Polydopamine-Functionalized Agarose Microspheres for 3D Engineered Neuronal Cultures

Abstract

Developing biomimetic 3D constructs to study neuronal networks and screen therapeutic agents in vitro is critical to address the limitations of conventional 2D cultures used in pre-clinical studies. Since substrate stiffness and culture dimensionality influence behaviour of neurons in vitro, using rigid 2D substrates limits translational potential and physiological accuracy. To improve physiological relevance of in vitro neuronal cultures, 3D constructs are engineered to recapitulate the material properties of the cerebral matrix which neuronal networks depend upon in vivo. Of particular importance is mimicking the low Young’s modulus of the extracellular matrix in the brain, while providing regions for neuronal adhesion to promote viability and growth. To this end, a 3D microsphere-based strategy was investigated, where multiple microspheres collectively acting as a scaffold with high interconnected porosity were used to provide adhesive, 3D growth area. Specifically, agarose—a natural polysaccharide—was used to fabricate soft microspheres which are subsequently functionalized with the bio-inspired and adhesive polymer, polydopamine. Microfluidic technology yielded an agarose microsphere population of highly monodisperse diameters. Successful functionalization of the microspheres with polydopamine was verified through identification of the characteristic Raman spectra of polydopamine. Measured through atomic force nanoindentations in water, the Young’s modulus of the agarose microsphere measured 4.9 ± 0.6 kPa –10⁶ times lower than conventional culture-ware. The Young’s modulus was found to increase to 26.8 ± 17.9 kPa after functionalization with polydopamine, where the large standard deviation was attributed to the nano- mechanical and -topographical non- homogeneity of the resulting surface due to polydopamine aggregation Microsphere stiffness followed the same trend while also increasing as a function of indentation depth. Functionalization of microspheres with polydopamine was also shown to significantly increase material surface adhesion compared to poly-D-lysine and laminin functionalized glass microspheres (a recently reported microsphere-based model). Finally, the microspheres were shown to provide embryonic cerebral neurons with 3D growth area while facilitating neural adhesion and neurite development. This work provides a 3D, soft and adhesive in vitro model for neuronal culture with improved biomimetics over current microsphere-based protocols, and ultimately rigid 2D culture-ware.