Extrusion-Based Bioprinting of Multilayered Nanocellulose Constructs for Cell Cultivation Using In Situ Freezing and Preprint CaCl₂ Cross-Linking

Abstract

Extrusion-based bioprinting with a preprint cross-linking agent and an in situ cooling stage provides a versatile method for the fabrication of 3D structures for cell culture. We added varying amounts of calcium chloride as a precross-linker into native nanofibrillated cellulose (NFC) hydrogel prior to 3D bioprinting to fabricate structurally stable multilayered constructs without the need for a separate cross-linking bath. To further enhance their stability, we bioprinted the multilayered structures onto an in situ temperature-controlled printing stage at 25, 0, and −10 °C. The extruded and subsequently freeze-dried volumetric constructs maintained their structures after being immersed into a cell culture medium. The ability to maintain the shape after immersion in cell media is an essential feature for the fabrication of stem cell-based artificial organs. We studied the viability and distribution of mouse embryonic fibroblast cells into the hydrogels using luminescence technique and confocal microscopy. Adding CaCl2 increased the stability of the multilayered nanocellulose structures, making them suitable for culturing cells inside the 3D hydrogel environment. Lower stage temperature considerably improved the structural stability of the 3D printed structures, however, had no effect on cell viability.

Bioprinting Scaffolds for Vascular Tissues and Tissue Vascularization

“…Extrusion printing is an economical and versatile method that enables printing of bioinks with high-cell numbers quickly. The force to extrude the bioink through the nozzle is commonly generated pneumatically [ 22 ], via a piston [ 23 ], or a helical screwblade [ 24 ]. Extrusion printing can be divided into direct extrusion, where the bioink is delivered directly from the syringe’s nozzle to the print bed, and indirect extrusion, in which bioink is deposited in combination with supportive material.…”

Synergistic coupling between 3D bioprinting and vascularization strategies

“…However, the employment of high pressure can be detrimental to cell viability [130]. As compared to other bioprinting techniques, EBB, on the other hand, enables faster bioprinting speed [131] with over 90% cell viability in general [132]. This strategy can also be combined with uniaxial or coaxial nozzles, which enable the generation of tissue construct with various structural configurations, such as hollow vascular constructs [133].…”

Nanocellulose-based porous materials: Regulation and pathway to commercialization in regenerative medicine

“…CNF-based structures have been reported for regenerative medicine. CNF aqueous suspension is similar to the extracellular matrix (ECM) and demonstrates sufficient mechanical stability for secondary cell culture purposes, even in their pure form and with no addition of polymers/particles [ [218] , [219] , [220] ]. However, to obtain properties beyond biocompatibility and cell interaction, polymers or (nano)particles are added to CNF suspension to fabricate hybrid or composite materials, allowing the incorporation of additional functionalities or the enhancement of specific properties [ 221 , 222 ].…”