Multi-organ immunity on a 3D-printed multi-tissue chip with tubing-free impeller pump.

Multi-organ-on-chip systems (MOOCs) have the potential to mimic communication between organ systems and reveal mechanisms of health and disease. However, many existing MOOCs are challenging for non-experts to implement, due to complex tubing, electronics, or pump mechanisms. In addition, few MOOCs have incorporated immune organs such as the lymph node (LN), limiting their applicability to critical events such as vaccination. Here we developed a 3D-printed, user-friendly device and companion tubing-free impeller pump to co-culture two or more tissue samples, including a LN, under a recirculating common media. Native tissue structure and immune function were incorporated by maintaining slices of murine LN tissue ex vivo in 3D- printed mesh supports for at least 24 hr. In a two-compartment model of a LN and an upstream injection site, vaccination of the multi-tissue chip was similar to in vivo vaccination in terms of locations of antigen accumulation and acute changes in activation markers and gene expression in the LN. We anticipate that in the future, this flexible platform will enable models of multi-organ immune responses throughout the body.

Parylene-C Coating Protects Resin-3D-Printed Devices from Material Erosion and Prevents Cytotoxicity toward Primary Cells.

Resin 3D printing is attractive for the rapid fabrication of microscale cell culture devices, but common resin materials are unstable and cytotoxic under culture conditions. Strategies such as leaching or overcuring are insufficient to protect sensitive primary cells such as white blood cells. Here, we evaluated the effectiveness of using a parylene C coating of commercially available clear resins to prevent cytotoxic leaching, degradation of microfluidic devices, and absorption of small molecules. We found that parylene C significantly improved both the cytocompatibility with primary murine white blood cells and the material integrity of prints while maintaining the favorable optical qualities held by clear resins.

Microscale impeller pump for recirculating flow in organs-on-chip and microreactors.

Fluid flow is an integral part of microfluidic and organ-on-chip technology, ideally providing biomimetic fluid, cell, and nutrient exchange as well as physiological or pathological shear stress. Currently, many of the pumps that actively perfuse fluid at biomimetic flow rates are incompatible with use inside cell culture incubators, require many tubing connections, or are too large to run many devices in a confined space. To address these issues, we developed a user-friendly impeller pump that uses a 3D-printed device and impeller to recirculate fluid and cells on-chip. Impeller rotation was driven by a rotating magnetic field generated by magnets mounted on a computer fan; this pump platform required no tubing connections and could accommodate up to 36 devices at once in a standard cell culture incubator. A computational model was used to predict shear stress, velocity, and changes in pressure throughout the device. The impeller pump generated biomimetic fluid velocities (50-6400 µm s-1) controllable by tuning channel and inlet dimensions and the rotational speed of the impeller, which were comparable to the order of magnitude of the velocities predicted by the computational model. Predicted shear stress was in the physiological range throughout the microchannel and over the majority of the impeller. The impeller pump successfully recirculated primary murine splenocytes for 1 h and Jurkat T cells for 24 h with no impact on cell viability, showing the impeller pump's feasibility for white blood cell recirculation on-chip. In the future, we envision that this pump will be integrated into single- or multi-tissue platforms to study communication between organs.

Engineering in vitro immune-competent tissue models for testing and evaluation of therapeutics.

Advances in 3D cell culture, microscale fluidic control, and cellular analysis have enabled the development of more physiologically-relevant engineered models of human organs with precise control of the cellular microenvironment. Engineered models have been used successfully to answer fundamental biological questions and to screen therapeutics, but these often neglect key elements of the immune system. There are immune elements in every tissue that contribute to healthy and diseased states. Including immune function will be essential for effective preclinical testing of therapeutics for inflammatory and immune-modulated diseases. In this review, we first discuss the key components to consider in designing engineered immune-competent models in terms of physical, chemical, and biological cues. Next, we review recent applications of models of immunity for screening therapeutics for cancer, preclinical evaluation of engineered T cells, modeling autoimmunity, and screening vaccine efficacy. Future work is needed to further recapitulate immune responses in engineered models for the most informative therapeutic screening and evaluation.

Modeling Immunity In Vitro: Slices, Chips, and Engineered Tissues.

Modeling immunity in vitro has the potential to be a powerful tool for investigating fundamental biological questions, informing therapeutics and vaccines, and providing new insight into disease progression. There are two major elements to immunity that are necessary to model: primary immune tissues and peripheral tissues with immune components. Here, we systematically review progress made along three strategies to modeling immunity: ex vivo cultures, which preserve native tissue structure; microfluidic devices, which constitute a versatile approach to providing physiologically relevant fluid flow and environmental control; and engineered tissues, which provide precise control of the 3D microenvironment and biophysical cues. While many models focus on disease modeling, more primary immune tissue models are necessary to advance the field. Moving forward, we anticipate that the expansion of patient-specific models may inform why immunity varies from patient to patient and allow for the rapid comprehension and treatment of emerging diseases, such as coronavirus disease 2019.

Visualisation of cholesterol and ganglioside GM1 in zebrafish models of Niemann-Pick type C disease and Smith-Lemli-Opitz syndrome using light sheet microscopy.

Lysosomal storage diseases are the most common cause of neurodegeneration in children. They are characterised at the cellular level by the accumulation of storage material within lysosomes. There are very limited therapeutic options, and the search for novel therapies has been hampered as few good small animal models are available. Here, we describe the use of light sheet microscopy to assess lipid storage in drug and morpholino induced zebrafish models of two diseases of cholesterol homeostasis with lysosomal dysfunction: First, Niemann-Pick type C disease (NPC), caused by mutations in the lysosomal transmembrane protein NPC1, characterised by intralysosomal accumulation of cholesterol and several other lipids. Second, Smith-Lemli-Opitz syndrome (SLOS), caused by mutations in 7-dehydrocholesterol reductase, which catalyses the last step of cholesterol biosynthesis and is characterised by intralysosomal accumulation of dietary cholesterol. This is the first description of a zebrafish SLOS model. We find that zebrafish accurately model lysosomal storage and disease-specific phenotypes in both diseases. Increased cholesterol and ganglioside GM1 were observed in sections taken from NPC model fish, and decreased cholesterol in SLOS model fish, but these are of limited value as resolution is poor, and accurate anatomical comparisons difficult. Using light sheet microscopy, we were able to observe lipid changes in much greater detail and identified an unexpected accumulation of ganglioside GM1 in SLOS model fish. Our data demonstrate, for the first time in zebrafish, the immense potential that light sheet microscopy has in aiding the resolution of studies involving lysosomal and lipid disorders.

Detrimental effect of zwitterionic buffers on lysosomal homeostasis in cell lines and iPSC-derived neurons.

Good's buffers are commonly used for cell culture and, although developed to have minimal to no biological impact, they cause alterations in cellular processes such as autophagy and lysosomal enzyme activity. Using Chinese hamster ovary cells and induced pluripotent stem cell-derived neurons, this study explores the effect of zwitterionic buffers, specifically HEPES, on lysosomal volume and Ca2+ levels. Certain zwitterionic buffers lead to lysosomal expansion and reduced lysosomal Ca2+. Care should be taken when selecting buffers for growth media to avoid detrimental impacts on lysosomal function.