"All-in-one" zwitterionic granular hydrogel bioink for stem cell spheroids production and 3D bioprinting

Based on "bottom-up" strategy, cell-laden 3D bioprinting is an important process of fabricating sophisticated biomimetic structures. Stem cell spheroids possess better biological properties and are advanced as printing units. However, efficient and convenient preparation, collection and printing of cell spheroids remains a challenge. The present study developed a composite granular hydrogel acted as "all-in-one" multi-functional bioink to realize Adipose-derived stem cell (ASC) spheroids Production and reorganization. Poly(sulfobetaine methacrylate) (PSBMA) based microspheres were fabricated and self-assembled to a granular hydrogel. Then, Nisopropylacrylamide (NIPAM) and SBMA were co-polymerized in-situ inside of assembled PSBMA microspheres to further crosslink the assembled microspheres, forming a composite granular hydrogel, which possesses bulk feature and shear-thinning, self-healing properties. Caves were created by compressing composite granular hydrogels into sugar particles that removed via dissolution in water. Due to the non-fouling feature and the porous structure, numerous ASC spheroids were formed spontaneously inside the porous composite granular hydrogel, which were transferred conveniently to 3D printer. The spheroids-laden composite granular hydrogels showed well-performed extrudability and fidelity, realizing reorganization of the produced spheroids to the stable 3D constructs for further in vitro culture. ASC spheroids in granular hydrogel after 3D printing showed high level of viability and stemness, as well as efficient chondrogenic/osteogenic/adipogenic differentiation. The composite granular hydrogel thus shows potential toward stem cell researches such as organoids construction. The results of both in vitro drug toxicity and in vivo cartilage regeneration experiments showed spheroids-laden granular hydrogel is promising in drug screening and tissue regeneration.