Bioprinting of cellular aggregates, such as tissue spheroids, to form three-dimensional (3D) complex-shaped arrangements, has posed a major challenge due to lack of robust, reproducible and practical bioprinting techniques. Here, we demonstrate 3D aspiration-assisted freeform bioprinting of tissue spheroids by precisely positioning them in self-healing yield-stress gels, enabling the self-assembly of spheroids for fabrication of tissues. The presented approach enables the traverse of spheroids directly from the cell media to the gel and freeform positioning of the spheroids on demand. We study 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 further 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.

Aspiration-assisted freeform bioprinting (AAfB) has emerged as a promising technique for precise placement of tissue spheroids in three-dimensional (3D) space enabling tissue fabrication. To achieve success in embedded bioprinting using AAfB, an ideal support bath should possess shear-thinning behavior and yield-stress to facilitate tight fusion and assembly of bioprinted spheroids forming tissues. Several studies have demonstrated support baths for embedded bioprinting in the past few years, yet a majority of these materials poses challenges due to their low biocompatibility, opaqueness, complex and prolonged preparation procedures, and limited spheroid fusion efficacy. In this study, to circumvent the aforementioned limitations, we present the feasibility of AAfB of human mesenchymal stem cell (hMSC) spheroids in alginate microgels as a support bath. Alginate microgels were first prepared with different particle sizes modulated by blending time and concentration, followed by determination of the optimal bioprinting conditions by the assessment of rheological properties, bioprintability, and spheroid fusion efficiency. The bioprinted and consequently self-assembled tissue structures made of hMSC spheroids were osteogenically induced for bone tissue formation. Alongside, we investigated the effects of peripheral blood monocyte-derived osteoclast incorporation into the hMSC spheroids in heterotypic bone tissue formation. We demonstrated that alginate microgels enabled unprecedented positional accuracy (~5%), transparency for visualization, and improved fusion efficiency (~97%) of bioprinted hMSC spheroids for bone fabrication. This study demonstrates the potential of using alginate microgels as a support bath for many different applications including but not limited to freeform bioprinting of spheroids, cell-laden hydrogels, and fugitive inks to form viable tissue constructs.

Engineered bone grafts require a vascular network to supply cells with oxygen, nutrients and remove waste. Using heterotypic mature cells to create these grafts in vivo has resulted in limited cell density, ectopic tissue formation and disorganized tissue. Despite evidence that progenitor cell aggregates, such as progenitor spheroids, are a potential candidate for fabrication of native-like pre-vascularized bone tissue, the factors dictating progenitor co-differentiation to create heterotypic pre-vascularized bone tissue remains poorly understood. In this study, we examined a three-dimensional heterotypic pre-vascularized bone tissue model, using osteogenic and endotheliogenic progenitor spheroids induced by miR-148b and miR-210 mimic transfection, respectively. Spheroids made of transfected cells were assembled into heterotypic structures to determine the impact on co-differentiation as a function of micro-RNA (miRNA) mimic treatment group and induction time. Our results demonstrated that miRNAs supported the differentiation in heterotypic structures, and that developing heterotypic structures is determined in part by progenitor maturity, as confirmed by gene and protein markers of osteogenic and endotheliogenic differentiation and the mineralization assay. As a proof of concept, miRNA-transfected spheroids were also bioprinted using aspiration-assisted bioprinting and organized into hollow structures to mimic the Haversian canal. Overall, the presented approach could be useful in fabrication of vascularized bone tissue using spheroids as building blocks.

Despite substantial advancements in development of cancer treatments, lack of standardized and physiologically-relevant in vitro testing platforms limit the early screening of anticancer agents. A major barrier is the complex interplay between the tumor microenvironment and immune response. To tackle this, a dynamic-flow based 3D bioprinted multi-scale vascularized breast tumor model, responding to chemo and immunotherapeutics is developed. Heterotypic tumors are precisely bioprinted at pre-defined distances from a perfused vasculature, exhibit tumor angiogenesis and cancer cell invasion into the perfused vasculature. Bioprinted tumors treated with varying dosages of doxorubicin for 72 h portray a dose-dependent drug response behavior. More importantly, a cell based immune therapy approach is explored by perfusing HER2-targeting chimeric antigen receptor (CAR) modified CD8 + T cells for 24 or 72 h. Extensive CAR-T cell recruitment to the endothelium, substantial T cell activation and infiltration to the tumor site, resulted in up to ≈70% reduction in tumor volumes. The presented platform paves the way for a robust, precisely fabricated, and physiologically-relevant tumor model for future translation of anti-cancer therapies to personalized medicine.

Engineering of osteochondral interfaces remains a challenge. MicroRNAs (miRs) have emerged as significant tools to regulate the differentiation and proliferation of osteogenic and chondrogenic formation in the human musculoskeletal system. Here, we describe a novel approach to osteochondral reconstruction based on three-dimensional (3D) bioprinting of miR-transfected adipose-derived stem cell (ADSC) spheroids to produce a heterotypic interface that addresses the intrinsic limitations of the traditional approach in inducing zonal differentiation via the use of diffusible cytokines. We evaluated the delivery of miR-148b for osteogenic differentiation and the codelivery of miR-140 and miR-21 for chondrogenic differentiation of ADSC spheroids. Our results demonstrated that miR-transfected ADSC spheroids exhibited upregulated expression of osteogenic and chondrogenic differentiation related gene and protein markers, and enhanced mineralization and cell proliferation compared to spheroids differentiated using commercially-available differentiation medium. Upon confirmation of osteogenic and chondrogenic potential of miR-transfected ADSC spheroids, using aspiration-assisted bioprinting, these spheroids were 3D bioprinted into a dual-layer heterotypic osteochondral interface with stratified arrangement of distinct osteogenic and chondrogenic zones. The proposed approach holds great promise in biofabrication of stratified tissues, not only for osteochondral interfaces presented in this work, but also for other composite tissues and tissue interfaces, such as but not limited to bone-tendon-muscle interface and craniofacial tissues.

Gene therapeutic applications combined with bio- and nano-materials have been used to address current shortcomings in bone tissue engineering due to their feasibility, safety and potential capability for clinical translation. Delivery of non-viral vectors can be altered using gene-activated matrices to improve their efficacy to repair bone defects. Ex-situ and in-situ delivery strategies are the most used methods for bone therapy, which have never been directly compared for their potency to repair critical-sized bone defects. In this regard, we first time explore the delivery of polyethylenimine (PEI) complexed plasmid DNA encoding bone morphogenetic protein-2 (PEI-pBMP-2) using the two delivery strategies, ex-situ and in-situ delivery. To realize these gene delivery strategies, we employed intraoperative bioprinting (IOB), enabling us to 3D bioprint bone tissue constructs directly into defect sites in a surgical setting. Here, we demonstrated IOB of an osteogenic bioink loaded with PEI-pBMP-2 for the in-situ delivery approach, and PEI-pBMP-2 transfected rat bone marrow mesenchymal stem cells (rBMSCs) laden bioink for the ex-situ delivery approach as alternative delivery strategies. We found that in-situ delivery of PEI-pBMP-2 significantly improved bone tissue formation compared to ex-situ delivery. Despite debates amongst individual advantages and disadvantages of ex-situ and in-situ delivery strategies, our results ruled in favor of the in-situ delivery strategy, which could be desirable to use for future clinical applications.

Aspiration-assisted freeform bioprinting (AAfB) has emerged as a promising technique for precise placement of tissue spheroids in three-dimensional (3D) space for fabrication of tissues. For successful embedded bioprinting using AAfB, an ideal support bath should possess shear-thinning behavior and yield-stress to obtain tightly fused assembly of bioprinted spheroids. Several studies have demonstrated support baths for embedded bioprinting, but these materials pose major challenges due to their low biocompatibility, opaqueness, complex and prolonged preparation procedures, and limited spheroid fusion efficacy. In this study, to circumvent the aforementioned limitations, we present the feasibility of AAfB of human mesenchymal stem cell (hMSC) spheroids in alginate microgels as a support bath. First, alginate microgels were prepared with different particle sizes modulated by blending time and concentration, followed by determination of the optimal bioprinting conditions by the assessment of rheological properties, bioprintability, and spheroid fusion efficiency. The bioprinted and consequently self-assembled tissue structures made of hMSC spheroids were osteogenically induced for bone tissue formation. Alongside, we investigated the effects of peripheral blood monocyte-derived osteoclast incorporation into the hMSC spheroids in heterotypic bone tissue formation. We demonstrated that alginate microgels enabled unprecedented positional accuracy (~5%), transparency for visualization, and improved fusion efficiency (~97%) of bioprinted hMSC spheroids for bone fabrication. This study demonstrates the feasibility of using alginate microgels as a support bath for many different applications including but not limited to freeform bioprinting of spheroids, cell-laden hydrogels, and fugitive inks to form viable tissue constructs.

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