Current animal and 2-D cell culture models employed in metastasis research and drug discovery remain poor mimics of human cancer physiology. Here we describe a “metastasis-on-a-chip” system allowing real time tracking of fluorescent colon cancer cells migrating from hydrogel-fabricated gut constructs to downstream liver constructs within a circulatory fluidic device system that responds to environmental manipulation and drug treatment. Devices consist of two chambers in which gut and liver constructs are housed independently, but are connected in series via circulating fluid flow. Constructs were biofabricated with a hyaluronic acid-based hydrogel system, capable of a variety of customizations, inside of which representative host tissue cells were suspended and metastatic colon carcinoma tumor foci were created. The host tissue of the constructs expressed normal epithelial markers, which the tumor foci failed to express. Instead, tumor regions lost membrane-bound adhesion markers, and expressed mesenchymal and proliferative markers, suggesting a metastatic phenotype. Metastatic tumor foci grew in size, eventually disseminating from the intestine construct and entering circulation, subsequently reaching in the liver construct, thus mimicking some of the migratory events observed during metastasis. Lastly, we demonstrated the ability to manipulate the system, including chemically modulating the hydrogel system mechanical properties and administering chemotherapeutic agents, and evaluated the effects of these parameters on invasive tumor migration. These results describe the capability of this early stage metastasis-on-a-chip system to model several important characteristics of human metastasis, thereby demonstrating the potential of the platform for making meaningful advances in cancer investigation and drug discovery.

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Current practices in drug development have led to therapeutic compounds being approved for widespread use in humans, only to be later withdrawn due to unanticipated toxicity. These occurrences are largely the result of erroneous data generated by in vivo and in vitro preclinical models that do not accurately recapitulate human physiology. Herein, a human primary cell-and stem cellderived 3D organoid technology is employed to screen a panel of drugs that were recalled from market by the FDA. The platform is comprised of multiple tissue organoid types that remain viable for at least 28 days, in vitro. For many of these compounds, the 3D organoid system was able to demonstrate toxicity. Furthermore, organoids exposed to non-toxic compounds remained viable at clinically relevant doses. Additional experiments were performed on integrated multi-organoid systems containing liver, cardiac, lung, vascular, testis, colon, and brain. These integrated systems proved to maintain viability and expressed functional biomarkers, long-term. Examples are provided that demonstrate how multi-organoid 'body-on-a-chip' systems may be used to model the interdependent metabolism and downstream effects of drugs across multiple tissues in a single platform. Such 3D in vitro systems represent a more physiologically relevant model for drug screening and will likely reduce the cost and failure rate associated with the approval of new drugs.

The current drug development pipeline takes approximately fifteen years and $2.6 billion to get a new drug to market. Typically, drugs are tested on two-dimensional (2D) cell cultures and animal models to estimate their efficacy before reaching human trials. However, these models are often not representative of the human body. The 2D culture changes the morphology and physiology of cells, and animal models often have a vastly different anatomy and physiology than humans. The use of bioengineered human cell-based organoids may increase the probability of success during human trials by providing human-specific preclinical data. They could also be deployed for personalized medicine diagnostics to optimize therapies in diseases such as cancer. However, one limitation in employing organoids in drug screening has been the difficulty in creating large numbers of homogeneous organoids in form factors compatible with high-throughput screening (e.g., 96-and 384-well plates). Bioprinting can be used to scale up deposition of such organoids and tissue constructs. Unfortunately, it has been challenging to 3D print hydrogel bioinks into small-sized wells due to well-bioink interactions that can result in bioinks spreading out and wetting the well surface instead of maintaining a spherical form. Here, we demonstrate an immersion printing technique to bioprint tissue organoids in 96-well plates to increase the throughput of 3D drug screening. A hydrogel bioink comprised of hyaluronic acid and collagen is bioprinted into a viscous gelatin bath, which blocks the bioink from interacting with the well walls and provides support to maintain a spherical form. This method was validated using several cancerous cell lines, and then applied to patient-derived glioblastoma (GBM) and sarcoma biospecimens for drug screening.

IntroductionEnvironmental toxins, such as lead and other heavy metals, pesticides, and other compounds, represent a significant health concern within the USA and around the world. Even in the twenty-first century, a plethora of cities and towns in the U.S. have suffered from exposures to lead in drinking water or other heavy metals in food or the earth, while there is a high possibility of further places to suffer such exposures in the near future.MethodsWe employed bioengineered 3D human liver and cardiac organoids to screen a panel of environmental toxins (lead, mercury, thallium, and glyphosate), and charted the response of the organoids to these compounds. Liver and cardiac organoids were exposed to lead (10 µM–10 mM), mercury (200 nM–200 µM), thallium (10 nM–10 µM), or glyphosate (25 µM–25 mM) for a duration of 48 h. The impacts of toxin exposure were then assessed by LIVE/DEAD viability and cytotoxicity staining, measuring ATP activity and determining IC50 values, and determining changes in cardiac organoid beating activity.ResultsAs expected, all of the toxins induced toxicity in the organoids. Both ATP and LIVE/DEAD assays showed toxicity in both liver and cardiac organoids. In particular, thallium was the most toxic, with IC50 values of 13.5 and 1.35 µM in liver and cardiac organoids, respectively. Conversely, glyphosate was the least toxic of the four compounds, with IC50 values of 10.53 and 10.85 mM in liver and cardiac organoids, respectively. Additionally, toxins had a negative influence on cardiac organoid beating activity as well. Thallium resulting in the most significant decreases in beating rate, followed by mercury, then glyphosate, and finally, lead. These results suggest that the 3D organoids have significant utility to be deployed in additional toxicity screening applications, and future development of treatments to mitigate exposures.Conclusion3D organoids have significant utility to be deployed in additional toxicity screening applications, such as future development of treatments to mitigate exposures, drug screening, and environmental toxin detection.

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Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening. Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

The therapeutic efficacy of clinically applied mesenchymal stromal cells (MSCs) is limited due to their injection into harsh in vivo environments, resulting in the significant loss of their secretory function upon transplantation. A potential strategy for preserving their full therapeutic potential is encapsulation of MSCs in a specialized protective microenvironment, for example hydrogels. However, commonly used injectable hydrogels for cell delivery fail to provide the bio-instructive cues needed to sustain and stimulate cellular therapeutic functions. Here we introduce a customizable collagen I-hyaluronic acid (COL-HA)-based hydrogel platform for the encapsulation of MSCs. Cells encapsulated within COL-HA showed a significant expansion of their secretory profile compared to MSCs cultured in standard (2D) cell culture dishes or encapsulated in other hydrogels. Functionalization of the COL-HA backbone with thiol-modified glycoproteins such as laminin led to further changes in the paracrine profile of MSCs. In depth profiling of more than 250 proteins revealed an expanded secretion profile of proangiogenic, neuroprotective and immunomodulatory paracrine factors in COL-HA-encapsulated MSCs with a predicted augmented pro-angiogenic potential. This was confirmed by increased capillary network formation of endothelial cells stimulated by conditioned media from COL-HA-encapsulated MSCs. Our findings suggest that encapsulation of therapeutic cells in a protective COL-HA hydrogel layer provides the necessary bio-instructive cues to maintain and direct their therapeutic potential. Our customizable hydrogel combines bioactivity and clinically applicable properties such as injectability, on-demand polymerization and tissue-specific elasticity, all features that will support and improve the ability to successfully deliver functional MSCs into patients.