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.

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.

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.

Towards spatially-organized organs-on-chip: Photopatterning cell-laden thiol-ene and methacryloyl hydrogels in a microfluidic device.

Micropatterning techniques for 3D cell cultures enable the recreation of tissue-level structures, but the combination of patterned hydrogels with organs-on-chip to generate organized 3D cultures under microfluidic perfusion remains challenging. To address this technological gap, we developed a user-friendly in-situ micropatterning protocol that integrates photolithography of crosslinkable, cell-laden hydrogels with a simple microfluidic housing, and tested the impact of crosslinking chemistry on stability and spatial resolution. Working with gelatin functionalized with photo-crosslinkable moieties, we found that inclusion of cells at high densities (≥ 107/mL) did not impede thiol-norbornene gelation, but decreased the storage moduli of methacryloyl hydrogels. Hydrogel composition and light dose were selected to match the storage moduli of soft tissues. To generate the desired pattern on-chip, the cell-laden precursor solution was flowed into a microfluidic chamber and exposed to 405 nm light through a photomask. The on-chip 3D cultures were self-standing and the designs were interchangeable by simply swapping out the photomask. Thiol-ene hydrogels yielded highly accurate feature sizes from 100 - 900 µm in diameter, whereas methacryloyl hydrogels yielded slightly enlarged features. Furthermore, only thiol-ene hydrogels were mechanically stable under perfusion overnight. Repeated patterning readily generated multi-region cultures, either separately or adjacent, including non-linear boundaries that are challenging to obtain on-chip. As a proof-of-principle, primary human T cells were patterned on-chip with high regional specificity. Viability remained high (> 85%) after 12-hr culture with constant perfusion. We envision that this technology will enable researchers to pattern 3D co-cultures to mimic organ-like structures that were previously difficult to obtain.

Applied tutorial for the design and fabrication of biomicrofluidic devices by resin 3D printing.

Resin 3D printing, especially digital light processing (DLP) printing, is a promising rapid fabrication method for bio-microfluidic applications such as clinical tests, lab-on-a-chip devices, and sensor integrated devices. The benefits of 3D printing lead many to believe this fabrication method will accelerate the use of microfluidics, but there are a number of potential obstacles to overcome for bioanalytical labs to fully utilize this technology. For commercially available printing materials, this includes challenges in producing prints with the print resolution and mechanical stability required for a particular design, along with cytotoxic components within many photopolymerizing resins and low optical compatibility for imaging experiments. Potential solutions to these problems are scattered throughout the literature and rarely available in head-to-head comparisons. Therefore, we present here a concise guide to the principles of resin 3D printing most relevant for fabrication of bioanalytical microfluidic devices. Intended to quickly orient labs that are new to 3D printing, the tutorial includes the results of selected systematic tests to inform resin selection, strategies for design optimization, and improvement of biocompatibility of resin 3D printed bio-microfluidic devices.

Platinum Chemotherapy Induces Lymphangiogenesis in Cancerous and Healthy Tissues That Can be Prevented With Adjuvant Anti-VEGFR3 Therapy.

Chemotherapy has been used to inhibit cancer growth for decades, but emerging evidence shows it can affect the tumor stroma, unintentionally promoting cancer malignancy. After treatment of primary tumors, remaining drugs drain via lymphatics. Though all drugs interact with the lymphatics, we know little of their impact on them. Here, we show a previously unknown effect of platinums, a widely used class of chemotherapeutics, to directly induce systemic lymphangiogenesis and activation. These changes are dose-dependent, long-lasting, and occur in healthy and cancerous tissue in multiple mouse models of breast cancer. We found similar effects in human ovarian and breast cancer patients whose treatment regimens included platinums. Carboplatin treatment of healthy mice prior to mammary tumor inoculation increased cancer metastasis as compared to no pre-treatment. These platinum-induced phenomena could be blocked by VEGFR3 inhibition. These findings have implications for cancer patients receiving platinums and may support the inclusion of anti-VEGFR3 therapy into treatment regimens or differential design of treatment regimens to alter these potential effects.

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.

Rapid Fabrication by Digital Light Processing 3D Printing of a SlipChip with Movable Ports for Local Delivery to Ex Vivo Organ Cultures.

SlipChips are two-part microfluidic devices that can be reconfigured to change fluidic pathways for a wide range of functions, including tissue stimulation. Currently, fabrication of these devices at the prototype stage requires a skilled microfluidic technician, e.g., for wet etching or alignment steps. In most cases, SlipChip functionality requires an optically clear, smooth, and flat surface that is fluorophilic and hydrophobic. Here, we tested digital light processing (DLP) 3D printing, which is rapid, reproducible, and easily shared, as a solution for fabrication of SlipChips at the prototype stage. As a case study, we sought to fabricate a SlipChip intended for local delivery to live tissue slices through a movable microfluidic port. The device was comprised of two multi-layer components: an enclosed channel with a delivery port and a culture chamber for tissue slices with a permeable support. Once the design was optimized, we demonstrated its function by locally delivering a chemical probe to slices of hydrogel and to living tissue with up to 120 µm spatial resolution. By establishing the design principles for 3D printing of SlipChip devices, this work will enhance the ability to rapidly prototype such devices at mid-scale levels of production.

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.

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.

Acute Lymph Node Slices Are a Functional Model System to Study Immunity Ex Vivo.

The lymph node is a highly organized and dynamic structure that is critical for facilitating the intercellular interactions that constitute adaptive immunity. Most ex vivo studies of the lymph node begin by reducing it to a cell suspension, thus losing the spatial organization, or fixing it, thus losing the ability to make repeated measurements. Live murine lymph node tissue slices offer the potential to retain spatial complexity and dynamic accessibility, but their viability, level of immune activation, and retention of antigen-specific functions have not been validated. Here we systematically characterized live murine lymph node slices as a platform to study immunity. Live lymph node slices maintained the expected spatial organization and cell populations while reflecting the 3D spatial complexity of the organ. Slices collected under optimized conditions were comparable to cell suspensions in terms of both 24-h viability and inflammation. Slices responded to T cell receptor cross-linking with increased surface marker expression and cytokine secretion, in some cases more strongly than matched lymphocyte cultures. Furthermore, slices processed protein antigens, and slices from vaccinated animals responded to ex vivo challenge with antigen-specific cytokine secretion. In summary, lymph node slices provide a versatile platform to investigate immune functions in spatially organized tissue, enabling well-defined stimulation, time-course analysis, and parallel read-outs.

Spatially resolved measurement of dynamic glucose uptake in live ex vivo tissues.

Highly proliferative cells depend heavily on glycolysis as a source of energy and biological precursor molecules, and glucose uptake is a useful readout of this aspect of metabolic activity. Glucose uptake is commonly quantified by using flow cytometry for cell cultures and positron emission tomography for organs in vivo. However, methods to detect spatiotemporally resolved glucose uptake in intact tissues are far more limited, particularly those that can quantify changes in uptake over time in specific tissue regions and cell types. Using lymph node metabolism as a case study, we developed an optimized method to detect dynamic and spatially resolved glucose uptake in living tissue by combining ex vivo tissue slice culture with a fluorescent glucose analogue. Live slices of murine lymph node were treated with the glucose analogue 2-[N-(7-nitrobenz-2-oxa-1,3-dia-xol-4-yl)amino]-2-deoxyglucose (2-NBDG). Incubation parameters were optimized to differentiate glucose uptake in activated versus naïve lymphocytes. Regional glucose uptake could be imaged at both the tissue level, by widefield microscopy, and at the cellular level, by confocal microscopy. Furthermore, the glucose assay was readily multiplexed with live immunofluorescence labelling to generate maps of 2-NBDG uptake across tissue regions, revealing highest uptake in T cell-dense regions. The signal was predominantly intracellular and localized to lymphocytes rather than stromal cells. Finally, we demonstrated that the assay was repeatable in the same slices, and imaged the dynamic distribution of glucose uptake in response to ex vivo T cell stimulation for the first time. We anticipate that this method will serve as a broadly applicable, user-friendly platform to quantify dynamic metabolic activities in complex tissue microenvironments.

Detergent wash improves vaccinated lymph node handling ex vivo.

Lymph nodes (LNs) are essential secondary immune organs where the adaptive immune response is generated against most infections and vaccines. We recently described the use of live ex vivo LN slices to study the dynamics of adaptive immunity. However, when working with reactive lymph nodes from vaccinated animals, the tissues frequently became dislodged from the supportive agarose matrix during slicing, leading to damage that prevented downstream analysis. Because reactive lymph nodes expand into the surrounding adipose tissue, we hypothesized that dislodging was a result of excess lipids on the collagen capsule of the LN, and that a brief wash with a mild detergent would improve LN interaction with the agarose without damaging tissue viability or function. Therefore, we tested the use of digitonin on improving slicing of vaccinated LNs. Prior to embedding, LNs were quickly dipped into a digitonin solution and washed in saline. Lipid droplets were visibly removed by this procedure. A digitonin wash step prior to slicing significantly reduced the loss of LN during slicing from 13 to 75% to 0-25%, without substantial impact on viability. Capture of fluorescent microparticles, uptake and processing of protein antigen, and cytokine secretion in response to a vaccine adjuvant, R848, were all unaffected by the detergent wash. This novel approach will enable ex vivo analysis of the generation of adaptive immune response in LNs in response to vaccinations and other immunotherapies.

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.