However, it is often challenging to print large free-form tissue structures owing to the inadequate structural integrity and mechanical stability of softhydrogel-based bioink. [4] For the past decade, considerable effort has been made to address this particular challenge. For example, Kang and co-workers proposed a hydrogel reinforcing strategy by coprinting cell-laden hydrogel bioinks with synthetic polycaprolactone (PCL) polymer that serves as a supporting framework. [5] Although this strategy allowed the fabrication of humanscale bone and cartilage tissue constructs, the mechanically robust PCL fibers often impeded the maturation of soft tissue such as the heart and liver. Alternatively, the embedded 3D bioprinting strategy has gained increasing popularity for constructing complex freeform structures. [6] In this approach, a suspension medium is utilized to support the deposition of bioinks in 3D space before crosslinking. The suspension medium undergoes rapid fluidization at yield stress and then solidification in the absence of stress due to its unique shear-thinning and self-healing properties. [7] The printed structures can easily be removed from the suspension medium by gently washing the suspension medium or raising the temperature. As an example of this strategy, Lee and co-workers printed a functional ventricle and full-size human heart model into a gelatin microparticles-based suspension medium by using a freeform reversible embedding of suspended hydrogels, also termed the FRESH technique. [8] Creating functional tissues and organs in vitro on demand is a major goal in biofabrication, but the ability to replicate the external geometry of specific organs and their internal structures such as blood vessels simultaneously remains one of the greatest impediments. Here, this limitation is addressed by developing a generalizable bioprinting strategy of sequential printing in a reversible ink template (SPIRIT). It is demonstrated that this microgel-based biphasic (MB) bioink can be used as both an excellent bioink and a suspension medium that supports embedded 3D printing due to its shear-thinning and self-healing behavior. When encapsulating human-induced pluripotent stem cells, the MB bioink is 3D printed to generate cardiac tissues and organoids by extensive stem cell proliferation and cardiac differentiation. By incorporating MB bioink, the SPIRIT strategy enables the effective printing of a ventricle model with a perfusable vascular network, which is not possible to fabricate using extant 3D printing strategies. This SPIRIT technique offers an unparalleled bioprinting capability to replicate the complex organ geometry and internal structure in a faster manner, which will accelerate the biofabrication and therapeutic applications of tissue and organ constructs.

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Ovary disease is a major cause of female infertility and could disrupt the hormone balance, which may cause multi-organ disorders in the long-term effect. However, conventional artificial ovary techniques usually encapsulate follicles in 3D hydrogels and block the ovulation ability. In this study, we fabricated a biomimetic micro-cavity ovary by a microsphere-templated technique to recapitulate the ovulation process, where sacrificial gelatin microspheres were mixed with photo-crosslinkable gelatin methacryloyl (GelMA) to engineer an open cavity niche for follicle growth. The microcavity’s parameters were optimized to be suitable for follicle growth. With that, we showed that the micro-cavity ovary could support the follicle growth to antral stage and the ovulation of meiosis-matured oocytes out of the micro-cavity ovary without extra manipulation. The ovulated oocyte was characterized in a great quality. This technique would be of great advantage to be further applied in clinics that recover natural conception after in vivo transplantation of the biomimetic ovary.