Multi-well plate lid for single-step pooling of 96 samples for high-throughput barcode-based sequencing.

High-throughput transcriptomics is of increasing fundamental biological and clinical interest. The generation of molecular data from large collections of samples, such as biobanks and drug libraries, is boosting the development of new biomarkers and treatments. Focusing on gene expression, the transcriptomic market exploits the benefits of next-generation sequencing (NGS), leveraging RNA sequencing (RNA-seq) as standard for measuring genome-wide gene expression in biological samples. The cumbersome sample preparation, including RNA extraction, conversion to cDNA and amplification, prevents high-throughput translation of RNA-seq technologies. Bulk RNA barcoding and sequencing (BRB-seq) addresses this limitation by enabling sample preparation in multi-well plate format. Sample multiplexing combined with early pooling into a single tube reduces reagents consumption and manual steps. Enabling simultaneous pooling of all samples from the multi-well plate into one tube, our technology relies on smart labware: a pooling lid comprising fluidic features and small pins to transport the liquid, adapted to standard 96-well plates. Operated with standard fluidic tubes and pump, the system enables over 90% recovery of liquid in a single step in less than a minute. Large scale manufacturing of the lid is demonstrated with the transition from a milled polycarbonate/steel prototype into an injection molded polystyrene lid. The pooling lid demonstrated its value in supporting high-throughput barcode-based sequencing by pooling 96 different DNA barcodes directly from a standard 96-well plate, followed by processing within the single sample pool. This new pooling technology shows great potential to address medium throughput needs in the BRB-seq workflow, thereby addressing the challenge of large-scale and cost-efficient sample preparation for RNA-seq.

Toward a Physiologically Relevant 3D Helicoidal-Oriented Cardiac Model: Simultaneous Application of Mechanical Stimulation and Surface Topography.

Myocardium consists of cardiac cells that interact with their environment through physical, biochemical, and electrical stimulations. The physiology, function, and metabolism of cardiac tissue are affected by this dynamic structure. Within the myocardium, cardiomyocytes' orientations are parallel, creating a dominant orientation. Additionally, local alignments of fibers, along with a helical organization, become evident at the macroscopic level. For the successful development of a reliable in vitro cardiac model, evaluation of cardiac cells' behavior in a dynamic microenvironment, as well as their spatial architecture, is mandatory. In this study, we hypothesize that complex interactions between long-term contraction boundary conditions and cyclic mechanical stimulation may provide a physiological mechanism to generate off-axis alignments in the preferred mechanical stretch direction. This off-axis alignment can be engineered in vitro and, most importantly, mirrors the helical arrangements observed in vivo. For this purpose, uniaxial mechanical stretching of dECM-fibrin hydrogels was performed on pre-aligned 3D cultures of cardiac cells. In view of the potential development of helical structures similar to those in native hearts, the possibility of generating oblique alignments ranging between 0° and 90° was explored. Indeed, our investigations of cell alignment in 3D, employing both mechanical stimulation and groove constraint, provide a reliable mechanism for the generation of helicoidal structures in the myocardium. By combining cyclic stretch and geometric alignment in grooves, an intermediate angle toward favored direction can be achieved experimentally: while cyclic stretch produces a perpendicular orientation, geometric alignment is associated with a parallel one. In our 2D and 3D culture conditions, nonlinear cellular addition of the strains and strain avoidance concept reliably predicted the preferred cellular alignment. The 3D dECM-fibrin model system in this study shows that cyclical stretching supports cell survival and development. Using mechanical stimulation of pre-aligned heart cells, maturation markers are augmented in neonatal cardiomyocytes, while the beating culture period is prolonged, indicating an improved model function. We propose a simplified theoretical model based on numerical simulation and nonlinear strain avoidance by cells to explain oblique alignment angles. Thus, this work lays a possible rational basis for understanding and engineering oblique cellular alignments, such as the helicoidal layout of the heart, using approaches that simultaneously enhance maturation and function.

A Three-Dimensional Engineered Cardiac In Vitro Model: Controlled Alignment of Cardiomyocytes in 3D Microphysiological Systems.

Cardiomyocyte alignment in myocardium tissue plays a significant role in the physiological, electrical, and mechanical functions of the myocardium. It remains, however, difficult to align cardiac cells in a 3D in vitro heart model. This paper proposes a simple method to align cells using microfabricated Polydimethylsiloxane (PDMS) grooves with large dimensions (of up to 350 µm in width), similar to the dimensions of trabeculae carneae, the smallest functional unit of the myocardium. Two cell groups were used in this work; first, H9c2 cells in combination with Nor10 cells for proof of concept, and second, neonatal cardiac cells to investigate the functionality of the 3D model. This model compared the patterned and nonpatterned 3D constructs, as well as the 2D cell cultures, with and without patterns. In addition to alignment, we assessed the functionality of our proposed 3D model by comparing beating rates between aligned and non-aligned structures. In order to assess the practicality of the model, the 3D aligned structures should be demonstrated to be detachable and alignable. This evaluation is crucial to the use of this 3D functional model in future studies related to drug screening, building blocks for tissue engineering, and as a heart-on-chip by integrating microfluidics.

Tools for manipulation and positioning of microtissues.

Complex three-dimensional (3D) in vitro models are emerging as a key technology to support research areas in personalised medicine, such as drug development and regenerative medicine. Tools for manipulation and positioning of microtissues play a crucial role in the microtissue life cycle from production to end-point analysis. The ability to precisely locate microtissues can improve the efficiency and reliability of processes and investigations by reducing experimental time and by providing more controlled parameters. To achieve this goal, standardisation of the techniques is of primary importance. Compared to microtissue production, the field of microtissue manipulation and positioning is still in its infancy but is gaining increasing attention in the last few years. Techniques to position microtissues have been classified into four main categories: hydrodynamic techniques, bioprinting, substrate modification, and non-contact active forces. In this paper, we provide a comprehensive review of the different tools for the manipulation and positioning of microtissues that have been reported to date. The working mechanism of each technique is described, and its merits and limitations are discussed. We conclude by evaluating the potential of the different approaches to support progress in personalised medicine.

Coplanar embedding of multiple 3D cell models in hydrogel towards high-throughput micro-histology.

Standardised and high-throughput methods have been developed for the production and experimental handling of some 3D in vitro models. However, adapted analytical tools are still missing for scientists and researchers to fully exploit the potential of complex cellular models in pre-clinical drug testing and precision medicine. Histology is the established, cost-effective and gold standard method for structural and functional tissue analysis. However, standard histological processes are challenging and costly to apply to 3D cell models, as their small size often leads to poor alignment of samples, which lowers analysis throughput. This body of work proposes a new approach: HistoBrick facilitates histological processing of spheroids and organoids by enabling gel embedding of 3D cell models with precise coplanar alignment, parallel to the sectioning plane, thus minimising the loss of sample material. HistoBrick's features are compatible with automation standards, potentially allowing automated sample transfer from a multi-well plate to the gel device. Moreover, HistoBrick's technology was validated by demonstrating the alignment of HepG2 cultured spheroids measuring 150-200 µm in diameter with a height precision of ± 80 µm. HistoBrick allows up to 96 samples to be studied across minimal sections, paving the way towards high-throughput micro-histology.

Effects of ionic strength and surface charge on protein adsorption at PEGylated surfaces.

PEGylated Nb2O5 surfaces were obtained by the adsorption of poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) copolymers, allowing control of the PEG surface density, as well as the surface charge. PEG (MW 2 kDa) surface densities between 0 and 0.5 nm(-2) were obtained by changing the PEG to lysine-mer ratio in the PLL-g-PEG polymer, resulting in net positive, negative and neutral surfaces. Colloid probe atomic force microscopy (AFM) was used to characterize the interfacial forces associated with the different surfaces. The AFM force analysis revealed interplay between electrical double layer and steric interactions, thus providing information on the surface charge and on the PEG layer thickness as a function of copolymer architecture. Adsorption of the model proteins lysozyme, alpha-lactalbumin, and myoglobin onto the various PEGylated surfaces was performed to investigate the effect of protein charge. In addition, adsorption experiments were performed over a range of ionic strengths, to study the role of electrostatic forces between surface charges and proteins acting through the PEG layer. The adsorbed mass of protein, measured by optical waveguide lightmode spectroscopy (OWLS), was shown to depend on a combination of surface charge, protein charge, PEG thickness, and grafting density. At high grafting density and high ionic strength, the steric barrier properties of PEG determine the net interfacial force. At low ionic strength, however, the electrical double layer thickness exceeds the thickness of the PEG layer, and surface charges "shining through" the PEG layer contribute to protein interactions with PLL-g-PEG coated surfaces. The combination of AFM surface force measurements and protein adsorption experiments provides insights into the interfacial forces associated with various PEGylated surfaces and the mechanisms of protein resistance.

Biosensors based on porous cellulose nanocrystal-poly(vinyl alcohol) scaffolds.

Cellulose nanocrystals (CNCs), which offer a high aspect ratio, large specific surface area, and large number of reactive surface groups, are well suited for the facile immobilization of high density biological probes. We here report functional high surface area scaffolds based on cellulose nanocrystals (CNCs) and poly(vinyl alcohol) (PVA) and demonstrate that this platform is useful for fluorescence-based sensing schemes. Porous CNC/PVA nanocomposite films with a thickness of 25-70 nm were deposited on glass substrates by dip-coating with an aqueous mixture of the CNCs and PVA, and the porous nanostructure was fixated by heat treatment. In a subsequent step, a portion of the scaffold's hydroxyl surface groups was reacted with 2-(acryloxy)ethyl (3-isocyanato-4-methylphenyl)carbamate to permit the immobilization of thiolated fluorescein-substituted lysine, which was used as a first sensing motif, via nucleophile-based thiol-ene Michael addition. The resulting sensor films exhibit a nearly instantaneous and pronounced change of their fluorescence emission intensity in response to changes in pH. The approach was further extended to the detection of protease activity by immobilizing a Förster-type resonance energy transfer chromophore pair via a labile peptide sequence to the scaffold. This sensing scheme is based on the degradation of the protein linker in the presence of appropriate enzymes, which separate the chromophores and causes a turn-on of the originally quenched fluorescence. Using a standard benchtop spectrometer to monitor the increase in fluorescence intensity, trypsin was detected at a concentration of 250 µg/mL, i.e., in a concentration that is typical for abnormal proteolytic activity in wound fluids.

Integrated optical biosensor for in-line monitoring of cell cultures.

An analytical detection platform was developed to evaluate the induced toxicity in cell cultures exposed to foreign agents like growth factors or nanoparticles. Connecting a biosensing detection device to the cell culture flasks allows analyzing the composition of cell medium in real-time. The analysis relies on the quantification of inflammatory cytokines released by cells into the cell culture medium, by means of solid-phase immunoassays analyzed with the wavelength interrogated optical sensing (WIOS) instrument. A fluidic system for in situ measurements allows detecting cytokines in real-time, with a sensitivity of 1-100 ng/mL depending on the cytokine. In addition, integration of an in-line optical absorbance measurement unit, in combination with the standard AB cell proliferation assay, provides information on the cell viability in the culture. Fluidic connections between the cell culture flasks, the optical biosensor and the absorbance measurement unit simultaneously allow quantifying up to three cytokines (interleukin 8, interleukin 6 and the monocyte chemotactic protein), assessing cellular proliferation, and thus discriminating between naïve cells and cells exposed to foreign agents such as growth factors (tumor necrosis factor alpha) or nanoparticles. This analytical tool presents a high potential for assessing the cytotoxicity of nanoparticles and other chemicals in vitro.

Waveguide interrogated optical immunosensor (WIOS) for detection of sulfonamide antibiotics in milk.

An immunosensor was developed for the detection of sulfonamide antibiotics in milk. Detection relied on a competitive immunoassay format, using immunoreagents previously developed for the generic detection of sulfonamide antibiotics and evaluated by enzyme-linked immunosorbent assay. The immunoassay was implemented onto a microsystem platform, the wavelength interrogated optical sensing device, which uses the evanescent field to probe changes at the interface of a waveguiding layer, and thus allows sensitive detection of biomolecule adsorption. The immunoreagents were immobilized onto the surface of the waveguide chip, and a fluidic cell allowed flowing analyte and detection solutions above the surface. Sulfapyridine was used as reference sulfonamide and detected with the immunosensor in buffer and in milk with a limit of detection (IC(90)) of 0.2+/-0.1 microg L(-1) and 0.5+/-0.1 microg L(-1), respectively. The analysis time was below 30 min, including regeneration of the sensing surface, with minimum sample preparation required. The reproducibility of the detection was better than 10%. A blind assay allowed identifying milk samples that were contaminated with different sulfonamide antibiotics at or above the maximum residue limits established by the European Union for these compounds (100 microg L(-1)). Thus, the developed immunosensor presents great potential as a generic sensing device for the fast and early detection of food contaminants on the field by non-skilled users.

High salt stability and protein resistance of poly(L-lysine)-g-poly(ethylene glycol) copolymers covalently immobilized via aldehyde plasma polymer interlayers on inorganic and polymeric substrates.

The electrostatic adsorption onto charged surfaces of comb copolymers comprising a polyelectrolyte backbone and pendent PEG side chains, such as poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), has in previous studies provided protein-repellent thin coatings, particularly on metal oxide surfaces. A drawback of this approach is, however, the instability of such adsorbed layers under extreme pH values or high ionic strength. We have overcome this limitation in the present study by covalently immobilizing PLL-g-PEG copolymers onto aldehyde plasma-modified substrates. Silicon wafers, optical waveguide chips, and perfluorinated ethylene-co-propylene (FEP) polymer substrates were first coated with a thin plasma polymer layer using a propionaldehyde plasma, followed by covalent immobilization of PLL-g-PEG via reductive amination between amine groups of the PLL backbone with aldehyde groups on the plasma-deposited interlayer. The stability in high salt media and the protein resistance of different molecular architectures of immobilized PLL-g-PEG layers were quantitatively investigated by XPS, an optical waveguide technique (OWLS), and ToF-SIMS. The adsorption of bovine serum albumin was found to be below the detection limit (<2 ng/cm(2)), as for electrostatically adsorbed PLL-g-PEG layers. However, after 24 h of exposure of covalently immobilized layers of PLL-g-PEG to high ionic strength buffer (2400 mM NaCl), no significant change in the protein resistance was observed, whereas under the same conditions electrostatically adsorbed PLL-g-PEG coatings lost their protein resistance. Moreover, covalent immobilization via an aldehyde plasma interlayer enabled the application of PLL-g-PEG layers onto substrates such as FEP onto which electrostatic binding is not possible. These findings create a generic platform for the covalent immobilization of PLL-g-PEG onto a wide variety of substrates.

Influence of PEG architecture on protein adsorption and conformation.

Poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) copolymers with various grafting ratios were adsorbed to niobium pentoxide-coated silicon wafers and characterized before and after protein adsorption using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Three proteins of different sizes, myoglobin (16 kD), albumin (67 kD), and fibrinogen (340 kD), were studied. XPS was used to quantify the amount of protein adsorbed to the bare and PEGylated surfaces. ToF-SIMS and principal component analysis (PCA) were used to study protein conformational changes on these surfaces. The smallest protein, myoglobin, generally adsorbed in higher numbers than the much larger fibrinogen. Protein adsorption was lowest on the surfaces with the highest PEG chain surface density and increased as the PEG layer density decreased. The highest adsorption was found on lysine-coated and bare niobium surfaces. ToF-SIMS and PCA data evaluation provided further information on the degree of protein denaturation, which, for a particular protein, were found to decrease with increasing PEG surface density and increase with decreasing protein size.

Relationship between interfacial forces measured by colloid-probe atomic force microscopy and protein resistance of poly(ethylene glycol)-grafted poly(L-lysine) adlayers on niobia surfaces.

Adsorbed layers of "comb-type" copolymers consisting of PEG chains grafted onto a poly(l-lysine) (PLL) backbone on niobium oxide substrates were studied by colloid-probe AFM in order to characterize the interfacial forces associated with coatings of varying architectures (PEG/PLL ratios and PEG chain lengths) and their relevance to protein resistance. The steric and electrostatic forces measured varied substantially with the architecture of the PLL-g-PEG copolymers. Varying the ionic strength of the buffer solutions enabled discrimination between electrostatic and steric-entropic contributions to the net interfacial force. For high PEG grafting densities the steric component was most prominent, but at low ionic strengths and high grafting densities, a repulsive electrostatic surface force was also observed; its origin was assigned to the niobia charges beneath the copolymer, as insufficient protonated amine groups in the PLL backbone were available for compensation of the oxide surface charges. For lower grafting densities and lower ionic strengths there was a substantial attractive electrostatic contribution arising from interaction of the electrical double layer arising from the protonated amine groups, with that of the silica probe surface (as under low ionic strength conditions, the electrical double layer was thicker than the PEG layer). For these PLL-g-PEG coatings the net interfacial force can thus be a markedly varying superposition of electrostatic and steric-entropic contributions, depending on various factors. The force curves correlate with protein adsorption data, demonstrating the utility of AFM colloid-probe force measurements for quantitative analysis of surface forces and how they determine interfacial interactions with proteins. Such characterization of the net interfacial forces is essential to elucidate the multiple types of interfacial forces relevant to the interactions between PLL-g-PEG coatings and proteins and to advance interpretation of protein adsorption or repellence beyond the oversimplified steric barrier model; in particular, our data demonstrate the importance of an ionic-strength-dependent minimum PEG layer thickness to screen the electrostatic interactions of charged interfaces.

Tuned graft copolymers as controlled coatings for DNA microarrays.

DNA microarrays have become a powerful tool for expression profiling and other genomics applications. A critical factor for their sensitivity is the interfacial coating between the chip substrate and the bound DNA. Such a coating has to embrace the divergent requirements of tightly binding the capture probe DNA during the spotting process and of minimizing the nonspecific binding of target DNA during the hybridization assay. To fulfill these conditions, most coatings require a passivation step. Here we demonstrate how the chain density of a graft copolymer with a polycationic backbone, poly(l-lysine)-graft-poly(ethylene glycol), can be tuned such that the binding capacity during capture probe deposition is maximized while the nonspecific binding during hybridization assays is kept to a minimum, thus alleviating the requirement for a separate passivation procedure. Evidence for the superior performance of such coatings in terms of signal-to-noise ratio and spot quality is presented using an evanescent field-based fluorescent sensing technique (the ZeptoREADER). The surface architecture is further characterized using optical waveguide lightmode spectroscopy and time-of-flight secondary ion mass spectrometry. Finally, in a model assay, we demonstrate that expression changes can be detected from 1 microg of total mRNA sample material with a limit of detectable differential expression of +/-1.5.

Reduction of friction at oxide interfaces upon polymer adsorption from aqueous solutions.

Reduction of the interfacial friction for the contact of a silicon oxide surface with sodium borosilicate in aqueous solutions has been accomplished through the adsorption of poly(L-lysine)-graft-poly(ethylene glycol) on one or both surfaces. Spontaneous polymer adsorption has been achieved via the electrostatic attraction of the cationic polylysine polymer backbone and a net negative surface charge, present for a specific range of solution pH values. Interfacial friction has been measured in aqueous solution, in the absence of wear, and on a microscopic scale with atomic force microscopy. The successful investigation of the polymer-coated interfaces has been aided by the use of sodium borosilicate microspheres (5.1 microm diameter) as the contacting probe tip. Measurements of interfacial friction as a function of applied load reveal a significant reduction in friction upon the adsorption of the polymer, as well as sensitivity to the coated nature of the interface (single-sided versus two-sided) and the composition of the adsorbed polymer. These measurements demonstrate the fundamental opportunity for lubrication in aqueous environments through the selective adsorption of polymer coatings.