Development of an intestinal mucosa ex vivo co-culture model to study viral infections.

Studying viral infections necessitates well-designed cell culture models to deepen our understanding of diseases and develop effective treatments. In this study, we present a readily available ex vivo 3D co-culture model replicating the human intestinal mucosa. The model combines fully differentiated human intestinal epithelium (HIE) with human monocyte-derived macrophages (hMDMs) and faithfully mirrors the in vivo structural and organizational properties of intestinal mucosal tissues. Specifically, it mimics the lamina propria, basement membrane, and the air-exposed epithelial layer, enabling the pioneering observation of macrophage migration through the tissue to the site of viral infection. In this study, we applied the HIE-hMDMs model for the first time in viral infection studies, infecting the model with two globally significant viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human norovirus GII.4. The results demonstrate the model's capability to support the replication of both viruses and show the antiviral role of macrophages, determined by their migration to the infection site and subsequent direct contact with infected epithelial cells. In addition, we evaluated the production of cytokines and chemokines in the intestinal niche, observing an increased interleukin-8 production during infection. A parallel comparison using a classical in vitro cell line model comprising Caco-2 and THP-1 cells for SARS-CoV-2 experiments confirmed the utility of the HIE-hMDMs model in viral infection studies. Our data show that the ex vivo tissue models hold important implications for advances in virology research.IMPORTANCEThe fabrication of intricate ex vivo tissue models holds important implications for advances in virology research. The co-culture model presented here provides distinct spatial and functional attributes not found in simplified models, enabling the evaluation of macrophage dynamics under severe acute respiratory syndrome coronavirus 2 and human norovirus (HuNoV) infections in the intestine. Moreover, these models, comprised solely of primary cells, facilitate the study of difficult-to-replicate viruses such as HuNoV, which cannot be studied in cell line models, and offer the opportunity for personalized treatment evaluations using patient cells. Similar co-cultures have been established for the study of bacterial infections and different characteristics of the intestinal tissue. However, to the best of our knowledge, a similar intestinal model for the study of viral infections has not been published before.

Quantitative Evaluation of the Cellular Uptake of Nanodiamonds by Monocytes and Macrophages.

Fluorescent nanodiamonds (FNDs) with negative nitrogen-vacancy (NV- ) defect centers are great probes for biosensing applications, with potential to act as biomarkers for cell differentiation. To explore this concept, uptake of FNDs (≈120 nm) by THP-1 monocytes and monocyte-derived M0-macrophages is studied. The time course analysis of FND uptake by monocytes confirms differing FND-cell interactions and a positive time-dependence. No effect on cell viability, proliferation, and differentiation potential into macrophages is observed, while cells saturated with FNDs, unload the FNDs completely by 25 cell divisions and subsequently take up a second dose effectively. FND uptake variations by THP-1 cells at early exposure-times indicate differing phagocytic capability. The cell fraction that exhibits relatively enhanced FND uptake is associated to a macrophage phenotype which derives from spontaneous monocyte differentiation. In accordance, chemical-differentiation of the THP-1 cells into M0-macrophages triggers increased and homogeneous FND uptake, depleting the fraction of cells that were non-responsive to FNDs. These observations imply that FND uptake allows for distinction between the two cell subtypes based on phagocytic capacity. Overall, FNDs demonstrate effective cell labeling of monocytes and macrophages, and are promising candidates for sensing biological processes that involve cell differentiation.

Heterogenous morphogenesis of Caco-2 cells reveals that flow induces three-dimensional growth and maturation at high initial seeding cell densities.

Here, we introduce a customized hanging insert fitting a six-well plate to culture Caco-2 cells on hydrogel membranes under flow conditions. The cells are cultured in the apical channel-like chamber, which provides about 1.3 dyn/cm2 shear, while the basolateral chamber is mixed when the device is rocked. The device was tested by investigating the functional impact of the initial seeding density in combination with flow applied at confluency. The low seeding density cultures grew in two dimensional (2D) irrespective of the flow. Flow and higher seeding density resulted in a mixture of three dimensional (3D) structures and 2D layers. Static culture and high cell seeding density resulted in 2D layers. The flow increased the height and ZO-1 expression of cells in 2D layers, which correlated with an improved barrier function. Cultures with 3D structures had higher ZO-1 expression than 2D cultures, but this did not correlate with an increased barrier function. 2D monolayers in static and dynamic cultures had similar morphology and heterogeneity in the expression of Mucin-2 and Villin, while the 3D structures had generally higher expression of these markers. The result shows that the cell density and flow determine 3D growth and that the highest barrier function was obtained with low-density cultures and flow.

cIAP1/2 antagonization by SMAC mimetic induces non-canonical NF-κB mediated TH 17 cell homotypic interactions and increases their resistance to shear stress.

SMAC antagonization of cIAP1/2 in TH 17 cells upregulates cell adhesion and cytoskeleton genes through the NIK-RelB and p52 axis. SMAC also increases the homotypic interactions of TH 17 cells through a non-canonical NF-κB- and integrin-mediated mechanism resulting in increased ability of TH 17 cells to withstand shear stress.

CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification.

False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal negative controls, performed simultaneously with amplification to monitor the background level in real-time, are therefore appealing in both research and clinic. Herein, we describe a nonspecific product-activated single-stranded DNA-cutting approach based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas12a (Cpf1) nuclease. The proposed approach, termed Cas12a-based internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout. The capability of CIRI as an internal negative control can potentially be extended to other amplification strategies and sensors, improving the performance of nucleic acid amplification-based methodologies.

Characterization of thin gelatin hydrogel membranes with balloon properties for dynamic tissue engineering.

Cell or tissue stretching and strain are present in any in vivo environment, but is difficult to reproduce in vitro. Here, we describe a simple method for casting a thin (about 500 µm) and soft (about 0.3 kPa) hydrogel of gelatin and a method for characterizing the mechanical properties of the hydrogel simply by changing pressure with a water column. The gelatin is crosslinked with mTransglutaminase and the area of the resulting hydrogel can be increased up 13-fold by increasing the radial water pressure. This is far beyond physiological stretches observed in vivo. Actuating the hydrogel with a radial force achieves both information about stiffness, stretchability, and contractability, which are relevant properties for tissue engineering purposes. Cells could be stretched and contracted using the gelatin membrane. Gelatin is a commonly used polymer for hydrogels in tissue engineering, and the discovered reversible stretching is particularly interesting for organ modeling applications.

Monitoring intra- and extracellular redox capacity of intact barley aleurone layers responding to phytohormones.

Redox regulation is important for numerous processes in plant cells including abiotic stress, pathogen defence, tissue development, seed germination and programmed cell death. However, there are few methods allowing redox homeostasis to be addressed in whole plant cells, providing insight into the intact in vivo environment. An electrochemical redox assay that applies the menadione-ferricyanide double mediator is used to assess changes in the intracellular and extracellular redox environment in living aleurone layers of barley (Hordeum vulgare cv. Himalaya) grains, which respond to the phytohormones gibberellic acid and abscisic acid. Gibberellic acid is shown to elicit a mobilisation of electrons as detected by an increase in the reducing capacity of the aleurone layers. By taking advantage of the membrane-permeable menadione/menadiol redox pair to probe the membrane-impermeable ferricyanide/ferrocyanide redox pair, the mobilisation of electrons was dissected into an intracellular and an extracellular, plasma membrane-associated component. The intracellular and extracellular increases in reducing capacity were both suppressed when the aleurone layers were incubated with abscisic acid. By probing redox levels in intact plant tissue, the method provides a complementary approach to assays of reactive oxygen species and redox-related enzyme activities in tissue extracts.

3D Printed Silicone-Hydrogel Scaffold with Enhanced Physicochemical Properties.

Scaffolds with multiple functionalities have attracted widespread attention in the field of tissue engineering due to their ability to control cell behavior through various cues, including mechanical, chemical, and electrical. Fabrication of such scaffolds from clinically approved materials is currently a huge challenge. The goal of this work was to fabricate a tissue engineering scaffold from clinically approved materials with the capability of delivering biomolecules and direct cell fate. We have used a simple 3D printing approach, that combines polymer casting with supercritical fluid technology to produce 3D interpenetrating polymer network (IPN) scaffold of silicone-poly(2-hydroxyethyl methacrylate)-co-poly(ethylene glycol) methyl ether acrylate (pHEMA-co-PEGMEA). The pHEMA-co-PEGMEA IPN materials were employed to support growth of human mesenchymal stem cells (hMSC), resulting in high cell viability and metabolic activity over a 3 weeks period. In addition, the IPN scaffolds support 3D tissue formation inside the porous scaffold with well spread cell morphology on the surface of the scaffold. As a proof of concept, sustained doxycycline (DOX) release from pHEMA-co-PEGMEA IPN was demonstrated and the biological activity of released drug from IPN was confirmed using a DOX regulated green fluorescent reporter (GFP) gene expression assay with HeLa cells. Given its unique mechanical and drug releasing characteristics, IPN scaffolds may be used for directing stem cell differentiation by releasing various chemicals from its hydrogel network.

Gold nanoparticle-based sensors activated by external radio frequency fields.

A novel molecular beacon (a nanomachine) is constructed that can be actuated by a radio frequency (RF) field. The nanomachine consists of the following elements arranged in molecular beacon configuration: a gold nanoparticle that acts both as quencher for fluorescence and a localized heat source; one reporter fluorochrome, and; a piece of DNA as a hinge and recognition sequence. When the nanomachines are irradiated with a 3 GHz RF field the fluorescence signal increases due to melting of the stem of the molecular beacon. A control experiment, performed using molecular beacons synthesized by substituting the gold nanoparticle by an organic quencher, shows no increase in fluorescence signal when exposed to the RF field. It may therefore be concluded that the increased fluorescence for the gold nanoparticle-conjugated nanomachines is not due to bulk heating of the solution, but is caused by the presence of the gold nanoparticles and their interaction with the RF field; however, existing models for heating of gold nanoparticles in a RF field are unable to explain the experimental results. Due to the biocompatibility of the construct and RF treatment, the nanomachines may possibly be used inside living cells. In a separate experiment a substantial increase in the dielectric losses can be detected in a RF waveguide setup coupled to a microfluidic channel when gold nanoparticles are added to a low RF loss liquid. This work sheds some light on RF heating of gold nanoparticles, which is a subject of significant controversy in the literature.

Refractometric monitoring of dissolution and fluid flow with distributed feedback dye laser sensor.

Monitoring the dissolution of solid material in liquids and monitoring of fluid flow is of significant interest for applications in chemistry, food production, medicine, and especially in the fields of microfluidics and lab on a chip. Here, real-time refractometric monitoring of dissolution and fast fluid flow with DFB dye laser sensors with an optical imaging spectroscopy setup is presented. The dye laser sensors provide both low detection limits and high spatial resolution. It is demonstrated how the materials NaCl, sucrose, and bovine serum albumin show characteristic dissolution patterns. The unique feature of the presented method is a high frame rate of up to 20 Hz, which is proven to enable the monitoring of fast flow of a sucrose solution jet into pure water.

Micro-flow-injection analysis (µFIA) immunoassay of herbicide residue 2,6-dichlorobenzamide ­ towards automated at-line monitoring using modular microfluidics.

As a part of developing new systems for continuously monitoring the presence of pesticides in groundwater, a microfluidic amperometric immunosensor was developed for detecting the herbicide residue 2,6-dichlorobenzamide (BAM) in water. A competitive immunosorbent assay served as the sensing mechanism and amperometry was applied for detection. Both the immunoreaction chip (IRC) and detection (D) unit are integrated on a modular microfluidic platform with in-built micro-flow-injection analysis (µFIA) function. The immunosorbent, immobilized in the channel of the IRC, was found to have high long-term stability and withstand many regeneration cycles, both of which are key requirements for systems utilized in continuous monitoring. The IRC was regenerated during 51 cycles in a heterogeneous competitive assay out of which 27 were without the analyte (the highest possible signal level) in order to assess the regeneration capability of the immunosorbent. Detection of BAM standard solutions was performed in the concentration range from 62.5 µg L(-1) to 0.0008 µg L(-1). Non-linear regression of the data using the four-parameter logistic equation generated a sigmoidal standard curve showing an IC50 value (concentration that reduces the signal by 50%) of 0.25 µg L(-1). The strongest signal variation is observed in the concentration range between 0.02 and 2.5 µg L(-1), which includes the 0.1 µg L(-1) threshold limit set by the European Commission for BAM in drinking water. The presented results demonstrate the potential of the constructed µFIA immunosensor as an at-line monitoring system for controlling the quality of ground water supply.

Standalone single- and bi-layered human skin 3D models supported by recombinant silk feature native spatial organization.

Physiologically relevant human skin models that include key skin cell types can be used for in vitro drug testing, skin pathology studies, or clinical applications such as skin grafts. However, there is still no golden standard for such a model. We investigated the potential of a recombinant function-alized spider silk protein, FN-silk, for the construction of a dermal, an epidermal, and a bilayered skin equivalent (BSE). Specifically, two formats of FN-silk (i.e., 3D network and nanomembrane) were evaluated. The 3D network was used as an elastic ECM-like support for the dermis, and the thin, permeable nanomembrane was used as a basement membrane to support the epidermal epi-thelium. Immunofluorescence microscopy and spatially resolved transcriptomics analysis demon-strated the secretion of key ECM components and the formation of microvascular-like structures. Furthermore, the epidermal layer exhibited clear stratification and the formation of a cornified layer, resulting in a tight physiologic epithelial barrier. Our findings indicate that the presented FN-silk-based skin models can be proposed as physiologically relevant standalone epidermal or dermal models, as well as a combined BSE.

Bioelectrochemical probing of intracellular redox processes in living yeast cells--application of redox polymer wiring in a microfluidic environment.

Conventionally, microbial bioelectrochemical assays have been conducted using immobilized cells on an electrode that is placed in an electrochemical batch cell. In this paper, we describe a developed microfluidic platform with integrated microelectrode arrays for automated bioelectrochemical assays utilizing a new double mediator system to map redox metabolism and screen for genetic modifications in Saccharomyces cerevisiae cells. The function of this new double mediator system based on menadione and osmium redox polymer (PVI-Os) is demonstrated. "Wiring" of S. cerevisiae cells using PVI-Os shows a significant improvement of bioelectrochemical monitoring in a microfluidic environment and functions as an effective immobilization matrix for cells that are not strongly adherent. The function of the developed microfluidic platform is demonstrated using two strains of S. cerevisiae, ENY.WA and its deletion mutant EBY44, which lacks the enzyme phosphoglucose isomerase. The cellular responses to introduced glucose and fructose were recorded for the two S. cerevisiae strains, and the obtained results are compared with previously published work when using an electrochemical batch cell, indicating that microfluidic bioelectrochemical assays employing the menadione-PVI-Os double mediator system provides an effective means to conduct automated microbial assays.

A quantitative meta-analysis comparing cell models in perfused organ on a chip with static cell cultures.

As many consider organ on a chip for better in vitro models, it is timely to extract quantitative data from the literature to compare responses of cells under flow in chips to corresponding static incubations. Of 2828 screened articles, 464 articles described flow for cell culture and 146 contained correct controls and quantified data. Analysis of 1718 ratios between biomarkers measured in cells under flow and static cultures showed that the in all cell types, many biomarkers were unregulated by flow and only some specific biomarkers responded strongly to flow. Biomarkers in cells from the blood vessels walls, the intestine, tumours, pancreatic island, and the liver reacted most strongly to flow. Only 26 biomarkers were analysed in at least two different articles for a given cell type. Of these, the CYP3A4 activity in CaCo2 cells and PXR mRNA levels in hepatocytes were induced more than two-fold by flow. Furthermore, the reproducibility between articles was low as 52 of 95 articles did not show the same response to flow for a given biomarker. Flow showed overall very little improvements in 2D cultures but a slight improvement in 3D cultures suggesting that high density cell culture may benefit from flow. In conclusion, the gains of perfusion are relatively modest, larger gains are linked to specific biomarkers in certain cell types.

A self-contained, programmable microfluidic cell culture system with real-time microscopy access.

Utilizing microfluidics is a promising way for increasing the throughput and automation of cell biology research. We present a complete self-contained system for automated cell culture and experiments with real-time optical read-out. The system offers a high degree of user-friendliness, stability due to simple construction principles and compactness for integration with standard instruments. Furthermore, the self-contained system is highly portable enabling transfer between work stations such as laminar flow benches, incubators and microscopes. Accommodation of 24 individual inlet channels enables the system to perform parallel, programmable and multiconditional assays on a single chip. A modular approach provides system versatility and allows many different chips to be used dependent upon application. We validate the system's performance by demonstrating on-chip passive switching and mixing by peristaltically driven flows. Applicability for biological assays is demonstrated by on-chip cell culture including on-chip transfection and temporally programmable gene expression.

Direct immobilization of DNA probes on non-modified plastics by UV irradiation and integration in microfluidic devices for rapid bioassay.

DNA microarrays have become one of the most powerful tools in the field of genomics and medical diagnosis. Recently, there has been increased interest in combining microfluidics with microarrays since this approach offers advantages in terms of portability, reduced analysis time, low consumption of reagents, and increased system integration. Polymers are widely used for microfluidic systems, but fabrication of microarrays on such materials often requires complicated chemical surface modifications, which hinders the integration of microarrays into microfluidic systems. In this paper, we demonstrate that simple UV irradiation can be used to directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on many different types of plastics without any surface modification. On average, five- and fourfold improvement in immobilization and hybridization efficiency have been achieved compared to surface-modified slides with aminated DNA probes. Moreover, the TC tag only costs 30% of the commonly used amino group modifications. Using this microarray fabrication technique, a portable cyclic olefin copolymer biochip containing eight individually addressable microfluidic channels was developed and used for rapid and parallel identification of Avian Influenza Virus by DNA hybridization. The one-step, cost-effective DNA-linking method on non-modified polymers significantly simplifies microarray fabrication procedures and permits great flexibility to plastic material selection, thus making it convenient to integrate microarrays into plastic microfluidic systems.

Accessible, fast and easy fabrication of hydrophilic-in-hydrophobic microdroplet arrays.

Microdroplet arrays (MDAs) are powerful tools for digital immunoassays, high-throughput screening and single cell analysis. However, MDAs are usually produced with cleanroom processes, which are associated with high costs and low availability. Furthermore, in order to obtain robust and stable MDAs based on hydrophilic spots surrounded by a hydrophobic background, the chemistry must be strictly controlled, which is challenging using shared equipment. Here, we developed a new method to fabricate MDA substrates independently from the cleanroom. A small and low-cost in-house built system to collimate the light source was assembled for photopatterning a negative resist, and spots with diameters down to 4 µm were obtained, with only 3% to 5% spot-to-spot variation across the same sample and high batch-to-batch reproducibility. The use of a negative photoresist enabled the formation of a hydrophobic coating in solution which yielded high-quality MDAs. The feasibility for carrying out digital assays was demonstrated by measuring anti-Tau antibody in sample buffers containing bovine serum albumin, with no noticeable surface fouling. The reported, robust, cost-effective, and fast process could hence lower the threshold to fabricate and use MDAs for digital immunoassays and other microcompartmentalization-based applications.

Customized 3D-printed stackable cell culture inserts tailored with bioactive membranes.

There is a high demand in various fields to develop complex cell cultures. Apart from titer plates, Transwell inserts are the most popular device because they are commercially available, easy to use, and versatile. While Transwell inserts are standardized, there are potential gains to customize inserts in terms of the number of layers, height between the layers and the size and composition of the bioactive membrane. To demonstrate such customization, we present a small library of 3D-printed inserts and a robust method to functionalize the inserts with hydrogel and synthetic membrane materials. The library consists of 24- to 96-well sized inserts as whole plates, strips, and singlets. The density of cultures (the number of wells per plate) and the number of layers was decided by the wall thickness, the capillary forces between the layers and the ability to support fluid operations. The highest density for a two-layer culture was 48-well plate format because the corresponding 96-well format could not support fluidic operations. The bottom apertures were functionalized with hydrogels using a new high-throughput dip-casting technique. This yielded well-defined hydrogel membranes in the apertures with a thickness of about 500 µm and a %CV (coefficient of variance) of < 10%. Consistent intestine barrier was formed on the gelatin over 3-weeks period. Furthermore, mouse intestinal organoid development was compared on hydrogel and synthetic filters glued to the bottom of the 3D-printed inserts. Condensation was most pronounced in inserts with filters followed by the gelatin membrane and the control, which were organoids cultured at the bottom of a titer plate well. This showed that the bottom of an insert should be chosen based on the application. All the inserts were fabricated using an easy-to-use stereolithography (SLA) printer commonly used for dentistry and surgical applications. Therefore, on demand printing of the customized inserts is realistic in many laboratory settings.

Photolithographic Patterning of FluorAcryl for Biphilic Microwell-Based Digital Bioassays and Selection of Bacteria.

FluorAcryl 3298 (FA) is a UV-curable fluoroacrylate polymer commonly employed as a chemically resistant, hydrophobic, and oleophobic coating. Here, FA was used in a cleanroom-based microstructuring process to fabricate hydrophilic-in-hydrophobic (HiH) micropatterned surfaces containing femtoliter-sized well arrays. A short protocol involving direct UV photopatterning, an etching step, and final recovery of the hydrophobic properties of the polymer produced patterned substrates with micrometer resolution. Specifically, HiH microwell arrays were obtained with a well diameter of 10 µm and various well depths ranging from 300 nm to 1 µm with high reproducibility. The 300 nm deep microdroplet array (MDA) substrates were used for digital immunoassays, which presented a limit of detection in the attomolar range. This demonstrated the chemical functionality of the hydrophilic and hydrophobic surfaces. Furthermore, the 1 µm deep wells could efficiently capture particles such as bacteria, whereas the 300 nm deep substrates or other types of flat HiH molecular monolayers could not. Capturing a mixture of bacteria expressing red- and green-fluorescent proteins, respectively, served as a model for screening and selection of specific phenotypes using FA-MDAs. Here, green-fluorescent bacteria were specifically selected by overlaying a solution of gelatin methacryloyl (GelMA) mixed with a photoinitiator and using a high-magnification objective, together with custom pinholes, in a common fluorescence microscope to cross-link the hydrogel around the bacteria of interest. In conclusion, due to the straightforward processing, versatility, and low-price, FA is an advantageous alternative to more commonly used fluorinated materials, such as CYTOP or Teflon-AF, for the fabrication of HiH microwell arrays and other biphilic microstructures.