Conceptual and Experimental Tools to Understand Spatial Effects and Transport Phenomena in Nonlinear Biochemical Networks Illustrated with Patchy Switching.

Many biochemical systems are spatially heterogeneous and exhibit nonlinear behaviors, such as state switching in response to small changes in the local concentration of diffusible molecules. Systems as varied as blood clotting, intracellular calcium signaling, and tissue inflammation are all heavily influenced by the balance of rates of reaction and mass transport phenomena including flow and diffusion. Transport of signaling molecules is also affected by geometry and chemoselective confinement via matrix binding. In this review, we use a phenomenon referred to as patchy switching to illustrate the interplay of nonlinearities, transport phenomena, and spatial effects. Patchy switching describes a change in the state of a network when the local concentration of a diffusible molecule surpasses a critical threshold. Using patchy switching as an example, we describe conceptual tools from nonlinear dynamics and chemical engineering that make testable predictions and provide a unifying description of the myriad possible experimental observations. We describe experimental microfluidic and biochemical tools emerging to test conceptual predictions by controlling transport phenomena and spatial distribution of diffusible signals, and we highlight the unmet need for in vivo tools.

Poly I:C vaccination drives transient CXCL9 expression near B cell follicles in the lymph node through type-I and type-II interferon signaling.

Subunit vaccines drive immune cell-cell interactions in the lymph node (LN), yet it remains unclear how distinct adjuvants influence the chemokines responsible for this interaction in the tissue. Here, we tested the hypothesis that classic Th1-polarizing vaccines elicit a unique chemokine signature in the LN compared to other adjuvants. Polyinosinic:polycytidylic acid (Poly I:C) vaccination resulted in dynamic upregulation of CXCL9 that was localized in the interfollicular region, a response not observed after vaccination with alum or a combination of alum and poly I:C. Experiments using in vivo mouse models and live ex vivo LN slices revealed that poly I:C vaccination resulted in a type-I IFN response in the LN that led to the secretion of IFNγ, and type-I IFN and IFNγ were required for CXCL9 expression in this context. CXCL9 expression in the LN was correlated with an IgG2c antibody polarization after vaccination; however, genetic depletion of the receptor for CXCL9 did not prevent the development of this polarization. Additionally, we measured secretion of CXCL9 from ex vivo LN slices after stimulation with a variety of adjuvants and confirmed that adjuvants that induced IFNγ responses also promoted CXCL9 expression. Taken together, these results identify a CXCL9 signature in a suite of Th1-polarizing adjuvants and determined the pathway involved in driving CXCL9 in the LN, opening avenues to target this chemokine pathway in future vaccines.

Spatially resolved quantification of oxygen consumption rate in ex vivo lymph node slices.

Cellular metabolism has been closely linked to activation state in cells of the immune system, and the oxygen consumption rate (OCR) in particular serves as a valuable metric for assessing metabolic activity. Several oxygen sensing assays have been reported for cells in standard culture conditions. However, none have provided a spatially resolved, optical measurement of local oxygen consumption in intact tissue samples, making it challenging to understand regional dynamics of consumption. Therefore, here we established a system to monitor the rates of oxygen consumption in ex vivo tissue slices, using murine lymphoid tissue as a case study. By integrating an optical oxygen sensor into a sealed perfusion chamber and incorporating appropriate correction for photobleaching of the sensor and of tissue autofluorescence, we were able to visualize and quantify rates of oxygen consumption in tissue. This method revealed for the first time that the rate of oxygen consumption in naïve lymphoid tissue was higher in the T cell region compared to the B cell and cortical regions. To validate the method, we measured OCR in the T cell regions of naïve lymph node slices using the optical assay and estimated the consumption rate per cell. The predictions from the optical assay were similar to reported values and were not significantly different from those of the Seahorse metabolic assay, a gold standard method for measuring OCR in cell suspensions. Finally, we used this method to quantify the rate of onset of tissue hypoxia for lymph node slices cultured in a sealed chamber and showed that continuous perfusion was sufficient to maintain oxygenation. In summary, this work establishes a method to monitor oxygen consumption with regional resolution in intact tissue explants, suitable for future use to compare tissue culture conditions and responses to stimulation.

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.

Selective Fluorination of the Surface of Polymeric Materials after Stereolithography 3D Printing.

With the microfluidics community embracing 3D resin printing as a rapid fabrication method, controlling surface chemistry has emerged as a new challenge. Fluorination of 3D-printed surfaces is highly desirable in many applications due to chemical inertness, low friction coefficients, antifouling properties, and the potential for selective hydrophobic patterning. Despite sporadic reports, silanization methods have not been optimized for covalent bonding with polymeric resins. As a case study, we tested the silanization of a commercially available (meth)acrylate-based resin (BV-007A) with a fluoroalkyl trichlorosilane. Interestingly, plasma oxidation was unnecessary for silanization of this resin and indeed was ineffective. Solvent-based deposition in a fluorinated oil (FC-40) generated significantly higher contact angles than deposition in ethanol or gas-phase deposition, yielding hydrophobic surfaces with contact angle >110° under optimized conditions. Attenuated total reflectance-Fourier transform infrared spectroscopy indicated that the increase in the contact angle correlated with consumption of a carbonyl moiety, suggesting covalent bonding of silane without plasma oxidation. Consistent with a covalent bond, silanization was resistant to mechanical damage and hydrolysis in methanol and was stable over long-term storage. When tested on a suite of photocrosslinkable resins, this silanization protocol generated highly hydrophobic surfaces (contact angle > 110°) on three resins and moderate hydrophobicity (90-100°) on the remainder. Selective patterning of hydrophobic regions in an open 3D-printed microchannel was possible in combination with simple masking techniques. Thus, this facile fluorination strategy is expected to be applicable for resin-printed materials in a variety of contexts including micropatterning and multiphase microfluidics.

Spatially Resolved Analytical Chemistry in Intact, Living Tissues.

Tissues are an exciting frontier for bioanalytical chemistry, one in which spatial distribution is just as important as total content. Intact tissue preserves the native cellular and molecular organization and the cell-cell contacts found in vivo. Live tissue, in particular, offers the potential to analyze dynamic events in a spatially resolved manner, leading to fundamental biological insights and translational discoveries. In this Perspective, we provide a tutorial on the four fundamental challenges for the bioanalytical chemist working in living tissue samples as well as best practices for mitigating them. The challenges include (i) the complexity of the sample matrix, which contributes myriad interfering species and causes nonspecific binding of reagents; (ii) hindered delivery and mixing; (iii) the need to maintain physiological conditions; and (iv) tissue reactivity. This framework is relevant to a variety of methods for spatially resolved chemical analysis, including optical imaging, inserted sensors and probes such as electrodes, and surface analyses such as sensing arrays. The discussion focuses primarily on ex vivo tissues, though many considerations are relevant in vivo as well. Our goal is to convey the exciting potential of analytical chemistry to contribute to understanding the functions of live, intact tissues.

Quantification of fractional and absolute functionalization of gelatin hydrogels by optimized ninhydrin assay and 1H NMR.

3D cell culture in protein-based hydrogels often begins with chemical functionalization of proteins with cross-linking agents such as methacryloyl or norbornene. An important and variable characteristic of these materials is the degree of functionalization (DoF), which controls the reactivity of the protein for cross-linking and therefore impacts the mechanical properties and stability of the hydrogel. Although 1H NMR has emerged as the most accurate technique for quantifying absolute DoF of chemically modified proteins, colorimetric techniques still dominate in actual use and may be more useful for quantifying fractional or percent DoF. In this work, we sought to develop an optimized colorimetric assay for DoF of common gelatin-based biomaterials and validate it versus NMR; along the way, we developed a set of best practices for both methods and considerations for their most appropriate use. First, the amine-reactive ninhydrin assay was optimized in terms of solvent properties, temperature, ninhydrin concentration, and range of gelatin standards. The optimized assay produced a linear response to protein concentration in a convenient, 96-well plate format and yielded a fractional DoF similar to NMR in most cases. In comparing with NMR, we identified that DoF can be expressed as fractional or absolute, and that fractional DoF can be inaccurate if the amino acid content of the parent protein is not properly accounted for. In summary, the fractional DoF of methacryloyl- and norbornene-functionalized gelatins was quantified by an optimized colorimetric ninhydrin assay and orthogonally by 1H NMR. These methods will be valuable for quality control analysis of protein-based hydrogels and 3D cell culture biomaterials. Graphical abstract.

Optimization of Enzymatic Antibody Fragmentation for Yield, Efficiency, and Binding Affinity.

Enzymatic antibody fragmentation has been well studied for various hosts and isotypes, but fragmentation patterns also vary unpredictably by clone, and optimizing Fab or F(ab')2 production by trial and error consumes large quantities of antibodies. Here, we report a systematic strategy for optimizing functional F(ab')2 production via pepsin digestion from small quantities of IgG. We tested three key parameters that affect fragmentation, pH, enzyme concentration (% pepsin w/w), and reaction time, and found that pH had the greatest impact on fragmentation yield and efficiency. We then developed a systematic approach to obtaining acceptable yields, digestion efficiency, and binding affinity. Three case studies are described to illustrate the approach. We anticipate that this work will provide a quick and cost-effective method for researchers to produce antibody fragments from whole IgG, avoiding haphazard trial and error.

Spatially resolved microfluidic stimulation of lymphoid tissue ex vivo.

The lymph node is a structurally complex organ of the immune system, whose dynamic cellular arrangements are thought to control much of human health. Currently, no methods exist to precisely stimulate substructures within the lymph node or analyze local stimulus-response behaviors, making it difficult to rationally design therapies for inflammatory disease. Here we describe a novel integration of live lymph node slices with a microfluidic system for local stimulation. Slices maintained the cellular organization of the lymph node while making its core experimentally accessible. The 3-layer polydimethylsiloxane device consisted of a perfusion chamber stacked atop stimulation ports fed by underlying microfluidic channels. Fluorescent dextrans similar in size to common proteins, 40 and 70 kDa, were delivered to live lymph node slices with 284 ± 9 µm and 202 ± 15 µm spatial resolution, respectively, after 5 s, which is sufficient to target functional zones of the lymph node. The spread and quantity of stimulation were controlled by varying the flow rates of delivery; these were predictable using a computational model of isotropic diffusion and convection through the tissue. Delivery to two separate regions simultaneously was demonstrated, to mimic complex intercellular signaling. Delivery of a model therapeutic, glucose-conjugated albumin, to specific regions of the lymph node indicated that retention of the drug was greater in the B-cell zone than in the T-cell zone. Together, this work provides a novel platform, the lymph node slice-on-a-chip, to target and study local events in the lymph node and to inform the development of new immunotherapeutics.

A 3D-printed multi-compartment organ-on-chip platform with a tubing-free pump models communication with the lymph node.

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 with the capacity 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 in mock tissue, vaccination of the multi-compartment 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.

Measurement of covalent bond formation in light-curing hydrogels predicts physical stability under flow.

Photocrosslinking hydrogels are promising for tissue engineering and regenerative medicine, but challenges in reaction monitoring often leave their optimization subject to trial and error. The stability of crosslinked gels under fluid flow, as in the case of a microfluidic device, is particularly challenging to predict, both because of obstacles inherent to solid-state macromolecular analysis that prevent accurate chemical monitoring, and because stability is dependent on size of the patterned features. To solve both problems, we obtained 1H NMR spectra of cured hydrogels which were enzymatically degraded. This allowed us to take advantage of the high-resolution that solution NMR provides. This unique approach enabled the measurement of degree of crosslinking (DoC) and prediction of material stability under physiological fluid flow. We showed that NMR spectra of enzyme-digested gels successfully reported on DoC as a function of light exposure and wavelength within two classes of photocrosslinkable hydrogels: methacryloyl-modified gelatin and a composite of thiol-modified gelatin and norbornene-terminated polyethylene glycol. This approach revealed that a threshold DoC was required for patterned features in each material to become stable, and that smaller features required a higher DoC for stability. Finally, we demonstrated that DoC was predictive of the stability of architecturally complex features when photopatterning, underscoring the value of monitoring DoC when using light-reactive gels. We anticipate that the ability to quantify chemical crosslinks will accelerate the design of advanced hydrogel materials for structurally demanding applications such as photopatterning and bioprinting.

Spatially resolved quantification of oxygen consumption rate in ex vivo lymph node slices.

Cellular metabolism has been closely linked to activation state in cells of the immune system, and the oxygen consumption rate (OCR) in particular serves as a valuable metric for assessing metabolic activity. Several oxygen sensing assays have been reported for cells in standard culture conditions. However, none have provided a spatially resolved, optical measurement of local oxygen consumption in intact tissue samples, making it challenging to understand regional dynamics of consumption. Therefore, here we established a system to monitor the rates of oxygen consumption in ex vivo tissue slices, using murine lymphoid tissue as a case study. By integrating an optical oxygen sensor into a sealed perfusion chamber and incorporating appropriate correction for photobleaching of the sensor and of tissue autofluorescence, we were able to visualize and quantify rates of oxygen consumption in tissue. This method revealed for the first time that the rate of oxygen consumption in naïve lymphoid tissue was higher in the T cell region compared to the B cell and cortical regions. To validate the method, we measured OCR in the T cell regions of naïve lymph node slices using the optical assay and estimated the consumption rate per cell. The predictions from the optical assay were similar to reported values and were not significantly different from those of the Seahorse metabolic assay, a gold standard method for measuring OCR in cell suspensions. Finally, we used this method to quantify the rate of onset of tissue hypoxia for lymph node slices cultured in a sealed chamber and showed that continuous perfusion was sufficient to maintain oxygenation. In summary, this work establishes a method to monitor oxygen consumption with regional resolution in intact tissue explants, suitable for future use to compare tissue culture conditions and responses to stimulation.

Mitigating reactive oxygen species production and increasing gel porosity improves lymphocyte motility and fibroblast spreading in photocrosslinked gelatin-thiol hydrogels.

On-chip 3D culture systems that incorporate immune cells such as lymphocytes and stromal cells are needed to model immune organs in engineered systems such as organs-on-chip. Photocrosslinking is a useful tool for creating such immune-competent hydrogel cultures with spatial cell organization. However, loss of viability and motility in photocrosslinked gels can limit its utility, especially when working with fragile primary cells. We hypothesized that optimizing photoexposure-induced ROS production, hydrogel porosity or a combination of both factors was necessary to sustain cell viability and motility during culture in photocrosslinked gelatin-thiol (GelSH) hydrogels. Jurkat T cells, primary human CD4+ T cells and human lymphatic fibroblasts were selected as representative lymphoid immune cells to test this hypothesis. Direct exposure of these cells to 385 nm light and LAP photoinitiator dramatically increased ROS levels. Pretreatment with an antioxidant, ascorbic acid (AA), protected the cells from light + LAP-induced ROS and was non-toxic at optimized doses. Furthermore, scanning electron microscopy showed that native GelSH hydrogels had limited porosity, and that adding collagen to GelSH precursor before crosslinking markedly increased gel porosity. Next, we tested the impact of AA pretreatment and increasing gel porosity, alone or in combination, on cell viability and function in 3D GelSH hydrogel cultures. Increasing gel porosity, rather than AA pretreatment, was more critical for rescuing viability of Jurkat T cells and spreading of human lymphatic fibroblasts in GelSH-based gels, but both factors improved the motility of primary human CD4+ T cells. Increased porosity enabled formation of spatially organized co-cultures of primary human CD4+ T cells and human lymphatic fibroblasts in photo-crosslinked gels in a multi-lane microfluidic chip, towards modeling the lymphoid organ microenvironment. Some optimization is still needed to improve homogeneity between regions on the chip. These findings will enable researchers utilizing photocrosslinking methods to develop immunocompetent 3D culture models that support viability and function of sensitive lymphoid cells.

Ex vivo model of breast cancer cell invasion in live lymph node tissue.

Lymph nodes (LNs) are common sites of metastatic invasion in breast cancer, often preceding spread to distant organs and serving as key indicators of clinical disease progression. However, the mechanisms of cancer cell invasion into LNs are not well understood. Existing in vivo models struggle to isolate the specific impacts of the tumor-draining lymph node (TDLN) milieu on cancer cell invasion due to the co-evolving relationship between TDLNs and the upstream tumor. To address these limitations, we used live ex vivo LN tissue slices with intact chemotactic function to model cancer cell spread within a spatially organized microenvironment. After showing that BRPKp110 breast cancer cells were chemoattracted to factors secreted by naïve LN tissue in a 3D migration assay, we demonstrated that ex vivo LN slices could support cancer cell seeding, invasion, and spread. This novel approach revealed dynamic, preferential cancer cell invasion within specific anatomical regions of LNs, particularly the subcapsular sinus (SCS) and cortex, as well as chemokine-rich domains of immobilized CXCL13 and CCL1. While CXCR5 was necessary for a portion of BRPKp110 invasion into naïve LNs, disruption of CXCR5/CXCL13 signaling alone was insufficient to prevent invasion towards CXCL13-rich domains. Finally, we extended this system to pre-metastatic TDLNs, where the ex vivo model predicted a lower invasion of cancer cells. The reduced invasion was not due to diminished chemokine secretion, but it correlated with elevated intranodal IL-21. In summary, this innovative ex vivo model of cancer cell spread in live LN slices provides a platform to investigate cancer invasion within the intricate tissue microenvironment, supporting time-course analysis and parallel read-outs. We anticipate that this system will enable further research into cancer-immune interactions and allow isolation of specific factors that make TDLNs resistant to cancer cell invasion, which are challenging to dissect in vivo.

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.

New tools for immunologists: models of lymph node function from cells to tissues.

The lymph node is a highly structured organ that mediates the body's adaptive immune response to antigens and other foreign particles. Central to its function is the distinct spatial assortment of lymphocytes and stromal cells, as well as chemokines that drive the signaling cascades which underpin immune responses. Investigations of lymph node biology were historically explored in vivo in animal models, using technologies that were breakthroughs in their time such as immunofluorescence with monoclonal antibodies, genetic reporters, in vivo two-photon imaging, and, more recently spatial biology techniques. However, new approaches are needed to enable tests of cell behavior and spatiotemporal dynamics under well controlled experimental perturbation, particularly for human immunity. This review presents a suite of technologies, comprising in vitro, ex vivo and in silico models, developed to study the lymph node or its components. We discuss the use of these tools to model cell behaviors in increasing order of complexity, from cell motility, to cell-cell interactions, to organ-level functions such as vaccination. Next, we identify current challenges regarding cell sourcing and culture, real time measurements of lymph node behavior in vivo and tool development for analysis and control of engineered cultures. Finally, we propose new research directions and offer our perspective on the future of this rapidly growing field. We anticipate that this review will be especially beneficial to immunologists looking to expand their toolkit for probing lymph node structure and function.

A Scalable, Modular Degasser for Passive In-Line Removal of Bubbles from Biomicrofluidic Devices.

Bubbles are a common cause of microfluidic malfunction, as they can perturb the fluid flow within the micro-sized features of a device. Since gas bubbles form easily within warm cell culture reagents, degassing is often necessary for biomicrofluidic systems. However, fabrication of a microscale degasser that can be used modularly with pre-existing chips may be cumbersome or challenging, especially for labs not equipped for traditional microfabrication, and current commercial options can be expensive. Here, we address the need for an affordable, accessible bubble trap that can be used in-line for continuous perfusion of organs-on-chip and other microfluidic cultures. We converted a previously described, manually fabricated PDMS degasser to allow scaled up, reproducible manufacturing by commercial machining or fused deposition modeling (FDM) 3D printing. After optimization, the machined and 3D printed degassers were found to be stable for >2 weeks under constant perfusion, without leaks. With a ~140 µL chamber volume, trapping capacity was extrapolated to allow for ~5-20 weeks of degassing depending on the rate of bubble formation. The degassers were biocompatible for use with cell culture, and they successfully prevented bubbles from reaching a downstream microfluidic device. Both degasser materials showed little to no leaching. The machined degasser did not absorb reagents, while the FDM printed degasser absorbed a small amount, and both maintained fluidic integrity from 1 µL/min to >1 mL/min of pressure-driven flow. Thus, these degassers can be fabricated in bulk and allow for long-term, efficient bubble removal in a simple microfluidic perfusion set-up.

Control of initiation, rate, and routing of spontaneous capillary-driven flow of liquid droplets through microfluidic channels on SlipChip.

This Article describes the use of capillary pressure to initiate and control the rate of spontaneous liquid-liquid flow through microfluidic channels. In contrast to flow driven by external pressure, flow driven by capillary pressure is dominated by interfacial phenomena and is exquisitely sensitive to the chemical composition and geometry of the fluids and channels. A stepwise change in capillary force was initiated on a hydrophobic SlipChip by slipping a shallow channel containing an aqueous droplet into contact with a slightly deeper channel filled with immiscible oil. This action induced spontaneous flow of the droplet into the deeper channel. A model predicting the rate of spontaneous flow was developed on the basis of the balance of net capillary force with viscous flow resistance, using as inputs the liquid-liquid surface tension, the advancing and receding contact angles at the three-phase aqueous-oil-surface contact line, and the geometry of the devices. The impact of contact angle hysteresis, the presence or absence of a lubricating oil layer, and adsorption of surface-active compounds at liquid-liquid or liquid-solid interfaces were quantified. Two regimes of flow spanning a 10(4)-fold range of flow rates were obtained and modeled quantitatively, with faster (mm/s) flow obtained when oil could escape through connected channels as it was displaced by flowing aqueous solution, and slower (micrometer/s) flow obtained when oil escape was mostly restricted to a micrometer-scale gap between the plates of the SlipChip ("dead-end flow"). Rupture of the lubricating oil layer (reminiscent of a Cassie-Wenzel transition) was proposed as a cause of discrepancy between the model and the experiment. Both dilute salt solutions and complex biological solutions such as human blood plasma could be flowed using this approach. We anticipate that flow driven by capillary pressure will be useful for the design and operation of flow in microfluidic applications that do not require external power, valves, or pumps, including on SlipChip and other droplet- or plug-based microfluidic devices. In addition, this approach may be used as a sensitive method of evaluating interfacial tension, contact angles, and wetting phenomena on chip.