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Metastatic breast cancer is one of the deadliest forms of malignancy, primarily driven by its characteristic microenvironment comprising of cancer cells interacting with stromal components. These interactions induce genetic and metabolic alterations creating a conducive environment for tumor growth. In this study, we developed a physiologically-relevant three dimensional (3D) vascularized breast cancer microenvironment comprising of metastatic MDA-MB-231 cells and human umbilical vein endothelial cells (HUVEC) loaded in human dermal fibroblasts (HDF) laden fibrin, representing the tumor stroma. The matrix as well as stromal cell density impacted the transcriptional profile of genes involved in tumor angiogenesis and cancer invasion, which are hallmarks of cancer. Cancer-specific canonical pathways and activated upstream regulators were also identified by the differential gene expression signatures of these composite cultures. Additionally, a tumor associated vascular bed of capillaries is established exhibiting dilated vessel diameters, representative of in vivo tumor physiology. Further, employing aspiration-assisted bioprinting, we identified cancer-endothelial crosstalk, in the form of collective angiogenesis of tumor spheroids bioprinted at close proximity. Overall, this bottom-up approach of tumor microenvironment fabrication provides an insight into the potential of in vitro tumor models and enables identification of novel therapeutic targets as a pre-clinical drug screening platform.

Extrusion-based bioprinting of hydrogels in a granular secondary gel enables the fabrication of cell-laden three-dimensional (3D) constructs in an anatomically accurate manner, which is challenging using conventional extrusion-based bioprinting processes. In this study, carbohydrazide-modified gelatin (Gel-CDH) was synthesized and deposited into a new multifunctional support bath consisting of gelatin microparticles suspended in an oxidized alginate (OAlg) solution. During extrusion, Gel-CDH and OAlg were rapidly cross-linked because of the Schiff base formation between aldehyde groups of OAlg and amino groups of Gel-CDH, which has not been demonstrated in the domain of 3D bioprinting before. Rheological results indicated that hydrogels with lower OAlg to Gel-CDH ratios possessed superior mechanical rigidity. Different 3D geometrically intricate constructs were successfully created upon the determination of optimal bioprinting parameters. Human mesenchymal stem cells and human umbilical vein endothelial cells were also bioprinted at physiologically relevant cell densities. The presented study has offered a novel strategy for bioprinting of natural polymer-based hydrogels into 3D complex-shaped biomimetic constructs, which eliminated the need for cytotoxic supplements as external cross-linkers or additional cross-linking processes, therefore expanding the availability of bioinks.

Current technologies for manufacturing of microfluidic devices include soft-lithography, wet and dry etching, thermoforming, micro-machining and three-dimensional (3D) printing. Among them, soft-lithography has been the mostly preferred one in medical and pharmaceutical fields due to its ability to generate polydimethylsiloxane (PDMS) devices with resin biocompatibility, throughput and transparency for imaging. It is a multi-step process requiring the preparation of a silicon wafer pattern, which is fabricated using photolithography according to a defined mask. Photolithography is a costly, complicated and time-consuming process requiring a clean-room environment, and the technology is not readily accessible in most of the developing countries. In addition, generated patterns on photolithography-made silicon wafers do not allow building 3D intricate shapes and silicon direct bonding is thus utilized for closed fluid channels and complex 3D structures. 3D Printing ofPDMS has recently gained significant interest due to its ability to define complex 3D shapes directly from user-defined designs. In this work, we investigated Carbopol as a sacrificial gel in order to create microfluidic channels inPDMS devices. Our study demonstrated that Carbopol ink possessed a shear-thinning behavior and enabled the extrusion-based printing of channel templates, which were overlaid with PDMS to create microfluidic devices upon curing ofPDMS and removal of the sacrificial Carbopol ink. To demonstrate the effectiveness of the fabricated devices, channels were lined up with human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cells (BMECs) in separate devices, where both HUVECs and BMECs demonstrated the formation of endothelium with highly aligned cells in the direction of fluid flow. Overall, we here present a highly affordable and practical approach in fabrication ofPDMS devices with closed fluid channels, which have great potential in a myriad of applications from cancer treatments to infectious disease diagnostics to artificial organs.