Localized apelin-17 analogue-bicelle interactions as a facilitator of membrane-catalyzed receptor recognition and binding.

The apelinergic system encompasses two peptide ligand families, apelin and apela, along with the apelin receptor (AR or APJ), a class A G-protein-coupled receptor. This system has diverse physiological effects, including modulating heart contraction, vasodilation/constriction, glucose regulation, and vascular development, with involvement in a variety of pathological conditions. Apelin peptides have been previously shown to interact with and become structured upon binding to anionic micelles, consistent with a membrane-catalyzed mechanism of ligand-receptor binding. To overcome the challenges of observing nuclear magnetic resonance (NMR) spectroscopy signals of a dilute peptide in biological environments, 19F NMR spectroscopy, including diffusion ordered spectroscopy (DOSY) and saturation transfer difference (STD) experiments, was used herein to explore the membrane-interactive behaviour of apelin. NMR-optimized apelin-17 analogues with 4-trifluoromethyl-phenylalanine at various positions were designed and tested for bioactivity through ERK activation in stably-AR transfected HEK 293 T cells. Far-UV circular dichroism (CD) spectropolarimetry and 19F NMR spectroscopy were used to compare the membrane interactions of these analogues with unlabelled apelin-17 in both zwitterionic/neutral and net-negative bicelle conditions. Each analogue binds to bicelles with relatively weak affinity (i.e., in fast exchange on the NMR timescale), with preferential interactions observed at the cationic residue-rich N-terminal and mid-length regions of the peptide leaving the C-terminal end unencumbered for receptor recognition, enabling a membrane-anchored fly-casting mechanism of peptide search for the receptor. In all, this study provides further insight into the membrane-interactive behaviour of an important bioactive peptide, demonstrating interactions and biophysical behaviour that cannot be neglected in therapeutic design.

Cell-contact-mediated assembly of contractile airway smooth muscle rings.

Microtissues in the shape of toroidal rings provide an ideal geometry to better represent the structure and function of the airway smooth muscle present in the small airways, and to better understand diseases such as asthma. Here, polydimethylsiloxane devices consisting of a series of circular channels surrounding central mandrels are used to form microtissues in the shape of toroidal rings by way of the self-aggregation and -assembly of airway smooth muscle cell (ASMC) suspensions. Over time, the ASMCs present in the rings become spindle-shaped and axially align along the ring circumference. Ring strength and elastic modulus increase over 14 d in culture, without significant changes in ring size. Gene expression analysis indicates stable expression of mRNA for extracellular matrix-associated proteins, including collagen I and lamininsα1 andα4 over 21 d in culture. Cells within the rings respond to TGF-ß1 treatment, leading to dramatic decreases in ring circumference, with increases in mRNA and protein levels for extracellular matrix and contraction-associated markers. These data demonstrate the utility of ASMC rings as a platform for modeling diseases of the small airways such as asthma.

Loading and Release of Quercetin from Contact-Drawn Polyvinyl Alcohol Fiber Scaffolds.

Polymeric drug releasing systems have numerous applications for the treatment of chronic diseases and traumatic injuries. In this study, a simple, cost-effective, and scalable method for dry spinning of crosslinked polyvinyl alcohol (PVA) fibers is presented. This method utilizes an entangled solution of PVA to form liquid bridges that are drawn into rapidly drying fibers through extensional flow. The fibers are crosslinked by a one-pot reaction in which glyoxal is introduced to the PVA solution prior to contact drawing. Failure analysis of fiber formation is used to understand the interplay of polymer concentration, glyoxal concentration, and crosslinking time to identify appropriate formulations for the production of glyoxal-crosslinked PVA fibers. The small molecule quercetin (an anti-inflammatory plant flavonoid) can be added to the one-pot reaction and is shown to be incorporated into the fibers in a concentration-dependent manner. Upon rehydration in an aqueous medium, the glyoxal-crosslinked PVA fiber scaffolds retain their morphology and slowly degrade, as measured over the course of 10 days. As the scaffolds degrade, they release the loaded quercetin, reaching a cumulative release of 56 ± 6% of the loaded drug after 10 days. The bioactivity of the released quercetin is verified by combining quercetin-loaded fibers with contact-drawn polyethylene oxide-type I collagen (PEO-Col) fibers and monitoring the growth of PC12 cells on the fibers. PC12 cells readily attach to the PEO-Col fibers and display increased nerve growth factor-induced elongation and neurite formation in the presence of quercetin-loaded PVA fibers relative to substrates formed from only PEO-Col fibers or PEO-Col and PVA fibers without quercetin.

Multi-pin contact drawing enables production of anisotropic collagen fiber substrates for alignment of fibroblasts and monocytes.

Type I collagen is the most abundant protein in the human body and is known to play important roles in numerous biological processes including tissue morphogenesis and wound healing. As such, it is one of the most frequently used substrates for cell culture, and there have been considerable efforts to develop collagen-based cell culture substrates that mimic the structural organization of collagen as it is found in native tissues, i.e., collagen fibers. However, producing collagen fibers from extracted collagen has been notoriously difficult, with existing methods providing only low throughput production of collagen fibers. In this study, we prepared collagen fibers using a highly efficient, bio-friendly, and cost-effective approach termed contact drawing, which uses an entangled polymer fluid to aid in fiber formation. Contact drawing technology has been demonstrated previously for collagen using highly concentrated dextran solutions with low concentrations of collagen. Here, we show that by replacing dextran with polyethylene oxide (PEO), high collagen content fibers may be readily formed from mixtures of soluble collagen and PEO, a polymer that readily forms fibers by contact drawing at concentrations as low as 0.5%wt. The presence of collagen and the formation of well-ordered collagen structures in the resulting fibers were characterized by attenuated total reflectance Fourier-transform infrared spectromicroscopy, Raman spectromicroscopy, and fluorescence microscopy. Corresponding to well-ordered collagen, the mechanical properties of the PEO-collagen fibers approximated those observed for native collagen fibers. Growth of cells on aligned PEO-collagen fibers attached to a polydimethyl siloxane support was examined for human dermal fibroblast (WS1) and human peripheral leukemia blood monocyte (THP-1) cell lines. WS1 and THP-1 cells readily attached, displayed alignment through migration and spreading, and proliferated on the collagen fiber substrate over the course of several days. We also demonstrated the retrieval of viable cells from the PEO-collagen fiber substrates through enzymatic digestion of the collagen substrate with collagenase IV.

Formation of Core-Sheath Polymer Fibers by Free Surface Spinning of Aqueous Two-Phase Systems.

Core-sheath fibers have numerous applications ranging from composite materials for advanced manufacturing to materials for drug delivery and regenerative medicine. Here, a simple and tunable approach for the generation of core-sheath fibers from immiscible solutions of dextran and polyethylene oxide is described. This approach exploits the entanglement of polymer molecules within the dextran and polyethylene oxide phases for free surface spinning into dry fibers. The mechanism by which these core-sheath fibers are produced after contact with a solid substrate (such as a microneedle) involves complex flows of the phase-separating polymer solutions, giving rise to a liquid-liquid core-sheath flow that is drawn into a liquid bridge. This liquid bridge then elongates into a core-sheath fiber through extensional flow as the contacting substrate is withdrawn. The core-sheath structure of the fibers produced by this approach is confirmed by attenuated total reflection Fourier-transform infrared spectroscopy and confocal microscopy. Tuning of the core diameter is also demonstrated by varying the weight percentage of dextran added to the reservoir from which the fibers are formed.

Predicting the Mixing Behavior of Aqueous Solutions Using a Machine Learning Framework.

The most direct approach to determining if two aqueous solutions will phase-separate upon mixing is to exhaustively screen them in a pair-wise fashion. This is a time-consuming process that involves preparation of numerous stock solutions, precise transfer of highly concentrated and often viscous solutions, exhaustive agitation to ensure thorough mixing, and time-sensitive monitoring to observe the presence of emulsion characteristics indicative of phase separation. Here, we examined the pair-wise mixing behavior of 68 water-soluble compounds by observing the formation of microscopic phase boundaries and droplets of 2278 unique 2-component solutions. A series of machine learning classifiers (artificial neural network, random forest, k-nearest neighbors, and support vector classifier) were then trained on physicochemical property data associated with the 68 compounds and used to predict their miscibility upon mixing. Miscibility predictions were then compared to the experimental observations. The random forest classifier was the most successful classifier of those tested, displaying an average receiver operator characteristic area under the curve of 0.74. The random forest classifier was validated by removing either one or two compounds from the input data, training the classifier on the remaining data and then predicting the miscibility of solutions involving the removed compound(s) using the classifier. The accuracy, specificity, and sensitivity of the random forest classifier were 0.74, 0.80, and 0.51, respectively, when one of the two compounds to be examined was not represented in the training data. When asked to predict the miscibility of two compounds, neither of which were represented in the training data, the accuracy, specificity, and sensitivity values for the random forest classifier were 0.70, 0.82 and 0.29, respectively. Thus, there is potential for this machine learning approach to improve the design of screening experiments to accelerate the discovery of aqueous two-phase systems for numerous scientific and industrial applications.

Polymer entanglement drives formation of fibers from stable liquid bridges of highly viscous dextran solutions.

Liquid bridges have been studied for over 200 years due to their occurrence in many natural and industrial phenomena. Most studies focus on millimeter scale liquid bridges of Newtonian liquids. Here, reptation theory was used to explain the formation of 10 cm long liquid bridges of entangled polymer solutions, which subsequently stabilize into polymer fibers with tunable diameters between 3 and 20 mm. To control the fiber formation process, a horizontal single-fiber contact drawing system was constructed consisting of a motorized stage, a micro-needle, and a liquid filled reservoir. Analyzing the liquid bridge rupture statistics as a function of elongation speed, solution concentration and dextran molecular weight revealed that the fiber formation process was governed by a single timescale attributed to the relaxation of entanglements within the polymer solution. Further characterization revealed that more viscous solutions produced fibers of larger diameters due to secondary flow dynamics. Verification that protein additives such as type I collagen had minimal effect on fiber formation demonstrates the potential application in biomaterial fabrication.

Material properties of disulfide-crosslinked hyaluronic acid hydrogels influence prostate cancer cell growth and metabolism.

Cells reside in vivo within three dimensional environments in which they interact with extracellular matrices (ECMs) that play an integral role in maintaining tissue homeostasis and preventing tumour growth. Thus, tissue culture approaches that more faithfully reproduce these interactions with the ECM are needed to study cancer development and progression. Many materials exist for modeling tissue environments, and the effects of differing mechanical, physical, and biochemical properties of such materials on cell behaviour are often intricately coupled and difficult to tease apart. Here, an optimized protocol was developed to generate low reaction volume disulfide-crosslinked hyaluronic acid (HA) hydrogels for use in cell culture applications to relate the properties of ECM materials to cell signalling and behaviour. Mechanically, HA hydrogels are comparable to other soft hydrogel materials such as Matrigel and agarose or to tissues lacking type I collagen and other fibrillar ECM components. The diffusion of soluble materials in these hydrogels is affected by unique mass transfer properties. Specifically, HA hydrogel concentration affects the diffusion of anionic particles above 500 kDa, whereas diffusion of smaller particles appears unimpeded by HA content, likely reflecting hydrogel pore size. The HA hydrogels have a strong exclusion effect that limits the movement of proteins into and out of the material once fully formed. Such mass transfer properties have interesting implications for cell culture, as they ultimately affect access to nutrients and the distribution of signalling molecules, affecting nutrient sensing and metabolic activity. The use of disulfide-crosslinked HA hydrogels for the culture of the model prostate cancer cell lines PC3 and LNCaP reveals correlations of protein activation linked to metabolic flux, which parallel and can thus potentially provide insights into cell survival mechanisms in response to starvation that occurs in cancer cell microenvironments.

Aqueous two-phase system antibody confinement enables cost-effective analysis of protein analytes by sandwich enzyme-linked immunosorbent assay with minimal optical crosstalk.

An aqueous two-phase system formed from polyethylene glycol and dextran was used to uniformly coat the bottom surfaces of the wells of standard 96-well assay plates with capture and detection antibodies to improve the performance and cost-effectiveness of sandwich enzyme-linked immunosorbent assay (ELISA). Using this approach, limits of detection and linear dynamic range values comparable to those obtained for conventional sandwich ELISA were obtained using considerably lower antibody quantities due to the much lower reagent volumes required when antibodies are applied in a dextran solution beneath a polyethylene glycol overlay. Confinement of the antibody reagents to the bottom surfaces of the wells within the dextran phase also dramatically decreased the optical crosstalk present between neighboring wells when using transparent microplates. Adaptation of the conventional single sandwich ELISA for aqueous two-phase system antibody confinement was demonstrated by analysis of standard curves for C-reactive protein, transforming growth factor beta 1, and the chemokine CXCL10.

SH-SY5Y and LUHMES cells display differential sensitivity to MPP+, tunicamycin, and epoxomicin in 2D and 3D cell culture.

SH-SY5Y and LUHMES cell lines are widely used as model systems for studying neurotoxicity. Most of the existing data regarding the sensitivity of these cell lines to neurotoxicants have been recorded from cells growing as two-dimensional (2D) cultures on the surface of glass or plastic. With the emergence of 3D culture platforms designed to better represent native tissue, there is a growing need to compare the toxicology of neurons grown in 3D environments to those grown in 2D to better understand the impact that culture environment has on toxicant sensitivity. Here, a simple 3D culture method was used to assess the impact of growth environment on the sensitivity of SH-SY5Y cells and LUHMES cells to MPP+, tunicamycin, and epoxomicin, three neurotoxicants that have been previously used to generate experimental models for studying Parkinson's disease pathogenesis. SH-SY5Y cell viability following treatment with these three toxicants was significantly lower in 2D cultures as compared to 3D cultures. On the contrary, LUHMES cells did not show significant differences between growth conditions for any of the toxicants examined. However, LUHMES cells were more sensitive to MPP+, tunicamycin, and epoxomicin than SH-SY5Y cells. Thus, both the choice of cell line and the choice of growth environment must be considered when interpreting in vitro neurotoxicity data.

Corrigendum: Confinement of Suspension-Cultured Cells in Polyethylene Glycol/Polyethylene Oxide-Albumin Aqueous Two-Phase Systems.

[This corrects the article DOI: 10.3389/fchem.2019.00441.].

Confinement of Suspension-Cultured Cells in Polyethylene Glycol/Polyethylene Oxide-Albumin Aqueous Two-Phase Systems.

Aqueous two-phase systems (ATPSs) have numerous applications in separation science, and more recently, in bioassays enabled by the solution micropatterning of cells. The most frequently used ATPS in these applications is the polyethylene glycol (PEG)-dextran (Dex) system, as the polymers that form this ATPS have been extensively characterized in terms of their physicochemical properties. However, in addition to this well-known system, there exist many other ATPSs with properties that may be exploited to improve upon the PEG-dextran system for specific applications. One of these underexplored systems is the ATPS formed from PEG/polyethylene oxide (PEO) and albumin. In this article, we characterize the phase separation of PEG (35 kDa) and polyethylene oxide (PEO) (200, 900, and 4,000 kDa) with bovine serum albumin (BSA). We describe the microscopic emulsion behavior of these systems in the presence of NaCl and compounds (NaHCO3, NaH2PO4, and HEPES) commonly used in buffer solutions and cell culture media. We further demonstrate that PEG- and PEO-albumin systems can be used in place of the PEG-dextran system for confinement of suspension-cultured cells (Jurkat T cells and RPMI-8226 B cells). Cell viability and morphology are examined for various polymer formulations relative to the commonly used PEG 35 kDa-Dex 500 kDa system and polymer-free cell culture medium. In addition, we examine cell activation for various phase-separating medium components by measuring IL-2 and IL-6 secretion. We demonstrate that we can confine immune cells and cytokines in the PEG-BSA system, and that this system can be employed to screen immune responses by enzyme-linked immunospot (ELISpot) assay. This new system represents a promising ATPS formulation for applications where low levels of baseline cell activation are required, for instance, when culturing immune cells.

Precision cell delivery in biphasic polymer systems enhances growth of keratinocytes in culture and promotes their attachment on acellular dermal matrices.

Current approaches for precision deposition of cells are not optimized for moist environments or for substrates with complex surface topographic features, for example, the surface of dermal matrices and other biomaterials. To overcome these challenges, an approach is presented that utilizes cell confinement in phase-separating polymer solutions of polyethylene glycol and dextran to precisely deliver keratinocytes in well-defined colonies. Using this approach, keratinocyte colonies are produced with superior viability, proliferative capacity, and barrier formation compared with the same number of cells dispersedly seeded across substrate surfaces. It is further demonstrated that keratinocytes delivered in colonies to the surface of acellular dermal matrices form an intact epidermal basal layer more rapidly and more completely than cells delivered by conventional dispersed seeding. These findings demonstrate that delivery of keratinocytes in phase-separating polymer solutions holds potential for enhancing growth of keratinocytes in culture and production of functional skin equivalents.

Fabrication of thin-layer matrigel-based constructs for three-dimensional cell culture.

Extracellular matrix-based hydrogels such as Matrigel are easy-to-use, commercially available, and offer environments for three-dimensional (3-D) cell culture that mimic native tissue. However, manipulating small volumes of these materials to produce thin-layer 3-D culture systems suitable for analysis is difficult because of air-liquid-substrate interfacial tension effects and evaporation. Here, we demonstrate two simple techniques that use standard liquid-handling tools and nontreated 96-well plates to produce uniform, thin-layer constructs for 3-D culture of cells in Matrigel. The first technique, the floating 3-D cell culture method, uses phase-separating polymers to form a barrier between the dispensed Matrigel, air, and cultureware surface to generate consistently thin hydrogels from volumes as low as 5 µL. These unanchored gels provide a useful assay for investigating airway smooth muscle cell contraction and may have future applications in studying asthma pathophysiology. The second technique, the fixed 3-D cell culture method, provides an anchored gel system for culturing noncontractile cells (e.g., neurons) where 20 µL of Matrigel is dispensed into the bottom of a well filled with culture medium to form a thin gel containing embedded cells. This technique has potential widespread applications as an accessible 3-D culture platform for high-throughput production of disease models for evaluation of novel drug therapies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2733, 2019.

Emerging Biotechnology Applications of Aqueous Two-Phase Systems.

Liquid-liquid phase separation between aqueous solutions containing two incompatible polymers, a polymer and a salt, or a polymer and a surfactant, has been exploited for a wide variety of biotechnology applications throughout the years. While many applications for aqueous two-phase systems fall within the realm of separation science, the ability to partition many different materials within these systems, coupled with recent advances in materials science and liquid handling, has allowed bioengineers to imagine new applications. This progress report provides an overview of the history and key properties of aqueous two-phase systems to lend context to how these materials have progressed to modern applications such as cellular micropatterning and bioprinting, high-throughput 3D tissue assembly, microscale biomolecular assay development, facilitation of cell separation and microcapsule production using microfluidic devices, and synthetic biology. Future directions and present limitations and design considerations of this adaptable and promising toolkit for biomolecule and cellular manipulation are further evaluated.

Microscopic evaluation of aqueous two-phase system emulsion characteristics enables rapid determination of critical polymer concentrations for solution micropatterning.

Aqueous two-phase systems have emerged as valuable tools for microscale analysis of cell growth and many other biotechnology applications. The most critical step in developing an aqueous two-phase system for a specific application is identifying the critical concentrations at which the polymer solutions phase-separate. Current techniques for determining these critical concentrations rely on laborious methods, highly specialized assays or computational methods that make this step difficult for non-specialists. To overcome these limitations, we present a simplified assay that uses only readily accessible laboratory instruments and consumables (e.g., multichannel micropipettes, 96-well plates and a simple compound microscope) to determine the critical concentrations of aqueous two-phase system-forming polymers. We demonstrate that formulations selected from phase diagrams that describe these critical concentrations can be applied for solution micropatterning of cells.

Liquid-in-liquid antibody confinement provides new possibilities for multiplexed diagnostics.

The accuracy of disease diagnosis and prognosis can be improved by measuring the concentrations of multiple biomarkers from patient samples. One of the most common and robust methods for detecting biomarkers from patient samples is the ELISA. Recently, there has been a push to improve the disease detection capabilities of immunoassays such as ELISA, as well as other diagnostic assays, by implementing multiplexing strategies. This perspective discusses recent progress using a unique multiplexing technology that takes advantage of phase-separating polymers to spatially confine antibody reagents for cross-reaction-free multiplexing of immunoassays.

Aqueous two-phase system-mediated antibody micropatterning enables multiplexed immunostaining of cell monolayers and tissues.

Conventional immunostaining methods consume large quantities of expensive antibodies and are limited in terms of the number of antigens that can be detected from a single sample. In order to achieve multiplexed immunostaining, we micropatterned antibodies using aqueous two-phase systems (ATPS) formed from polyethylene glycol (PEG) and dextran. Multiple antigens can be detected on a single fixed sample by incorporating antibodies within dextran solutions, which are then patterned by micropipetting at specific sites on the sample in a solution of PEG. The antibodies are retained within the dextran phase due to biomolecular partitioning, allowing multiple protein markers to be visualized simultaneously by way of chromogenic, chemiluminescent, or immunofluorescent detection. This aqueous two-phase system-mediated antibody micropatterning approach allows antibody dilutions to be easily optimized, reduces the consumption of expensive primary antibodies and can prevent antibody cross-reactions, since the antibodies are retained at separate sites within the dextran microdroplets.

Elongation of fibers from highly viscous dextran solutions enables fabrication of rapidly dissolving drug carrying fabrics.

A simple method is presented for forming thread-like fibers from highly viscous dextran solutions. Based on the cohesive and adhesive forces between a dextran solution and the substrate to which it is applied, multiple fibers of approximately 10 µm in diameter can be elongated simultaneously. These fibers can be woven into multiple layers to produce fabrics of varying fiber orientations and mechanical properties. Various bioactive agents can be incorporated into the dextran solution prior to fiber formation, including hemostatic and antibiotic agents. Fabrics containing thrombin are capable of coagulating human platelet poor plasma in vitro. Fabrics containing antibiotics are capable of suppressing bacterial growth in a disk diffusion assay. These data suggest that this new material composed entirely of dextran has promise as a drug delivery component in wound dressings.