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.

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.

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.

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.

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.

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.

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.

Peptide Inhibitors of the α-Cobratoxin-Nicotinic Acetylcholine Receptor Interaction.

Venomous snakebites cause >100 000 deaths every year, in many cases via potent depression of human neuromuscular signaling by snake α-neurotoxins. Emergency therapy still relies on antibody-based antivenom, hampered by poor access, frequent adverse reactions, and cumbersome production/purification. Combining high-throughput discovery and subsequent structure-function characterization, we present simple peptides that bind α-cobratoxin (α-Cbtx) and prevent its inhibition of nicotinic acetylcholine receptors (nAChRs) as a lead for the development of alternative antivenoms. Candidate peptides were identified by phage display and deep sequencing, and hits were characterized by electrophysiological recordings, leading to an 8-mer peptide that prevented α-Cbtx inhibition of nAChRs. We also solved the peptide:α-Cbtx cocrystal structure, revealing that the peptide, although of unique primary sequence, binds to α-Cbtx by mimicking structural features of the nAChR binding pocket. This demonstrates the potential of small peptides to neutralize lethal snake toxins in vitro, establishing a potential route to simple, synthetic, low-cost antivenoms.

Large-scale spontaneous self-organization and maturation of skeletal muscle tissues on ultra-compliant gelatin hydrogel substrates.

Cellular self-organization is the fundamental driving force behind the complex architectures of native tissue. Yet, attempts at replicating native tissue architectures in vitro often involve complex micro-fabrication methods and materials. While impressive progress has been made within engineered models of striated muscle, the wide adaptation of these models is held back by the need for specific tools and knowhow. In this report, we show that C2C12 myoblasts spontaneously organize into highly aligned myotube tissues on the mm to cm scale, when cultured on sufficiently soft yet fully isotropic gelatin hydrogel substrates. Interestingly, we only observed this phenomenon for hydrogels with Young's modulus of 6 kPa and below. For slightly more rigid compositions, only local micrometer-scale myotube organization was observed, similar to that seen in conventional polystyrene dishes. The hydrogel-supported myotubes could be cultured for multiple weeks and matured into highly contractile phenotypes with notable upregulation of myosin heavy chain, as compared to myotubes developed in conventional petri dishes. The procedure for casting the ultra-soft gelatin hydrogels is straight forward and compatible with standardized laboratory tools. It may thus serve as a simple, yet versatile, approach to generating skeletal muscle tissue of improved physiological relevance for applied and basic research.

Homogeneous circle-to-circle amplification for real-time optomagnetic detection of SARS-CoV-2 RdRp coding sequence.

Circle-to-circle amplification (C2CA) is a specific and precise cascade nucleic acid amplification method consisting of more than one round of padlock probe ligation and rolling circle amplification (RCA). Although C2CA provides a high amplification efficiency with a negligible increase of false-positive risk, it contains several step-by-step operation processes. We herein demonstrate a homogeneous and isothermal nucleic acid quantification strategy based on C2CA and optomagnetic analysis of magnetic nanoparticle (MNP) assembly. The proposed homogeneous circle-to-circle amplification eliminates the need for additional monomerization and ligation steps after the first round of RCA, and combines two amplification rounds in a one-pot reaction. The second round of RCA produces amplicon coils that anneal to detection probes grafted onto MNPs, resulting in MNP assembly that can be detected in real-time using an optomagnetic sensor. The proposed methodology was applied for the detection of a synthetic complementary DNA of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2, also known as 2019-nCoV) RdRp (RNA-dependent RNA polymerase) coding sequence, achieving a detection limit of 0.4 fM with a dynamic detection range of 3 orders of magnitude and a total assay time of ca. 100 min. A mathematical model was set up and validated to predict the assay performance. Moreover, the proposed method was specific to distinguish SARS-CoV and SARS-CoV-2 sequences with high similarity.

3D Printed Stackable Titer Plate Inserts Supporting Three Interconnected Tissue Models for Drug Transport Studies.

Current in vitro drug screening methods often rely on single-cell models and are therefore imprecise in predicting drug absorption, distribution, metabolism, excretion, and toxicity. This study presents a method to fabricate 3D printed inserts that are compatible with commercially available titer plates. Hydrogels can be casted into the inserts and cells can be cultured either in or on the hydrogels. Once individual cell cultures are fully differentiated, the three different cell cultures are stacked on top of each other for biological experiments. To show the possibilities of this approach, three tissue models representing the first pass metabolism is used. The three tissue models are based on gelatin hydrogels and Caco-2, HUVEC, and HepG2 cells to simulate the small intestine, vascular endothelium, and liver, respectively. The device is simple to fabricate, user friendly, and an alternative to microfluidic-based organ on a chip systems. The presented first pass metabolism study allows for gaining information on drug absorption, distribution, metabolism, and, in the future, excretion in one compact device complying the micro titer plate format.

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.

The FAST Pump, a low-cost, easy to fabricate, SLA-3D-printed peristaltic pump for multi-channel systems in any lab.

With the increasing interest in high throughput screening and parallel assays, laboratories around the world inevitably find themselves in need of driving a multitude of fluid lines to facilitate their large scale studies. The comparatively low cost and no-fluid-contact design of peristaltic pumps make them the go-to systems for such ventures, but using commercially available pumping systems this still becomes a costly endeavor at typically $250-$1000 per pump line. Here we have developed an alternative, a peristaltic pump that can be fabricated in most research laboratories using 3D-printing and readily available off-the-shelf parts. The pump features 8 parallel channels with linear ranges spanning from 0.7 µL/min to 6 mL/min. The pump can be fabricated and assembled by anyone with access to a 3D-printer at a cost of less than $45 per channel and is driven by a stepper motor that connects directly to any computer. This device has the potential to be disruptive in areas such as drug screening and assay development, as well as lab-on-a-chip applications and cell cultivation, where it significantly reduces hardware expenses and allows for construction of more comprehensive fluidic systems at a fraction of current costs.

Microcontainers for oral insulin delivery - In vitro studies of permeation enhancement.

Oral delivery of peptides is challenging due to their low uptake through the small intestinal epithelium. Tight junctions, connecting the enterocytes, impede permeability, often necessitating the use of permeation enhancers in the formulation. Loading of peptide and permeation enhancer into micro-scale devices, such as microcontainers, can potentially confine the effective absorptive area through unidirectional release and thereby enhance absorption. This concept is investigated by in vitro permeation studies of insulin across Caco-2 cell and Caco-2/HT29-MTX-E12 co-culture monolayers mimicking the intestinal absorption barrier. The importance of proximity between the microcontainers and the barrier is assessed, by keeping the amounts of insulin and sodium caprate fixed throughout all experiments, while collectively orienting the unidirectional release towards the cell monolayers. Increasing the distance is observed to have a negative effect on insulin permeation matching a one-phase exponential decay function, while no difference in insulin transport is observed between Caco-2 and co-culture monolayers. Although there are no signs of cytotoxicity caused by the microcontainer material, reversible cell deterioration, as a consequence of high local concentrations of sodium caprate, becomes evident upon qualitative assessment of the cell monolayers. These results both suggest a potential of increasing oral bioavailability of peptides by the use of microcontainers, while simultaneously visualising the ability of regaining monolayer integrity upon local permeation enhancer induced toxicity.

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.

Controlling fluid flow to improve cell seeding uniformity.

Standard methods for seeding monolayer cell cultures in a multiwell plate or dish do not uniformly distribute cells on the surface. With traditional methods, users find aggregation around the circumference, in the centre, or a combination of the two. This variation is introduced due to the macro scale flow of the cell seeding suspension, and movement of the dish before cells can settle and attach to the surface. Reproducibility between labs, users, and experiments is hampered by this variability in cell seeding. We present a simple method for uniform and user-independent the cell seeding using an easily produced uniform cell seeder (UCS) device. This allows precise control of cell density in a reproducible manner. By containing the cell seeding suspension in a defined volume above the culture surface with the UCS, fluctuations in cell density are minimised. Seeding accuracy, as defined by the actual cell density versus the target seeding density is improved dramatically across users with various levels of expertise. We go on to demonstrate the impact of local variation in cell density on the lineage commitment of human embryonic stem cells (hESCs) towards pancreatic endoderm (PE). Variations in the differentiation profile of cells across a culture well closely mirror variations in cell density introduced by seeding method-with the UCS correcting variations in differentiation efficiency. The UCS device provides a simple and reproducible method for uniform seeding across multiple culture systems.

Droplet-based microfluidics as a future tool for strain improvement in lactic acid bacteria.

Strain development is frequently used to improve the performance and functionality of industrially important microbes. As traditional mutagenesis screen is especially utilized by the food industry to improve strains used in food fermentation, high-throughput and cost-effective screening tools are important in mutant selection. The emerging droplet-based microfluidics technology miniaturizes the volume for cell cultivation and phenotype interrogation down to the picoliter scales, which facilitates screening of microbes for improved phenotypical properties tremendously. In this mini review, we present recent application of the droplet-based microfluidics in microbial strain improvement with a focus on its potential use in the screening of lactic acid bacteria.

Micro-droplet arrays for micro-compartmentalization using an air/water interface.

Here we present a water-in-air droplet platform for micro-compartmentalization for single molecule guided synthesis and analysis consisting of a flow-system hosting dense arrays of aqueous microdroplets on a glass surface surrounded by air. The droplets are formed in a few seconds by passing a waterfront over the array of hydrophilic spots surrounded by a hydrophobic coating, thus forming a micro-droplet array (MDA). The droplet volumes are tunable from approximately 50 femtoliter to 20 picoliter by adjusting the size of the hydrophilic spots. MDAs consisting of femtoliter volume droplets were stable for more than 24 hours in air at 37 °C in a reversibly sealed flow-system, thus allowing us to perform assays that require long incubations in the droplets. Using differently fluorescing liquids, it was further shown that droplets can be reformed on the same MDA several times by passing a new liquid plug over the surface, and that fluorescence from one reaction can be washed away with little to no carry-over, hence allowing for multistep reactions to be carried out on the system. The MDA created by an air/water interface supported digital immunoassays as was demonstrated by measuring the Aß42 peptide in cerebrospinal fluid of Alzheimers patients and control patients. To demonstrate a two step droplet assay, first, histidine tagged peptides were expressed in the droplets and bound to the droplet-enclosed surface. Subsequently, the his-tagged peptides were detected using enzyme-conjugated antibodies in a second droplet generation step. As such, the chip demonstrates features necessary for library preparations for high throughput screening applications.