Intercellular Mitochondrial Transfer Using 3D Bioprinting

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.https://digitalcommons.odu.edu/gradposters2022_healthsciences/1010/thumbnail.jp

The Revolution Will Be Open-Source: How 3D Bioprinting Can Change 3D Cell Culture

(First paragraph) The development of three-dimensional culture scaffolds represents a revolutionary step forward for in vitro culture systems. Various synthetic and naturally occurring substrates have been developed that support 3D growth of cells. In most fields, including mammary gland biology and tumorigenesis, the two most common substrates used are the basement membrane rich extracellur matrix (ECM) isolated from EngelbrethHolm-Swarm (EHS) mouse sarcomas (e.g. Matrigel) and collagen extracted from rat-tails. The processes of 3D culture in these two substrates has remained unchanged for nearly half a century: cells are either mixed with unpolymerized matrix to disperse them randomly throughout the substrate upon polymerization or overlaid randomly on top of a preformed hydrogel. While effective in generating organoid/tumoroid structures, the random nature of these processes has many drawbacks that limit the reproducibility and tunability of the experimental design. Furthermore, random cellular distributions limit the utility of these substrates for studying interactions within the cellular microenvironment, which have been shown to be critical for the control of stem and cancer cell function [1]

Tissue Specific Microenvironments: A Key Tool for Tissue Engineering and Regenerative Medicine

The accumulated evidence points to the microenvironment as the primary mediator of cellular fate determination. Comprised of parenchymal cells, stromal cells, structural extracellular matrix proteins, and signaling molecules, the microenvironment is a complex and synergistic edifice that varies tissue to tissue. Furthermore, it has become increasingly clear that the microenvironment plays crucial roles in the establishment and progression of diseases such as cardiovascular disease, neurodegeneration, cancer, and ageing. Here we review the historical perspectives on the microenvironment, and how it has directed current explorations in tissue engineering. By thoroughly understanding the role of the microenvironment, we can begin to correctly manipulate it to prevent and cure diseases through regenerative medicine techniques

3D Bioprinted Structures from Cells of Non-Epithelial Mesodermal and Endodermal Lineage Using a Custom Accessible 3D Bioprinting Platform

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\u27s 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 systemshttps://digitalcommons.odu.edu/gradposters2022_engineering/1001/thumbnail.jp

3D Bioprinting and Implantation of Mouse Mammary Epithelial Structures Using a Custom Accessible 3D Bioprinting Platform

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.https://digitalcommons.odu.edu/gradposters2022_engineering/1000/thumbnail.jp

Stem Cell Differentiation and Effects of Three-Dimensional Cellular Microenvironments

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.https://digitalcommons.odu.edu/engineering_batten/1016/thumbnail.jp

Accessible Bioprinting: Adaptation of a Low-Cost 3D-Printer for Precise Cell Placement and Stem Cell Differentiation

The precision and repeatability offered by computer-aided design and computer-numerically controlled techniques in biofabrication processes is quickly becoming an industry standard. However, many hurdles still exist before these techniques can be used in research laboratories for cellular and molecular biology applications. Extrusion-based bioprinting systems have been characterized by high development costs, injector clogging, difficulty achieving small cell number deposits, decreased cell viability, and altered cell function post-printing. To circumvent the high-price barrier to entry of conventional bioprinters, we designed and 3D printed components for the adaptation of an inexpensive \u27off-the-shelf\u27 commercially available 3D printer. We also demonstrate via goal based computer simulations that the needle geometries of conventional commercially standardized, \u27luer-lock\u27 syringe-needle systems cause many of the issues plaguing conventional bioprinters. To address these performance limitations we optimized flow within several microneedle geometries, which revealed a short tapered injector design with minimal cylindrical needle length was ideal to minimize cell strain and accretion. We then experimentally quantified these geometries using pulled glass microcapillary pipettes and our modified, low-cost 3D printer. This systems performance validated our models exhibiting: reduced clogging, single cell print resolution, and maintenance of cell viability without the use of a sacrificial vehicle. Using this system we show the successful printing of human induced pluripotent stem cells (hiPSCs) into Geltrex and note their retention of a pluripotent state 7 d post printing. We also show embryoid body differentiation of hiPSC by injection into differentiation conducive environments, wherein we observed continuous growth, emergence of various evaginations, and post-printing gene expression indicative of the presence of all three germ layers. These data demonstrate an accessible open-source 3D bioprinter capable of serving the needs of any laboratory interested in 3D cellular interactions and tissue engineering

Combined 3D Bioprinting and Tissue-Specific ECM System Reveals the Influence of Brain Matrix on Stem Cell Differentiation

We have previously shown that human and murine breast extracellular matrix (ECM) can significantly impact cellular behavior, including stem cell fate determination. It has been established that tissue-specific extracellular matrix from the central nervous system has the capacity to support neuronal survival. However, the characterization of its influence on stem cell differentiation and its adaptation to robust 3D culture models is underdeveloped. To address these issues, we combined our 3D bioprinter with hydrogels containing porcine brain extracellular matrix (BMX) to test the influence of the extracellular matrix on stem cell differentiation. Our 3D bioprinting system generated reproducible 3D neural structures derived from mouse embryonic stem cells (mESCs). We demonstrate that the addition of BMX preferentially influences 3D bioprinted mESCs towards neural lineages compared to standard basement membrane (Geltrex/Matrigel) hydrogels alone. Furthermore, we demonstrate that we can transplant these 3D bioprinted neural cellular structures into a mouse’s cleared mammary fat pad, where they continue to grow into larger neural outgrowths. Finally, we demonstrate that direct injection of human induced pluripotent stem cells (hiPSCS) and neural stem cells (NSCs) suspended in pure BMX formed neural structures in vivo. Combined, these findings describe a unique system for studying brain ECM/stem cell interactions and demonstrate that BMX can direct pluripotent stem cells to differentiate down a neural cellular lineage without any additional specific differentiation stimuli

Preferential Lineage-Specific Differentiation of Osteoblast-Derived Induced Pluripotent Stem Cells into Osteoprogenitors

While induced pluripotent stem cells (iPSCs) hold great clinical promise, one hurdle that remains is the existence of a parental germ-layer memory in reprogrammed cells leading to preferential differentiation fates. While it is problematic for generating cells vastly different from the reprogrammed cells\u27 origins, it could be advantageous for the reliable generation of germ-layer specific cell types for future therapeutic use. Here we use human osteoblast-derived iPSCs (hOB-iPSCs) to generate induced osteoprogenitors (iOPs). Osteoblasts were successfully reprogrammed and demonstrated by endogenous upregulation of Oct4, Sox2, Nanog, TRA-1-81, TRA-16-1, SSEA3, and confirmatory hPSC Scorecard Algorithmic Assessment. The hOB-iPSCs formed embryoid bodies with cells of ectoderm and mesoderm but have low capacity to form endodermal cells. Differentiation into osteoprogenitors occurred within only 2-6 days, with a population doubling rate of less than 24 hrs; however, hOB-iPSC derived osteoprogenitors were only able to form osteogenic and chondrogenic cells but not adipogenic cells. Consistent with this, hOB-iOPs were found to have higher methylation of PPAR gamma but similar levels of methylation on the RUNX2 promoter. These data demonstrate that iPSCs can be generated from human osteoblasts, but variant methylation patterns affect their differentiation capacities. Therefore, epigenetic memory can be exploited for efficient generation of clinically relevant quantities of osteoprogenitor cells

A 3D Bioprinter Platform for Mechanistic Analysis of Tumoroids and Chimeric Mammary Organoids

The normal mammary microenvironment can suppress tumorigenesis and redirect cancer cells to adopt a normal mammary epithelial cell fate in vivo. Understanding of this phenomenon offers great promise for novel treatment and detection strategies in cancer, but current model systems make mechanistic insights into the process difficult. We have recently described a low-cost bioprinting platform designed to be accessible for basic cell biology laboratories. Here we report the use of this system for the study of tumorigenesis and microenvironmental redirection of breast cancer cells. We show our bioprinter significantly increases tumoroid formation in 3D collagen gels and allows for precise generation of tumoroid arrays. We also demonstrate that we can mimic published in vivo findings by co-printing cancer cells along with normal mammary epithelial cells to generate chimeric organoids. These chimeric organoids contain cancer cells that take part in normal luminal formation. Furthermore, we show for the first time that cancer cells within chimeric structures have a significant increase in 5-hydroxymethylcytosine levels as compared to bioprinted tumoroids. These results demonstrate the capacity of our 3D bioprinting platform to study tumorigenesis and microenvironmental control of breast cancer and highlight a novel mechanistic insight into the process of microenvironmental control of cancer