Embedded 3D printing in self-healing annealable composites for precise patterning of functionally mature human neural constructs

Abstract

Human in vitro models of neural tissue with controllable cellular composition, tunable microenvironment, and defined spatial patterning are needed to facilitate studies of brain development and disease. Towards this end, bioprinting has emerged as a promising strategy. However, precise and programmable printing of extremely soft and compliant materials that are permissive for stem cell differentiation and functional neuronal growth has been a major challenge. Therefore, solutions for engineering structurally and functionally defined human neural constructs remain scarce. Here, we present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self-Healing Annealable Particle-Extracellular matrix (SHAPE) composites. The approach combines rheological benefits of granular soft microgel supports with the versatile biomimicry of bulk hydrogels to simultaneously enable precise freeform patterning of stem cells, and consequent generation and long-term maintenance of subtype-specific neurons within engineered networks that extend into the bulk of the annealed support. The developed approach further allows multi-ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular channels. Due to its modularity, SHAPE biomanufacturing toolbox not only offers a solution for functional modeling of mechanically sensitive neural constructs, but also has potential to be applied to a wide range of biomaterials with different crosslinking mechanisms to model tissues and diseases where recapitulation of complex architectural features and topological cues is essential.

A dive into the bath: embedded 3D bioprinting of freeform in vitro models

“…Annealing of these composite materials for "locking" the structure after high fidelity bioprinting generates a stable and cell-interactive matrix for long term functional development of target tissues models. 25 This concept allows to provide functional housing devices or living environments, the typical microfluidic bioreactor of organs-on-chip, to the printed constructs for their long-term maturation/mainten- ance and screening. Several methods can be applied for annealing the bath depend on its nature, 62 including e.g., thermally induced crosslinking of ECM proteins exiting in its composition, 25 enzymatic crosslinking of gelatin-based hydrogels (e.g.…”

“…25 This concept allows to provide functional housing devices or living environments, the typical microfluidic bioreactor of organs-on-chip, to the printed constructs for their long-term maturation/mainten- ance and screening. Several methods can be applied for annealing the bath depend on its nature, 62 including e.g., thermally induced crosslinking of ECM proteins exiting in its composition, 25 enzymatic crosslinking of gelatin-based hydrogels (e.g. by microbial transglutaminase), 65 or by promoting nanoparticles self-assembly with addition of biocompatible ions (e.g.…”

“…Importantly, a key advantage of bioprinting in support baths is the possibility of using low viscosity bioinks, 21 which favors the viability of printed cells and its general biological performance in printed constructs. [22][23][24][25] Similarly, compared to traditional unsupported strategies, these systems further facilitate the bioprinting of multimaterial 26 and multicellular structures. 27 Moreover, since the crosslinking process takes place when the bioprinting of all the material is finished 28 and not layer-by-layer as in conventional methods, 29 an additional advantage of bioprinting in support baths is the possibility of accelerating the manufacturing process of cellularized constructs, opening the door for the manufacturing escalation 30,31 of the bioprinting process, reducing printing times and costs.…”

Balance between the cell viability and death in 3D

“…cell aggregates, and biocompatible polymer-based bioinks to print layer-by-layer structures, expected to heal the tissue. Popular techniques include inkjet [42], laser-assisted [43], and extrusion bioprinting [44,45], which can be used to generate different 'tissue building blocks' assembled via a bottom-up approach. In contrast to bioprinting, the bioassembly is a top-down approach in which cells and cell aggregates are seeded in porous scaffolds with a 2D or 3D organization, allowing them to self-assemble into tissue constructs [46].…”

In Situ Endothelialization of Free‐Form 3D Network of Interconnected Tubular Channels via Interfacial Coacervation by Aqueous‐in‐Aqueous Embedded Bioprinting

“…Such support medium can be granular microgels, [ 24 ] cell spheroids, [ 25 ] healable bulk hydrogels, [ 26–28 ] as well as composites of granular gels and viscous solutions. [ 29 ] Typically, viscous inks are required for these gel mediums, which could inhibit cell motility and induce high shear strain on cells at a fine printing resolution. Although liquid mediums permit the use of low‐viscosity ink, aqueous structures constructed in viscous oils are not suitable for cell culturing.…”