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Prior work has shown that our bioprinting system can reliably produce human mammary organoids and tumoroids with high precision. However, this was not previously applied to mouse models, which are also important with respect to translational research in cancer drug development. To address this, we have produced protocols for the development of in vitro structures from murine mammary epithelial and tumor cells. Additionally, we assessed the translatability of both human and murine bioprinted organoids into mouse mammary fat pads over a period of 6 weeks. Our lab found that our produced organoids are reliable, they can survive in vivo, and meaningfully integrate within host systems. Therefore, we have demonstrated that our system is adaptable to both human and murine models, as it offers a unique methodology for in vivo transplantation of human or murine organoids into mice, which can boost research efforts in cancer therapy research.

Prior work within our lab has demonstrated the ability to print both murine and human mammary organoids and tumoroids in vitro that can also be reliably transplanted into a murine host for translational studies. Peripherally, this bioprinting system has also been used for 3D printing neurons, stem cells, cancer cells, and a primary cell line rich with fibroblasts, but each of these efforts were with cells of ectodermal lineage. Thus, the system's capacity for use on cells of other origins had been untested. To address this, we have now developed protocols for cells of endodermal and non-epithelial mesodermal/mesenchymal lineage. In this work, we find that we can produce reliable organoids, tumoroids, and other in vitro structures from them, thus expanding the functional range of our open 3D bioprinting platform. Therefore, we demonstrate that our system is versatile for adaptation to multiple cellular systems and can be applied to the work of labs that wish to study development and pathologies in other organ systems

Mitochondria are one of the most complex and vital organelles in eukaryotic cells. In recent years, it has been shown that through intercellular mitochondrial transfer, this important organelle provides a critical role in tissue homeostasis, damaged tissue repair, and tumor progression under physiological conditions. However, the mechanism of mitochondrial transfer and its effect on various cellular microenvironments has not yet been defined. Understanding the metabolic effects of mitochondrial transfer between cells and exploring the signaling leading to the intercellular mechanisms could provide advancements in both translational medicine and cell therapy for cancer progression and age-related diseases. Our group has studied the ability of the normal mammary microenvironment to redirect cancer cells to a normal mammary epithelial cell fate both in vivo and in vitro using our 3D bioprinting system. Therefore, we sought to determine if mitochondrial transfer may play a role in mammary epithelium induced redirection of cancer cells. We used MCF-7 breast cancer cells and MCF-12a epithelial breast cells for experimentation. Using a fluorescent GFP-MITO lentivirus, we were able to mark mitochondrial protein in the MCF-12a epithelial cells to track mitochondrial transfer activity. The MCF-7 cells were labeled red to distinguish the two cell types. The cells were then co-cultured in 2D tissue flasks and printed into hydrogels using the 3D bioprinter. Using fluorescent microscopy, mitochondrial protein was observed traveling from epithelial to mammary cancer cells. We hypothesize this is done for cancer cells to stabilize mitochondria and improve metabolic function and ATP production. Further research to establish mitochondrial transfer, its mechanism(s), and molecular effects could lead insight into how this cellular communication rescues and normalizes metabolic factors of the mammary and stem cell microenvironment leading to potential fate redirection and cellular revitalization.

The cellular microenvironment has been shown to play a fundamental role in the regulation of cell function, stem cell fate determination, maintenance of cell potency and tissue homeostasis. Our laboratory focuses on the study of the effects of cellular microenvironment in the context of cancer and neurological models, based on the observation that a healthy environment can induce the suppression of tumorigenesis in mouse models. Insights concerning the molecular mechanisms that drive these processes are very limited, partly due to the inability of the current traditional methods of investigation, such as two-dimensional cell cultures and animal models, to accurately represent the human in vivo cellular microenvironment. Three-dimensional cell cultures allow to overcome the structural limitations posed by monolayer cultures, and maintain the ease of experiment design, monitoring and data analysis associated with in vitro procedures. Our laboratory has established systems to overcome some of these limitations and rely on the strengths of three-dimensional culture methods to elucidate mechanisms that govern stem cell differentiation. A customized 3D extrusion-based bioprinter was developed starting from a commercially available model, allowing for precise and controlled injection of cells within three-dimensional substrates. This tool allows for design of highly controlled experiments, in which the effects of cellular microenvironment on stem cell differentiation can be studied at a single-cell resolution. For increased levels of biomimicry, tissue specific substrates are generated from extracted tissue. Collected tissue is subjected to a chemical decellularization process, followed by lyophilization, enzymatic digestion and neutralization, to generate a self-gelling product upon incubation at 37°C. Mammary and brain extracellularmatrix-derived substrates have been shown to support the growth of cells of the epithelial and neuronal lineages, respectively. Here, we apply these established systems to study the effects of the environment constituted by the three-dimensional substrates on the differentiation of injected stem cells.