Suspended Collagen Hydrogels to Replicate Human Colonic Epithelial Cell Interactions with Immune Cells.

The human colon plays a critical role in fluid and salt absorption and harbors the largest immune compartment. There is a widespread need for in vitro models of human colon physiology with its innate immune system. A method is described to produce a cassette with a network of struts supporting a suspended, non-chemically cross-linked collagen hydrogel scaffold compatible with the co-culture of primary gastrointestinal epithelium and migratory inflammatory cells. The epithelial monolayer cultured on the suspended collagen possesses a population of polarized and differentiated cells similar to that present in vivo. This epithelial layer displays proper barrier function with a transepithelial electrical resistance (TEER) ≥ 1,500 Ω cm2 and an apparent permeability ≤10-5 cm2 s-1 . Immune cells plated on the basal face of the scaffold transmigrated over a period of 24 h to the epithelial layer in response to epithelial production of IL-8 induced by luminal stimulation of Clostridium difficile Toxin A. These studies demonstrate that this in vitro platform possesses a functional primary colonic epithelial layer with an immune cell compartment capable of recruitment in response to pro-inflammatory cues coming from the epithelium.

Stem/Proliferative and Differentiated Cells within Primary Murine Colonic Epithelium Display Distinct Intracellular Free Ca2+ Signal Codes.

The second messenger, intracellular free calcium (Ca2+ ), acts to transduce mitogenic and differentiation signals incoming to the colonic epithelium. A self-renewing monolayer of primary murine colonic epithelial cells is formed over a soft, transparent hydrogel matrix for the scalable analysis of intracellular Ca2+ transients. Cultures that are enriched for stem/proliferative cells exhibit repetitive, high frequency (≈25 peaks h-1 ), and short pulse width (≈25 s) Ca2+ transients. Upon cell differentiation the transient frequency declines by 50% and pulse width widens by 200%. Metabolites and growth factors that are known to modulate stem cell proliferation and differentiation through Wnt and Notch signaling pathways, including CHIR-99021, N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), bone morphogenetic proteins (BMPs), and butyrate, also modulate Ca2+ oscillation patterns in a consistent manner. Increasing the stiffness of the supportive matrix from 200 Pa to 3 GPa shifts Ca2+ transient patterns toward those resembling differentiated cells. The ability to monitor Ca2+ oscillations with the spatial and temporal resolution offered by this platform, combined with its amenability to high-content screens, provides a powerful tool for investigating real-time communication within a wide range of primary tissues in addition to the colonic epithelium.

Magnetically-propelled fecal surrogates for modeling the impact of solid-induced shear forces on primary colonic epithelial cells.

The colonic epithelium is continuously exposed to an array of biological and mechanical stimuli as its luminal contents are guided over the epithelial surface through regulated smooth muscle contraction. In this report, the propulsion of solid fecal contents over the colonic epithelium is recapitulated through noninvasive actuation of magnetic agarose hydrogels over primary intestinal epithelial cultures, in contrast to the vast majority of platforms that apply shear forces through liquid microflow. Software-controlled magnetic stepper motors enable experimental control over the frequency and velocity of these events to match in vivo propulsive contractions, while the integration of standardized well plate spacing facilitates rapid integration into existing assay pipelines. The application of these solid-induced shear forces did not deleteriously affect cell monolayer surface coverage, viability, or transepithelial electrical resistance unless the device parameters were raised to a 50× greater contraction frequency and 4× greater fecal velocity than those observed in healthy humans. At a frequency and velocity that is consistent with average human colonic motility, differentiation of the epithelial cells into absorptive and goblet cell phenotypes was not affected. Protein secretion was modulated with a two-fold increase in luminal mucin-2 secretion and a significant reduction in basal interleukin-8 secretion. F-actin, zonula occludens-1, and E-cadherin were each present in their proper basolateral locations, similar to those of static control cultures. While cellular height was unaffected by magnetic agarose propulsion, several alterations in lateral morphology were observed including decreased circularity and compactness, and an increase in major axis length, which align with surface epithelial cell morphologies observed in vivo and may represent early markers of luminal exfoliation. This platform will be of widespread utility for the investigation of fecal propulsive forces on intestinal physiology, shedding light on how the colonic epithelium responds to mechanical cues.

Hyperglycemia minimally alters primary self-renewing human colonic epithelial cells while TNFα-promotes severe intestinal epithelial dysfunction.

Hyperglycemia is thought to increase production of inflammatory cytokines and permeability of the large intestine. Resulting intestinal inflammation is then often characterized by excess secretion of tumor necrosis factor alpha (TNFα). Thus, hyperglycemia in hospitalized patients suffering from severe trauma or disease is frequently accompanied by TNFα secretion, and the combined impact of these insults on the intestinal epithelium is poorly understood. This study utilized a simple yet elegant model of the intestinal epithelium, comprised of primary human intestinal stem cells and their differentiated progeny, to investigate the impact of hyperglycemia and inflammatory factors on the colonic epithelium. When compared to epithelium cultured under conditions of physiologic glucose, cells under hyperglycemic conditions displayed decreased mucin-2 (MUC2), as well as diminished alkaline phosphatase (ALP) activity. Conditions of 60 mM glucose potentiated secretion of the cytokine IL-8 suggesting that cytokine secretion during hyperglycemia may be a source of tissue inflammation. TNFα measurably increased secretion of IL-8 and IL-1ß, which was enhanced at 60 mM glucose. Surprisingly, intestinal permeability and paracellular transport were not altered by even extreme levels of hyperglycemia. The presence of TNFα increased MUC2 presence, decreased ALP activity, and negatively impacted monolayer barrier function. When TNFα hyperglycemia and ≤30 mM glucose and were combined, MUC2 and ALP activity remained similar to that of TNFα alone, although synergistic effects were seen at 60 mM glucose. An automated image analysis pipeline was developed to assay changes in properties of the zonula occludens-1 (ZO-1)-demarcated cell boundaries. While hyperglycemia alone had little impact on cell shape and size, cell morphologic properties were extraordinarily sensitive to soluble TNFα. These results suggest that TNFα acted as the dominant modulator of the epithelium relative to glucose, and that control of inflammation rather than glucose may be key to maintaining intestinal homeostasis.

In vitro generation of self-renewing human intestinal epithelia over planar and shaped collagen hydrogels.

The large intestine, with its array of crypts lining the epithelium and diverse luminal contents, regulates homeostasis throughout the body. In vitro crypts formed from primary human intestinal epithelial stem cells on a 3D shaped hydrogel scaffold replicate the functional and architectural features of in vivo crypts. Collagen scaffolding assembly methods are provided, along with the microfabrication and soft lithography protocols necessary to shape these hydrogels to match the dimensions and density of in vivo crypts. In addition, stem-cell scale-up protocols are provided so that even ultrasmall primary samples can be used as starting material. Initially, these cells are seeded as a proliferative monolayer over the shaped scaffold and cultured as stem/proliferative cells to expand them and cover the scaffold surface with the crypt-shaped structures. To convert these immature crypts into fully polarized, functional units with a basal stem cell niche and luminal differentiated cell zone, stable, linear gradients of growth factors are formed across the crypts. This platform supports the formation of chemical gradients across the crypts, including those of growth and differentiation factors, inflammatory compounds, bile and food metabolites and bacterial products. All microfabrication and device assembly steps are expected to take 8 d, with the primary cells cultured for 12 d to form mature in vitro crypts.

Microphysiological System Design: Simplicity Is Elegance.

Design parameters for microphysiological systems (MPS) are driven by the need for new tools to answer questions focusing on human physiology in a robust and reliable manner. Within this perspective, engineering benchmarks and principles are identified to guide the construction of new devices in the MPS field, with emphasis placed on the design principles common to all tissues, as well as those unique to a subset of tissues. Leading organ replica technologies that recapitulate various functions of the brain, heart, intestine, and lung are highlighted as examples that meet the identified benchmarks and standards, with current barriers for large scale production and commercialization discussed. To reach their full potential and achieve widespread use, MPS will have to be recognized officially by government agencies, and toward this end, considerations of MPS as a potential regulatory tool are presented.

Photopatterned Membranes and Chemical Gradients Enable Scalable Phenotypic Organization of Primary Human Colon Epithelial Models.

Biochemical gradients across the intestinal epithelium play a major role in governing intestinal stem cell compartmentalization, differentiation dynamics, and organ-level self-renewal. However, scalable platforms that recapitulate the architecture and gradients present in vivo are absent. We present a platform in which individually addressable arrays of chemical gradients along the intestinal crypt long axis can be generated, enabling scalable culture of primary in vitro colonic epithelial replicas. The platform utilizes standardized well plate spacing, maintains access to basal and luminal compartments, and relies on a photopatterned porous membrane to act as diffusion windows while supporting the in vitro crypts. Simultaneous fabrication of 3875 crypts over a single membrane was developed. Growth factor gradients were modeled and then experimentally optimized to promote long-term health and self-renewal of the crypts which were assayed in situ by confocal fluorescence microscopy. The cultured in vitro crypt arrays successfully recapitulated the architecture and luminal-to-basal phenotypic polarity observed in vivo. Furthermore, known signaling regulators (e.g., butyrate and DAPT) produced measurable and predictable effects on the organized cell compartments, each decreasing crypt proliferation in the basal regions to negligible values. This platform is readily adaptable to the screening of tissue from individual patients to assay the impact of food and bacterial metabolites and/or drugs on colonic crypt dynamics. Importantly, the cassette is compatible with a wide range of sensing/detection modalities, and the developed fabrication methods should find applications for other cell and tissue types.

Antifouling Lipid Membranes over Protein A for Orientation-Controlled Immunosensing in Undiluted Serum and Plasma.

An important advance in biosensor research is the extension and application of laboratory-developed methodologies toward clinical diagnostics, though the propensity toward nonspecific binding of materials in clinically relevant matrices, such as human blood serum and plasma, frequently leads to compromised assays. Several surface chemistries have been developed to minimize nonspecific interactions of proteins and other biological components found within blood and serum samples, though these often exhibit substantially variable outcomes. Herein we report a surface chemistry consisting of a charged-matched supported lipid membrane that has been tailored to form over a gold surface functionalized with protein A. Fine tuning of the interfacial charge of this membrane, along with rational selection of a backfilling self-assembled monolayer, allows for high surface coverage with retention of orientation-controlled capture antibody attachment. We demonstrate using surface-plasmon resonance (SPR) that this highly charged lipid membrane is antifouling, allowing for complete removal of nonspecific human serum and plasma components using only a mild buffer rinse, which we attribute to unique steric interactions with the underlying surface. Furthermore, this surface chemistry is successfully applied for specific detection of IgG and cholera toxin in undiluted human biofluids with negligible sacrifice of SPR signal compared to buffered analysis. This novel lipid membrane interface over protein A may open new avenues for direct biosensing of disease markers within clinical samples.

Primary Cell-Derived Intestinal Models: Recapitulating Physiology.

The development of physiologically relevant intestinal models fueled by breakthroughs in primary cell-culture methods has enabled successful recapitulation of key features of intestinal physiology. These advances, paired with engineering methods, for example incorporating chemical gradients or physical forces across the tissues, have yielded ever more sophisticated systems that enhance our understanding of the impact of the host microbiome on human physiology as well as on the genesis of intestinal diseases such as inflammatory bowel disease and colon cancer. In this review we highlight recent advances in the development and usage of primary cell-derived intestinal models incorporating monolayers, organoids, microengineered platforms, and macrostructured systems, and discuss the expected directions of the field.

Bioengineered Systems and Designer Matrices That Recapitulate the Intestinal Stem Cell Niche.

The relationship between intestinal stem cells (ISCs) and the surrounding niche environment is complex and dynamic. Key factors localized at the base of the crypt are necessary to promote ISC self-renewal and proliferation, to ultimately provide a constant stream of differentiated cells to maintain the epithelial barrier. These factors diminish as epithelial cells divide, migrate away from the crypt base, differentiate into the postmitotic lineages, and end their life span in approximately 7 days when they are sloughed into the intestinal lumen. To facilitate the rapid and complex physiology of ISC-driven epithelial renewal, in vivo gradients of growth factors, extracellular matrix, bacterial products, gases, and stiffness are formed along the crypt-villus axis. New bioengineered tools and platforms are available to recapitulate various gradients and support the stereotypical cellular responses associated with these gradients. Many of these technologies have been paired with primary small intestinal and colonic epithelial cells to re-create select aspects of normal physiology or disease states. These biomimetic platforms are becoming increasingly sophisticated with the rapid discovery of new niche factors and gradients. These advancements are contributing to the development of high-fidelity tissue constructs for basic science applications, drug screening, and personalized medicine applications. Here, we discuss the direct and indirect evidence for many of the important gradients found in vivo and their successful application to date in bioengineered in vitro models, including organ-on-chip and microfluidic culture devices.

Efficient label-free chemiluminescent immunosensor based on dual functional cupric oxide nanorods as peroxidase mimics.

Dual-functional cupric oxide nanorods (CuONRs) as peroxidase mimics are proposed for the development of a flow-through, label-free chemiluminescent (CL) immunosensor. Forming the basis of this cost-efficient, label-free immunoassay, CuONRs, synthesized using a simple hydrothermal method, were deposited onto epoxy-activated standard glass slides, followed by immobilization of biotinylated capture antibodies through a streptavidin bridge. The CuONRs possess excellent catalytic activity, along with high stability as a solid support. Antigens could then be introduced to the sensing system, forming large immunocomplexes that prevent CL substrate access to the surface, thereby reducing the CL signal in a concentration dependent fashion. Using carcinoembryonic antigen (CEA) as a model analyte, the proposed label-free immunosensor was able to rapidly determine CEA with a wide linear range of 0.1-60ngmL-1 and a low detection limit of 0.05ngmL-1. This nanozyme-based immunosensor is simple, sensitive, cost-efficient, and has the potential to be a very promising platform for fast and efficient biosensing applications.

Plasmonic Sensing with 3D Printed Optics.

Three-dimensional (3D) printing has undergone an exponential growth in popularity due to its revolutionary and near limitless manufacturing capabilities. Recent trends have seen this technology utilized across a variety of scientific disciplines, including the measurement sciences, but precise fabrication of optical components for high-performance biosensing has not yet been demonstrated. We report here 3D printing of high-quality, custom prisms by stereolithography that enable Kretschmann-configured plasmonic sensing of bacterial toxins. Simple benchtop polishing procedures render a smooth surface that supports propagation of surface plasmon polaritons with a deposited gold layer, which exhibit high bulk refractive index sensitivities and are capable of discriminating trace levels of cholera toxin on a supported lipid membrane interface. Further evidence of the flexibility of this manufacturing technique is demonstrated with printed prisms of varied geometries and in situ monitoring of nanoparticle growth by total internal reflection spectroscopy. This work represents the first example of 3D printed light-guiding sensing platforms and demonstrates the versatility and broad perspective of 3D printing in optical detection.

Selective protein recognition in supported lipid bilayer arrays by tailored, dual-mode deep cavitand hosts.

Self-folding deep cavitands with variably functionalized upper rims are able to selectively immobilize proteins at a biomimetic supported lipid bilayer surface. The immobilization process takes advantage of the dual-mode binding capabilities of the hosts, combining a defined binding pocket with upper rim charged/H-bonding groups. A variety of proteins can be selectively immobilized at the bilayer interface, either via complementary charge/H-bonding interactions, cavity-based molecular recognition, or a combination of both. The immobilization process can be used to bind unmodified native proteins, epitopes for bioadhesion, or proteins covalently modified with suitable RNMe3+ binding "handles" and charged groups that can either match or mismatch with the cavitand rim. The immobilization process can be monitored in real time using surface plasmon resonance (SPR) spectroscopy, and applied to the construction of cavitand:lipid arrays using the hosts and trehalose vitrified phospholipid vesicles. The selective, dual-mode protein recognition is maintained in the arrays, and can be visualized using SPR imaging.

DNA Linkers and Diluents for Ultrastable Gold Nanoparticle Bioconjugates in Multiplexed Assay Development.

A novel bioconjugation strategy leading to ultrastable gold nanoparticles (AuNPs), utilizing DNA linkers and diluents in place of traditional self-assembled monolayers, is reported. The protective capacity of DNA confers straightforward biomolecular attachment and multistep derivatization capabilities to these nanoparticles and, more significantly, substantially enhances their stability in demanding and complex sensing environments. The DNA/AuNPs were assembled through pH-assisted thiol-gold bonding of single stranded DNA and salt aging, with preconjugated biotin moieties facing outward from the gold surface. These nanoparticles remain a stable colloidal suspension under a wide range of buffers and ionic strengths and can endure multiple rounds of lyophilization while retaining high biological activity. Furthermore, the high stability of the DNA/AuNPs allows for multiple reactions and conjugations to be performed within the colloidal suspensions (i.e., Protein A and antibody binding) for tailored and specific recognition to take place. We have demonstrated the applications of the DNA/AuNPs for colorimetric assays and ELISA feasibility; additionally, SPR imaging analysis of a supported membrane microarray shows excellent results with DNA/AuNPs as the enhancing agent. Together, the properties imparted by this interface render the material suitable for clinical and point-of-care applications where stability, throughput, and extended shelf lives are needed.

Plasmonic nanodisc arrays on calcinated titania for multimodal analysis of phosphorylated peptides.

A hybrid material of gold nanodiscs on a calcinated titania nanofilm that allows for selective quantitative and qualitative characterization of surface-enriched phosphopeptides has been designed and reported. Fabrication was realized through a combination of layer-by-layer deposition and high temperature calcination for the titania, and hole-mask colloidal lithography for the plasmonic nanostructures. The morphology of the resulting titania material was rigorously characterized, exhibiting substantially decreased surface roughness, which allows for lithographic fabrication of plasmonic nanostructures. Moreover, high specificity in adsorption and enrichment of phosphopeptides was exhibited, which was verified by LSPR shifts and matching peaks under mass spectrometric analysis. The construction of these biochips should inform other combinatorial nanofabrication techniques, in addition to allowing future phosphoproteomic analyses to be performed in a time and resource-efficient manner.

Mix and Match: Coassembly of Amphiphilic Dendrimers and Phospholipids Creates Robust, Modular, and Controllable Interfaces.

Self-assembly of supramolecular structures has become an attractive means to create new biologically inspired materials and interfaces. We report the first robust hybrid bilayer systems readily coassembled from amphiphilic dendrimers and a naturally occurring phospholipid. Both concentration and generation of the dendrimers have direct impacts on the biophysical properties of the coassemblies. Raising the dendrimer concentration increases the hybrid bilayer stability, while changes in the generation and the concentration of the embedded dendrimers impact the fluidity of the coassembled systems. Multivalent dendrimer amine terminals allow for nondestructive in situ derivatization, providing a convenient approach to decorate and modulate the local environment of the hybrid bilayer. The coassembly of lipid/dendrimer interfaces offers a unique platform for the creation of hybrid systems with modular and precisely controllable behavior for further applications in sensing and drug delivery.

Calcinated gold nanoparticle arrays for on-chip, multiplexed and matrix-free mass spectrometric analysis of peptides and small molecules.

A patterned gold nanoparticle microarray, functionalized with a nanoscale silicate coating, has been developed for on-chip, high-throughput mass spectrometric analyses of biomolecules with minimal sample preparation and reagent costs. Fabrication was realized by the combination of layer-by-layer functionalization of the nanoparticles with suitable polyelectrolytes, followed by fluidic patterning of the glass microarray support and calcination for permanent fixation of the nano-coating. Performance of the microarray was evaluated for surface-assisted laser-desorption/ionization mass spectrometry (SALDI-MS), where the nano-silicate coating was found to enhance SALDI efficiency, resulting in comparable performance to some common organic matrices for small and medium sized molecules. Performance contributing factors of this material have been discussed; heat confinement and interband transition/plasmonic resonance may play important roles. Taking the accessibility of fabrication, performance, and reusability of this substrate together, the material developed here provides a new tool for multiplexed and chip-based mass spectrometric analysis.

Bioinspired Assemblies and Plasmonic Interfaces for Electrochemical Biosensing.

Electrochemical biosensing represents a collection of techniques that may be utilized for capture and detection of biomolecules in both simple and complex media. While the instrumentation and technological aspects play important roles in detection capabilities, the interfacial design aspects are of equal importance, and often, those inspired by nature produce the best results. This review highlights recent material designs, recognition schemes, and method developments as they relate to targeted electrochemical analysis for biological systems. This includes the design of electrodes functionalized with peptides, proteins, nucleic acids, and lipid membranes, along with nanoparticle mediated signal amplification mechanisms. The topic of hyphenated surface plasmon resonance assays is also discussed, as this technique may be performed concurrently with complementary and/or confirmatory measurements. Together, smart materials and experimental designs will continue to pave the way for complete biomolecular analyses of complex and technically challenging systems.

On-Demand Formation of Supported Lipid Membrane Arrays by Trehalose-Assisted Vesicle Delivery for SPR Imaging.

The fabrication of large-scale, solid-supported lipid bilayer (SLB) arrays has traditionally been an arduous and complex task, primarily due to the need to maintain SLBs within an aqueous environment. In this work, we demonstrate the use of trehalose vitrified phospholipid vesicles that facilitate on-demand generation of microarrays, allowing each element a unique composition, for the label-free and high-throughput analysis of biomolecular interactions by SPR imaging (SPRi). Small, unilamellar vesicles (SUVs) are suspended in trehalose, deposited in a spatially defined manner, with the trehalose vitrifying on either hydrophilic or hydrophobic SPR substrates. SLBs are subsequently spontaneously formed on-demand simply by in situ hydration of the array in the SPR instrument flow cell. The resulting SLBs exhibit high lateral mobility, characteristic of fluidic cellular lipid membranes, and preserve the biological function of embedded cell membrane receptors, as indicated by SPR affinity measurements. Independent fluorescence and SPR imaging studies show that the individual SLBs stay localized at the area of deposition, without any encapsulating matrix, confining coral, or boundaries. The introduced methodology allows individually addressable SLB arrays to be analyzed with excellent label-free sensitivity in a real-time, high-throughput manner. Various protein-ganglioside interactions have been selected as a model system to illustrate discrimination of strong and weak binding responses in SPRi sensorgrams. This methodology has been applied toward generating hybrid bilayer membranes on hydrophobic SPR substrates, demonstrating its versatility toward a range of surfaces and membrane geometries. The stability of the fabricated arrays, over medium to long storage periods, was evaluated and found to be good. The highly efficient and easily scalable nature of the method has the potential to be applied to a variety of label-free sensing platforms requiring lipid membranes for high-throughput analysis of their properties and constituents.