Bioprinting is an emerging technique for the fabrication of three-dimensional (3D) cell-laden constructs. However, the progress for generating a 3D complex physiological microenvironment has been hampered by a lack of advanced cell-responsive bioinks that enable bioprinting with high structural fidelity, particularly in the case of extrusion-based bioprinting. Herein, we report a novel strategy to directly bioprint cell-laden constructs using bioinks made of gelatin methacryloyl (GelMA) physical gels (GPGs). Attributed to their shear-thinning and self-healing properties, the GPG bioinks could retain the shape and form integral structures after deposition, allowing for subsequent UV crosslinking for permanent stabilization. We showed the structural fidelity by bioprinting various 3D structures that are typically challenging to fabricate using conventional bioinks under extrusion modes. Moreover, the use of the GPG bioinks enabled direct bioprinting of highly porous and soft constructs at relatively low concentrations (down to 3%) of GelMA. We also demonstrated that the bioprinted constructs not only permitted cell survival but also enhanced cell proliferation as well as spreading at lower concentrations of the GPG bioinks. We believe our strategy of bioprinting will provide many opportunities in convenient fabrication of 3D cell-laden constructs for applications in tissue engineering, regenerative medicine, and pharmaceutical screening.

The development of a multimaterial extrusion bioprinting platform is reported. This platform is capable of depositing multiple coded bioinks in a continuous manner with fast and smooth switching among different reservoirs for rapid fabrication of complex constructs, through digitally controlled extrusion of bioinks from a single printhead consisting of bundled capillaries synergized with programmed movement of the motorized stage.

Over the last decades, the fabrication of 3D tissues has become commonplace in tissue engineering and regenerative medicine. However, conventional 3D biofabrication techniques such as scaffolding, microengineering, and fiber and cell sheet engineering are limited in their capacity to fabricate complex tissue constructs with the required precision and controllability that is needed to replicate biologically relevant tissues. To this end, 3D bioprinting offers great versatility to fabricate biomimetic, volumetric tissues that are structurally and functionally relevant. It enables precise control of the composition, spatial distribution, and architecture of resulting constructs facilitating the recapitulation of the delicate shapes and structures of targeted organs and tissues. This Review systematically covers the history of bioprinting and the most recent advances in instrumentation and methods. It then focuses on the requirements for bioinks and cells to achieve optimal fabrication of biomimetic constructs. Next, emerging evolutions and future directions of bioprinting are discussed, such as freeform, high-resolution, multimaterial, and 4D bioprinting. Finally, the translational potential of bioprinting and bioprinted tissues of various categories are presented and the Review is concluded by exemplifying commercially available bioprinting platforms. BioprintingThe ORCID identification number(s) for the author(s) of this article can be found under https://doi.

The complex microenvironment in which malignant tumor cells grow is crucial for cancer progression. The physical and biochemical characteristics of this niche are involved in controlling cancer cell differentiation, proliferation, invasion, and metastasis. It is therefore essential to understand how cancer cells interact and communicate with their surrounding tissuethe so-called tumor stromaand how this interplay regulates disease progression. To mimic the tumor microenvironment (TME), 3D in vitro models are widely used because they can incorporate different patient-derived tissues/cells and allow longitudinal readouts, thus permitting deeper understanding of cell interactions. These models are therefore excellent tools to bridge the gap between oversimplified 2D systems and unrepresentative animal models. We present an overview of state-of-the-art 3D models for studying tumor-stroma interactions, with a focus on understanding why the TME is a key target in cancer therapy. Towards Biologically Relevant Tumor Models The composition of the tumor microenvironment (TME, see Glossary) and tumor stromal interactions are major factors that aggravate tumor growth and metastasis, leading to poor clinical outcomes [1-5]. There is increasing evidence that the activated stroma is a disease-defining factor, highlighting it as an important player in cancer cell invasion/extravasation, migration, angiogenesis, drug resistance [6,7], cancer stem cell maintenance [8], and immunosurveillance evasion [6,9,10]. Highlights Complex tumor-stroma interactions, a key feature of most solid tumors, drive tumor progression, metastasis, and drug resistance, and ultimately lead to treatment failure. Clinically relevant 3D in vitro models are necessary to recapitulate the complex interactions between tumor and stromal cells, and provide tools to better understand the molecular mechanisms as well as for testing anticancer therapies. Novel bioengineered models, organoid systems, and microfabrication technologies, such as 3D bioprinting, have delivered key innovations towards more advanced platforms for tumor modeling in vitro.

Glioblastoma-associated macrophages (GAMs) play a crucial role in the progression and invasiveness of glioblastoma multiforme (GBM); however, the exact crosstalk between GAMs and glioblastoma cells is not fully understood. Furthermore, there is a lack of relevant in vitro models to mimic their interactions. Here, novel 3D-bioprinted mini-brains consisting of glioblastoma cells and macrophages are presented as tool to study the interactions between these two cell types and to test therapeutics that target this interaction. It is demonstrated that in the mini-brains, glioblastoma cells actively recruit macrophages and polarize them into a GAM-specific phenotype, showing clinical relevance to transcriptomic and patient survival data. Furthermore, it is shown that macrophages induce glioblastoma cell progression and invasiveness in the mini-brains. Finally, it is demonstrated how therapeutics can inhibit the interaction between GAMs and tumor cells resulting in reduced tumor growth and more sensitivity to chemotherapy. It is envisioned that this 3D-bioprinted tumor model is used to improve the understanding of tumor biology and for evaluating novel cancer therapeutics.

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Embedded extrusion bioprinting allows for the generation of complex structures that otherwise cannot be achieved with conventional layer-by-layer deposition from the bottom, by overcoming the limits imposed by gravitational force. By taking advantage of a hydrogel bath, serving as a sacrificial printing environment, it is feasible to extrude a bioink in freeform until the entire structure is deposited and crosslinked. The bioprinted structure can be subsequently released from the supporting hydrogel and used for further applications. Combining this advanced three-dimensional (3D) bioprinting technique with a multimaterial extrusion printhead setup enables the fabrication of complex volumetric structures built from multiple bioinks. The work described in this paper focuses on the optimization of the experimental setup and proposes a workflow to automate the bioprinting process, resulting in a fast and efficient conversion of a virtual 3D model into a physical, extruded structure in freeform using the multimaterial embedded bioprinting system. It is anticipated that further development of this technology will likely lead to widespread applications in areas such as tissue engineering, pharmaceutical testing, and organs-on-chips.

Pancreatic stellate cells (PSCs) are the precursors of cancer-associated fibroblasts (CAFs), which potentiate pancreatic tumor growth and progression. In this study, we investigated whether Lipoxin A4 (LXA4), an endogenous bioactive lipid, can inhibit the differentiation of human PSCs (hPSCs) into CAF-like myofibroblasts and thereby hPSC-induced pro-tumorigenic effects. LXA4 significantly inhibited TGF-β-mediated differentiation of hPSCs by inhibiting pSmad2/3 signalling. Furthermore, treatment with LXA4 abolished the paracrine effects (proliferation and migration of Panc-1 tumor cells) of hPSCs in vitro. These data demonstrated that LXA4 can interrupt pro-tumoral paracrine signalling of hPSCs. Furthermore, LXA4 treatment significant decreased the size and growth rate of 3D-heterospheroids comprised of hPSC and Panc-1 and these effects were exhibited due to inhibition of hPSC-induced collagen1 expression. In vivo, we examined the therapeutic efficacy of LXA4 in a co-injection (Panc-1 and hPSCs) subcutaneous tumor model. Intriguingly, LXA4 significantly abolished the tumor growth (either injected intratumor or intraperitoneally), attributed to a significant reduction in fibrosis, shown with collagen1 expression. Altogether, this study proposes LXA4 as a potent inhibitor for hPSCs which can be applied to reprogram tumor stroma in order to treat pancreatic cancer.