Aspiration-assisted Freeform Bioprinting of Tissue Spheroids in a Yield-stress Gel

Abstract

Bioprinting of cellular aggregates, such as tissue spheroids or organoids, in complex threedimensional (3D) arrangements has been a major obstacle for scaffold-free fabrication of tissues and organs. In this research, we unveiled a new approach to the bioprinting of tissue spheroids in a yield stress granular gel, which exhibited unprecedented capabilities in freeform positioning of spheroids in 3D. Due to its Herschel-Bulkley and self-healing properties as well as its biological inertness, the granular gel supported both the positioning and self-assembly of tissue spheroids.We studied the underlying physical mechanism of the approach to elucidate the interactions between the aspirated spheroids and the gel's yield-stress during the transfer of spheroids from cell media to the gel. We demonstrate the application of the proposed approach in the realization of various freeform shapes and self-assembly of human mesenchymal stem cell spheroids for the construction of cartilage and bone tissues.Three-dimensional (3D) bioprinting within granular gels or suspension baths exhibiting Herschel-Bulkley or Bingham plastic properties has recently become a powerful approach to create complex-shaped anatomically-accurate tissues and organs 1-9 . Carbopol microgels have been one of the popular granular gel medium due to its shear thinning and self-healing properties, in which the granular gel transforms from a stable solid state into a flowing fluid phase when exposed to an external stress that exceeds its yield stress 6,10-12 . As the nozzle moves inside the granular gel, the gel locally fluidizes when in contact with the nozzle but then rapidly solidifies after the nozzle has passed thus supporting the bioprinted tissue constructs 1,7 . In most cases of bioprinting in a granular gel, cells are bioprinted while encapsulated within a hydrogel formulation, resulting in limited cell densities. 13 Hence, cellular aggregates, such as tissue spheroids, possess greater promise due to their favorable properties in building native-like tissues [14][15][16] .Bioprinting of spheroids is an attractive approach, in which spheroids are used as building blocks for fabrication of tissues that mimic the native counterparts in terms of histology and physiology 14,16,17 . Several spheroid bioprinting techniques have been reported. The first technique is extrusion-based bioprinting 18 , in which spheroids are loaded in a syringe barrel and extruded in a delivery gel medium one by one. However, spheroids self-assemble readily in the syringe and are prone to break apart during the extrusion process. Concurrently, support structures need to be 3D printed to facilitate the aggregation of extruded spheroids. An important advance has been made by utilizing the Kenzan method 19 , where spheroids are skewered on a needle array. Since the position of each spheroid depends on the needle size, location and arrangement, freeform (i.e., complex-shaped) bioprinting of spheroids is quite challenging as the spheroid positioning of spheroids along the z-axis (dire...