A microfluidic model of human brain (μHuB) for assessment of blood brain barrier

Microfluidic cellular models, commonly referred to as “organs‐on‐chips,” continue to advance the field of bioengineering via the development of accurate and higher throughput models, captivating the essence of living human organs. This class of models can mimic key in vivo features, including shear stresses and cellular architectures, in ways that cannot be realized by traditional two‐dimensional in vitro models. Despite such progress, current organ‐on‐a‐chip models are often overly complex, require highly specialized setups and equipment, and lack the ability to easily ascertain temporal and spatial differences in the transport kinetics of compounds translocating across cellular barriers. To address this challenge, we report the development of a three‐dimensional human blood brain barrier (BBB) microfluidic model (μHuB) using human cerebral microvascular endothelial cells (hCMEC/D3) and primary human astrocytes within a commercially available microfluidic platform. Within μHuB, hCMEC/D3 monolayers withstood physiologically relevant shear stresses (2.73 dyn/cm2) over a period of 24 hr and formed a complete inner lumen, resembling in vivo blood capillaries. Monolayers within μHuB expressed phenotypical tight junction markers (Claudin‐5 and ZO‐1), which increased expression after the presence of hemodynamic‐like shear stress. Negligible cell injury was observed when the monolayers were cultured statically, conditioned to shear stress, and subjected to nonfluorescent dextran (70 kDa) transport studies. μHuB experienced size‐selective permeability of 10 and 70 kDa dextrans similar to other BBB models. However, with the ability to probe temporal and spatial evolution of solute distribution, μHuBs possess the ability to capture the true variability in permeability across a cellular monolayer over time and allow for evaluation of the full breadth of permeabilities that would otherwise be lost using traditional end‐point sampling techniques. Overall, the μHuB platform provides a simplified, easy‐to‐use model to further investigate the complexities of the human BBB in real‐time and can be readily adapted to incorporate additional cell types of the neurovascular unit and beyond.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149762/1/btm210126_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149762/2/btm210126.pd

Bioengineered Efficacy Models of Skin Disease: Advances in the Last 10 Years

Models of skin diseases, such as psoriasis and scleroderma, must accurately recapitulate the complex microenvironment of human skin to provide an efficacious platform for investigation of skin diseases. Skin disease research has been shifting from less complex and less relevant 2D (two-dimensional) models to significantly more relevant 3D (three-dimensional) models. Three-dimensional modeling systems are better able to recapitulate the complex cell–cell and cell–matrix interactions that occur in vivo within skin. Three-dimensional human skin equivalents (HSEs) have emerged as an advantageous tool for the study of skin disease in vitro. These 3D HSEs can be highly complex, containing both epidermal and dermal compartments with integrated adnexal structures. The addition of adnexal structures to 3D HSEs has allowed researchers to gain more insight into the complex pathology of various hereditary and acquired skin diseases. One method of constructing 3D HSEs, 3D bioprinting, has emerged as a versatile and useful tool for generating highly complex HSEs. The development of commercially available 3D bioprinters has allowed researchers to create highly reproducible 3D HSEs with precise integration of multiple adnexal structures. While the field of bioengineered models for study of skin disease has made tremendous progress in the last decade, there are still significant efforts necessary to create truly biomimetic skin disease models. In future studies utilizing 3D HSEs, emphasis must be placed on integrating all adnexal structures relevant to the skin disease under investigation. Thorough investigation of the intricate pathology of skin diseases and the development of effective treatments requires use of highly efficacious models of skin diseases

Rapid Assessment of Migration and Proliferation: A Novel 3D High-Throughput Platform for Rational and Combinatorial Screening of Tissue-Specific Biomaterials

Designing an ideal biomaterial supportive of multicellular tissue repair is challenging, especially with a poor understanding of the synergy between constituent proteins and growth factors. A brute-force approach, based on screening all possible combinations of proteins and growth factors, is inadequate due to the prohibitively large experimental space coupled with current low-throughput screening techniques. A high-throughput screening platform based on rational and combinatorial strategies for design and testing of proteins and growth factors can significantly impact the discovery of novel tissue-specific biomaterials. Here, we report the development of a flexible high-throughput screening platform, Rapid Assessment of Migration and Proliferation (RAMP), to rapidly investigate cell viability, proliferation, and migration in response to highly miniaturized three-dimensional biomaterial cultures (4–20 μL) with sparingly low cell densities (63–1000 cells per μL for cell arrays; 1 μL of 1000–10,000 cells per μL for migration arrays). The predictions made by RAMP on the efficacy and potency of the biomaterials are in agreement with the predictions made by conventional assays but at a throughput that is at least 100–1000-fold higher. The RAMP assay is therefore a novel approach for the rapid discovery of tissue-specific biomaterials for tissue engineering and regenerative medicine

Molecular Architecture of the Blood Brain Barrier Tight Junction Proteins–A Synergistic Computational and <i>In Vitro</i> Approach

The blood-brain barrier (BBB) constituted by claudin-5 tight junctions is critical in maintaining the homeostasis of the central nervous system, but this highly selective molecular interface is an impediment for therapeutic interventions in neurodegenerative and neurological diseases. Therapeutic strategies that can exploit the paracellular transport remain elusive due to lack of molecular insights of the tight junction assembly. This study focuses on analyzing the membrane driven <i>cis</i> interactions of claudin-5 proteins in the formation of the BBB tight junctions. We have adopted a synergistic approach employing <i>in silico</i> multiscale dynamics and <i>in vitro</i> cross-linking experiments to study the claudin-5 interactions. Long time scale simulations of claudin-5 monomers, in seven different lipid compositions, show formation of <i>cis</i> dimers that subsequently aggregate into strands. <i>In vitro</i> formaldehyde cross-linking studies also conclusively show that <i>cis</i>-interacting claudin-5 dimers cross-link with short methylene spacers. Using this synergistic approach, we have identified five unique dimer interfaces in our simulations that correlate with the cross-linking experiments, four of which are mediated by transmembrane (TM) helices and the other mediated by extracellular loops (ECL). Potential of mean force calculations of these five dimers revealed that the TM mediated interfaces, which can have distinctive leucine zipper interactions in some cases, are more stable than the ECL mediated interface. Additionally, simulations show that claudin-5 dimerization is significantly influenced by the lipid microenvironment. This study captures the fundamental interactions responsible for the BBB tight junction assembly and offers a framework for extending this work to other tight junctions found in the body

Specificity of Growth Inhibitors and their Cooperative Effects in Calcium Oxalate Monohydrate Crystallization

The molecular recognition and interactions governing site-specific adsorption of growth inhibitors on crystal surfaces can be tailored in order to control the anisotropic growth rates and physical properties of crystalline materials. Here we examine this phenomenon in calcium oxalate monohydrate (COM) crystallization, a model system of calcification with specific relevance for pathological mineralization. We analyzed the effect of three putative growth inhibitorschondroitin sulfate, serum albumin, and transferrinusing analytical techniques capable of resolving inhibitor–crystal interactions from interfacial to bulk scales. We observed that each inhibitor alters surface growth by adsorbing on to distinct steps emanating from screw dislocations on COM surfaces. Binding of inhibitors to different crystallographic faces produced morphological modifications that are consistent with classical mechanisms of layer-by-layer crystal growth inhibition. The site-specific adsorption of inhibitors on COM surfaces was confirmed by bulk crystallization, fluorescent confocal microscopy, and atomic force microscopy. Kinetic studies of COM growth at varying inhibitor concentrations revealed marked differences in their efficacy and potency. Systematic analysis of inhibitor combinations, quantified via the combination index, identified various binary pairings capable of producing synergistic, additive, and antagonistic effects. Collectively, our investigation of physiologically relevant biomolecules suggests potential roles of COM inhibitors in pathological crystallization and provides guiding principles for biomimetic design of molecular modifiers for applications in crystal engineering

Specificity of Growth Inhibitors and their Cooperative Effects in Calcium Oxalate Monohydrate Crystallization

The molecular recognition and interactions governing site-specific adsorption of growth inhibitors on crystal surfaces can be tailored in order to control the anisotropic growth rates and physical properties of crystalline materials. Here we examine this phenomenon in calcium oxalate monohydrate (COM) crystallization, a model system of calcification with specific relevance for pathological mineralization. We analyzed the effect of three putative growth inhibitorschondroitin sulfate, serum albumin, and transferrinusing analytical techniques capable of resolving inhibitor–crystal interactions from interfacial to bulk scales. We observed that each inhibitor alters surface growth by adsorbing on to distinct steps emanating from screw dislocations on COM surfaces. Binding of inhibitors to different crystallographic faces produced morphological modifications that are consistent with classical mechanisms of layer-by-layer crystal growth inhibition. The site-specific adsorption of inhibitors on COM surfaces was confirmed by bulk crystallization, fluorescent confocal microscopy, and atomic force microscopy. Kinetic studies of COM growth at varying inhibitor concentrations revealed marked differences in their efficacy and potency. Systematic analysis of inhibitor combinations, quantified via the combination index, identified various binary pairings capable of producing synergistic, additive, and antagonistic effects. Collectively, our investigation of physiologically relevant biomolecules suggests potential roles of COM inhibitors in pathological crystallization and provides guiding principles for biomimetic design of molecular modifiers for applications in crystal engineering

Natural Promoters of Calcium Oxalate Monohydrate Crystallization

Crystallization is often facilitated by modifiers that interact with specific crystal surfaces and mediate the anisotropic rate of growth. Natural and synthetic modifiers tend to function as growth inhibitors that hinder solute attachment and impede the advancement of layers on crystal surfaces. There are fewer examples of modifiers that operate as growth promoters, whereby modifier–crystal interactions accelerate the kinetic rate of crystallization. Here, we examine two proteins, lysozyme and lactoferrin, which are observed in the organic matrix of three types of pathological stones: renal, prostatic, and pancreatic stones. This work focuses on the role of these proteins in the crystallization of calcium oxalate monohydrate (COM), the most prominent constituent of human kidney stones. Using a combination of experimental techniques, we show that these proteins, which are rich in l-arginine and l-lysine amino acids, promote COM growth. The synthesis and testing of peptides derived from contiguous segments of lysozyme’s primary amino acid sequence revealed subdomains within the protein that operate either as an inhibitor or promoter of COM growth, with the latter exhibiting efficacies that nearly match that of the protein. We observed that cationic proteins promote COM growth over a wide range of modifier concentration, which differs from calcification promoters in the literature that exhibit dual roles as promoters and inhibitors at low and high concentration, respectively. This seems to suggest a unique mechanism of action for lysozyme and lactoferrin. Possible explanations for their effects on COM growth and crystal habit are proposed on the basis of classical colloidal theories and the physicochemical properties of peptide subdomains, including the number and spatial location of charged or hydrogen-bonding moieties