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2605 Background: Breast cancer (BC) is the second leading cause of cancer death following lung cancer. Bioprinting, the use of computer aided process to print biological living and non-living material to create patterns in 2D or 3D structures, is a novel technique that has been proposed to be used to develop tissue engineered solutions for a wide array of clinical applications, e.g., skin grafting. We investigate here if bioprinted breast cancer cells show some of the hallmarks of cancer tissues, and thus may represent good in vitro models for drug discovery. Methods: For this study, MCF-7 BC cells were cultured, stained, counted and turned into a bioink solution by suspending in phosphate buffered saline solution. The cells were bioprinted over a 96-well plate and pre-incubated for 18 hours in DMEM and RPMI media with 10% Fetal Bovine Serum and Charcoal Stripped Serum, respectively. After 18 hours of incubation the media was supplemented with Tamoxifen at 5µM, 10µM, 50µM, 90µM and 110µM concentrations. Cytotoxicity was measured 24 hours post-treatment using a differential nuclear staining assay and an INCell 2000 bioimager system. Results: Bioprinted cells exposed to high concentrations of Tamoxifen (90 µM and 110µM) exhibited a viability of 8.2% and 10.8%, respectively. Whereas viability of manually seeded cells at those concentrations was 0.11% and 0.05%. Viability of negative and positive controls was at 7.6% and 97.0% for the bioprinted samples and for the normally seeded cells was 4.9% and 98.8% respectively. Conclusions: In our study, we have established a novel 2D/3D breast tumor model applying bioprinting technology for drug discovery. The higher cell viability of MCF-7’s at high concentrations of Tamoxifen could be attributed to the hormesis effect and activation of chaperone proteins, e.g., HSP70 and HSP90, possibly caused by bioprinting. We hypothesize that bioprinted MCF-7 cells also show increased levels of chaperone proteins, which may in a way mimic their in vivo behavior. In this novel in vitro 2D/3D model, the bioprinted cells show a more biological relevant behavior than normally cultured cells. Insights into the cell behavior after bioprinting may elucidate how to build improved in vitro models for BC research.

Bioprinting is an emerging technique used to layer extrudable materials and cells into simple constructs to engineer tissue or arrive at in vitro organ models. Although many examples of bioprinted tissues exist, many lack the biochemical complexity found in the native extracellular matrix. Therefore, the resulting tissues may be less competent than native tissues—this can be especially problematic for tissues that need strong mechanical properties, such as cardiac or those found in the great vessels. Decellularization of native tissues combined with processing for bioprinting may improve the cellular environment for proliferation, biochemical signaling, and improved mechanical characteristics for better outcomes. Whole porcine hearts were decellularized using a series of detergents, followed by lyophilization and mechanical grinding in order to produce a fine powder. Temperature-controlled enzymatic digestion was done to allow for the resuspension of the decellularized extracellular matrix into a pre-gel solution. Using a commercial extrusion bioprinter with a temperature-controlled printhead, a 1:1 scale model of a human ascending aorta and dog bone shaped structures were printed into a reservoir of alginate and xanthium gum then allowed to crosslink at 37C. The bioengineered aortic construct was monitored for cell adhesion, survival, and proliferation through fluorescent microscopy. The dog bone structure was subjected to tensile mechanical testing in order to determine structural and mechanical patterns for comparison to native tissue structures. The stability of the engineered structure was maintained throughout the printing process, allowing for a final structure that upheld the dimensions of the original Computer-Aided Design model. The decellularized ECM (Ē = 920 kPa) exhibited almost three times greater elasticity than the porcine cardiac tissue (Ē = 330 kPa). Similarly, the porcine cardiac tissue displayed two times the deformation than that of the printed decellularized ECM. Cell proliferation and attachment were observed during the in vitro cell survivability assessment of human aortic smooth muscle cells within the extracellular matrix, along with no morphological abnormalities to the cell structure. These observations allow us to report the ability to bioprint mechanically stable, cell-laden structures that serve as a bridge in the current knowledge gap, which could lead to future work involving complex, large-scale tissue models.

The rapidly growing field of tissue engineering hopes to soon address the shortage of transplantable tissues, allowing for precise control and fabrication that could be made for each specific patient. The protocols currently in place to print large-scale tissues have yet to address the main challenge of nutritional deficiencies in the central areas of the engineered tissue, causing necrosis deep within and rendering it ineffective. Bioprinted microvasculature has been proposed to encourage angiogenesis and facilitate the mobility of oxygen and nutrients throughout the engineered tissue. An implant made via an inkjet printing process containing human microvascular endothelial cells was placed in both B17-SCID and NSG-SGM3 animal models to determine the rate of angiogenesis and degree of cell survival. The implantable tissues were made using a combination of alginate and gelatin type B; all implants were printed via previously published procedures using a modified HP inkjet printer. Histopathological results show a dramatic increase in the average microvasculature formation for mice that received the printed constructs within the implant area when compared to the manual and control implants, indicating inkjet bioprinting technology can be effectively used for vascularization of engineered tissues.