB) magnetic bead for actuation. By this combination, a magnetically deformable 3D culture substrate is created to study cells in an environment that mimics soft, curved, and dynamic properties of tissues in vivo. The structure is actuated via an external magnetic field and bends back and forth along its longest axis. Lastly, preliminary cell culture trials are conducted showing the proliferation of cells on the structures.