Biology subjects

Ingber, D. E.

Publications and source records attributed to Ingber, D. E..

36 records · Page 2Linked to original sources

bioRxiv2022

Target-agnostic discovery of Rett Syndrome therapeutics by coupling computational network analysis and CRISPR-enabled in vivo disease modeling

Many neurodevelopmental genetic disorders, such as Rett syndrome, are caused by a single gene mutation but trigger changes in expression and regulation of numerous other genes. This severely impair functions of multiple organs and organ systems beyond the central nervous system (CNS), adding to the challenge of developing broadly effective treatments based on a single drug target. This challenge is further complicated by the lack of sufficiently broad and biologically relevant drug screens, and the inherent complexity in identifying clinically relevant targets responsible for diverse phenotypes. Here, we combined human gene regulatory network-based computational drug prediction with in vivo screening in a population-level diversity, CRISPR-edited, Xenopus laevis tadpole model of Rett syndrome to carry out target-agnostic drug discovery, which rapidly led to the identification of the FDA-approved drug vorinostat as a potential repurposing candidate. Vorinostat broadly improved both CNS and non-CNS (e.g., gastrointestinal, respiratory, inflammatory) abnormalities in a pre-clinical mouse model of Rett syndrome. This is the first Rett syndrome treatment to demonstrate pre-clinical efficacy across multiple organ systems when dosed after the onset of symptoms, and network analysis revealed a putative therapeutic mechanism for its cross-organ normalizing effects based on its impact on acetylation metabolism and post-translational modifications of microtubules. Although traditionally considered an inhibitor of histone deacetylases (HDAC), vorinostat unexpectedly restored protein acetylation across both hypo- and hyperacetylated tissues, suggesting non-HDAC-mediated therapeutic mechanisms supported by proteomic analysis.

neuroscience↗

bioRxiv2022

Establishment of physiologically relevant oxygen gradients in microfluidic organ chips

In vitro models of human organs must accurately reconstitute oxygen concentrations and gradients that are observed in vivo to mimic gene expression, metabolism, and host-microbiome interactions. Here we describe a simple strategy to achieve physiologically relevant oxygen tension in a two-channel human small intestine-on-a-chip (Intestine Chip) lined with primary human duodenal epithelium and intestinal microvascular endothelium in parallel channels separated by a porous membrane while both channels are perfused with oxygenated medium. This strategy was developed using computer simulations that predicted lowering the oxygen permeability of poly-dimethlysiloxane (PDMS) chips in specified locations using a gas impermeable film will allow the cells to naturally decrease the oxygen concentration through aerobic respiration and reach steady-state oxygen levels < 36 mm Hg (< 5%) within the epithelial lumen. The approach was experimentally confirmed using chips with embedded oxygen sensors that maintained this stable oxygen gradient. Furthermore, Intestine Chips cultured with this approach supported formation of a villus epithelium interfaced with a continuous endothelium and maintained intestinal barrier integrity for 72 h. This strategy recapitulates in vivo functionality in an efficient, inexpensive, and scalable format that improves the robustness and translatability of Organ Chip technology for studies on microbiome as well as oxygen sensitivity.

bioengineering↗

bioRxiv2021

Qualifying a human Liver-Chip for predictive toxicology: Performance assessment and economic implications

Human organ-on-a-chip (Organ-Chip) technology has the potential to disrupt preclinical drug discovery and improve success in drug development pipelines as it can recapitulate organ-level pathophysiology and clinical responses. The Innovation and Quality (IQ) consortium formed by multiple pharmaceutical and biotechnology companies to confront this challenge has published guidelines that define criteria for qualifying preclinical models, however, systematic and quantitative evaluation of the predictive value of Organ-Chips has not yet been reported. Here, 870 Liver-Chips were analyzed to determine their ability to predict drug-induced liver injury (DILI) caused by small molecules identified as benchmarks by the IQ consortium. The Liver-Chip met the qualification guidelines across a blinded set of 27 known hepatotoxic and non-toxic drugs with a sensitivity of 87% and a specificity of 100%. A computational economic value analysis suggests that with this performance the Liver-Chip could generate $3 billion annually for the pharmaceutical industry due to increased R&D productivity.

pharmacology and toxicology↗

bioRxiv2021

Self-assembling short immunostimulatory duplex RNAs with broad spectrum antiviral activity

The current COVID-19 pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III), in a wide range of human cell types. These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique conserved sequence motif (sense strand: 5-C, antisense strand: 3-GGG) that mediates end-to-end dimer self-assembly of these RNAs by Hoogsteen G-G base-pairing. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory siRNAs, their activity is independent of TLR7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad spectrum inhibition of infections by many respiratory viruses with pandemic potential, including SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza A, as well as the common cold virus HCoV-NL63 in both cell lines and human Lung Chips that mimic organ-level lung pathophysiology. These short dsRNAs can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics at low cost.

immunology↗

bioRxiv2021

Enhancers of host immune tolerance to bacterial infection discovered using linked computational and experimental approaches

Current therapeutic strategies against bacterial infections focus on reduction of pathogen load through antibiotics; however, stimulation of host tolerance to infection might offer an alternative approach. Here we used computational transcriptomics and a Xenopus embryo infection model to rapidly discover infection response pathways, identify potential tolerance inducer drugs, and validate their ability to induce broad tolerance. Xenopus embryos exhibit natural tolerance to A. baumanii, K. pneumoniae, S. aureus, and S. pneumoniae bacteria, whereas A. hydrophila and P. aeruginosa produce infection that leads to death. Transcriptional profiling led to definition of a 20-gene signature that allows for discrimination between tolerant and susceptible states, as well as identification of active and passive tolerance responses based on the degree of engagement of gene transcription modulation. Upregulation of metal ion transport and hypoxia pathways reminiscent of responses observed in primate and mouse infection models were identified as tolerance mediators, and drug screening in the susceptible A. hydrophila infection model confirmed that a metal chelator (deferoxamine) and HIF-1 agonist (1,4-DPCA) increase embryo survival despite high pathogen load. These data demonstrate the value of combining the Xenopus embryo infection model with multi-omics analyses for mechanistic discovery and drug repurposing to induce host tolerance to bacterial infection.

immunology↗

bioRxiv2021

Enteric coronavirus infection and treatment modeled with an immunocompetent human intestine-on-a-chip

Many patients infected with coronaviruses, such as SARS-CoV-2 and NL63 that use ACE2 receptors to infect cells, exhibit gastrointestinal symptoms and viral proteins are found in the human gastrointestinal tract, yet little is known about the inflammatory and pathological effects of coronavirus infection on the human intestine. Here, we used a human intestine-on-a-chip (Intestine Chip) microfluidic culture device lined by patient organoid-derived intestinal epithelium interfaced with human vascular endothelium to study host cellular and inflammatory responses to infection with NL63 coronavirus. These organoid-derived intestinal epithelial cells dramatically increased their ACE2 protein levels when cultured under flow in the presence of peristalsis-like mechanical deformations in the Intestine Chips compared to when cultured statically as organoids or in Transwell inserts. Infection of the intestinal epithelium with NL63 on-chip led to inflammation of the endothelium as demonstrated by loss of barrier function, increased cytokine production, and recruitment of circulating peripheral blood mononuclear cells (PMBCs). Treatment of NL63 infected chips with the approved protease inhibitor drug, nafamostat, inhibited viral entry and resulted in a reduction in both viral load and cytokine secretion, whereas remdesivir, one of the few drugs approved for COVID19 patients, was not found to be effective and it also was toxic to the endothelium. This model of intestinal infection was also used to test the effects of other drugs that have been proposed for potential repurposing against SARS-CoV-2. Taken together, these data suggest that the human Intestine Chip might be useful as a human preclinical model for studying coronavirus related pathology as well as for testing of potential anti-viral or anti-inflammatory therapeutics.

bioengineering↗

bioRxiv2021

Mechanical control of innate immune responses against viral infection revealed in a human Lung Alveolus Chip

Mechanical forces associated with breathing play a fundamental role in lung development and disease but the molecular pathways remain largely unknown. Here, we used a mechanically actuatable Human Lung Alveolus Chip that recapitulates human lung alveolar type I and type II cell differentiation, alveolar-capillary interface formation, and genome-wide gene expression profiles characteristic of the distal lung to investigate the role of physical forces associated with cyclic breathing motions in lung innate immune responses to viral infection. When the mechanically active Alveolus Chips are infected with the influenza H3N2 virus, a cascade of host responses is elicited on-chip, including increased production of cytokines and expression of inflammation-associated genes in pulmonary epithelial and endothelial cells, resulting in enhanced recruitment of circulating immune cells as occurs during viral infection in vivo. Surprisingly, studies carried out in parallel with static chips revealed that physiological breathing motions suppress viral replication by activating protective innate immune responses in epithelial and endothelial cells. This is mediated at least in part through upregulation of S100 calcium-binding protein A7 (S100A7), which binds to the Receptor for Advanced Glycation End Products (RAGE), an inflammatory mediator that is most highly expressed in the lung alveolus in vivo. This mechano-immunological control mechanism is further supported by the finding that existing RAGE inhibitor drugs can suppress the production of inflammatory cytokines in response to influenza virus infection in this model. S100A7-RAGE interactions and modulation of mechanical ventilation parameters could therefore serve as new targets for therapeutic intervention in patients infected with influenza and other potential pandemic viruses that cause life-threatening lung inflammation.

immunology↗

bioRxiv2020

Harnessing Colon Chip technology to identify commensal bacteria that promote host tolerance to infection

Commensal bacteria within the gut microbiome contribute to development of host tolerance to infection, however, identifying specific microbes responsible for this response is difficult. Here we describe methods for developing microfluidic organ-on-a-chip models of small and large intestine lined with epithelial cells isolated from duodenal, jejunal, ileal, or colon organoids derived from wild type or transgenic mice. To focus on host-microbiome interactions, we carried out studies with the mouse Colon Chip and demonstrated that it can support co-culture with living gut microbiome and enable assessment of effects on epithelial adhesion, tight junctions, barrier function, mucus production, and cytokine release. Moreover, infection of the Colon Chips with the pathogenic bacterium, Salmonella typhimurium, resulted in epithelial detachment, decreased tight junction staining, and increased release of cytokines (CXCL1, CXCL2, and CCL20) that closely mimicked changes previously seen in mice. Symbiosis between microbiome bacteria and the intestinal epithelium was also recapitulated by populating Colon Chips with complex living mouse or human microbiome. By taking advantage of differences in the composition between complex microbiome samples cultured on each chip using 16s sequencing, we were able to identify Enterococcus faecium as a positive contributor to host tolerance, confirming past findings obtained in mouse experiments. Thus, mouse Intestine Chips may represent new experimental in vitro platforms for identifying particular bacterial strains that modulate host response to pathogens, as well as for investigating the cellular and molecular basis of host-microbe interactions.

bioengineering↗

bioRxiv2020

Human organs-on-chips as tools for repurposing approved drugs as potential influenza and COVID19 therapeutics in viral pandemics

The rising threat of pandemic viruses, such as SARS-CoV-2, requires development of new preclinical discovery platforms that can more rapidly identify therapeutics that are active in vitro and also translate in vivo. Here we show that human organ-on-a-chip (Organ Chip) microfluidic culture devices lined by highly differentiated human primary lung airway epithelium and endothelium can be used to model virus entry, replication, strain-dependent virulence, host cytokine production, and recruitment of circulating immune cells in response to infection by respiratory viruses with great pandemic potential. We provide a first demonstration of drug repurposing by using oseltamivir in influenza A virus-infected organ chip cultures and show that co-administration of the approved anticoagulant drug, nafamostat, can double oseltamivirs therapeutic time window. With the emergence of the COVID-19 pandemic, the Airway Chips were used to assess the inhibitory activities of approved drugs that showed inhibition in traditional cell culture assays only to find that most failed when tested in the Organ Chip platform. When administered in human Airway Chips under flow at a clinically relevant dose, one drug - amodiaquine - significantly inhibited infection by a pseudotyped SARS-CoV-2 virus. Proof of concept was provided by showing that amodiaquine and its active metabolite (desethylamodiaquine) also significantly reduce viral load in both direct infection and animal-to-animal transmission models of native SARS-CoV-2 infection in hamsters. These data highlight the value of Organ Chip technology as a more stringent and physiologically relevant platform for drug repurposing, and suggest that amodiaquine should be considered for future clinical testing.

microbiology↗

bioRxiv2020

Modular biomaterials vaccine technology protects against multiple pathogens and septic shock

Broad spectrum vaccines could provide a solution to the emergence of antibiotic resistant microbes, pandemics and engineered biothreat agents. Here, we describe a modular vaccine (composite infection vaccine technology (ciVAX)) which can be rapidly assembled and in which 4 of the 5 components are already approved for human use. ciVAX consists of an injectable biomaterial scaffold with factors to recruit and activate dendritic cells (DC) in vivo and microbeads conjugated with the broad-spectrum opsonin Fc-Mannose-binding Lectin (FcMBL) that is pre-bound to polysaccharide-rich cell wall antigens, such as the pathogen-associated molecular patterns (PAMPs) fractions, captured from whole inactivated bacteria. Vaccination of mice and rabbits with ciVAX generates potent humoral and T cell responses to PAMPs isolated from native antibiotic-resistant E. coli and S. aureus, and ciVAX protects mice and pigs against lethal E coli challenge in sepsis and septic shock models. In addition to the efficacy of ciVAX against homologous challenge, PAMPS isolated from an infected animal protects other animals against infection by heterologous challenge using different E. coli serotypes - demonstrating the potential for use of ciVAX in controlling pandemics. The advantage of the ciVAX technology is the strong immunogenicity with limited reactogenicity, the use of inactivated pathogens, and the modular manufacture using cGMP approved products which can be stockpiled ready for the next pandemic. One Sentence SummaryBiomaterial vaccine induces strong immunogenicity, weak reactogenicity, and protects from E. coli sepsis in rodents and pigs, and MRSA skin abscess.

immunology↗

bioRxiv2019

Lymph node follicle formation and vaccination responses reconstituted in vitro in a human Organ Chip

Lymphoid follicles (LFs) are responsible for generation of adaptive immune responses in secondary lymphoid organs and form ectopically during chronic inflammation. A human model of LF formation would provide a tool to understand LF development and an alternative to non-human primate models for preclinical evaluation of vaccines. Here, we show that primary human blood B- and T-lymphocytes autonomously assemble into ectopic LFs when cultured in a three-dimensional (3D) extracellular matrix gel within an organ-on-a-chip microfluidic device. Dynamic fluid flow is required for LF formation and prevention of lymphocyte autoactivation. These germinal center-like LFs contain B cells expressing Activation-Induced Cytidine Deaminase and exhibit plasma cell (PC) differentiation upon activation. To explore their utility for vaccine testing, autologous monocyte-derived dendritic cells were integrated into LF Chips. The human LF chips demonstrated improved antibody responses to split virion influenza vaccination compared to 2D cultures, which were enhanced by addition of a squalene-in-water emulsion adjuvant, and this was accompanied by increases in LF size and number. When inoculated with commercial influenza vaccine, PC formation and production of anti-hemagglutinin IgG were observed, as well as secretion of cytokines similar to those observed in vaccinated humans over clinically relevant timescales.

immunology↗

bioRxiv2019

Human colon-on-a-chip enables continuous in vitro analysis of colon mucus layer accumulation and physiology

Background & AimsThe mucus layer in the human colon protects against commensal bacteria and pathogens, and defects in its unique bilayered structure contribute to intestinal disorders, such as ulcerative colitis. However, our understanding of colon physiology is limited by the lack of in vitro models that replicate human colonic mucus layer structure and function. Here, we investigated if combining organ-on-a-chip and organoid technologies can be leveraged to develop a human-relevant in vitro model of colon mucus physiology. MethodsA human colon-on-a-chip (Colon Chip) microfluidic device lined by primary patient-derived colonic epithelial cells was used to recapitulate mucus bilayer formation, and to visualize mucus accumulation in living cultures non-invasively. ResultsThe Colon Chip supports spontaneous goblet cell differentiation and accumulation of a mucus bilayer with impenetrable and penetrable layers, and a thickness similar to that observed in human colon, while maintaining a subpopulation of proliferative epithelial cells. Live imaging of the mucus layer formation on-chip revealed that stimulation of the colonic epithelium with prostaglandin E2, which is elevated during inflammation, causes rapid mucus volume expansion via an NKCC1 ion channel-dependent increase in its hydration state, but no increase in de novo mucus secretion. ConclusionThis study is the first to demonstrate production of colonic mucus with a physiologically relevant bilayer structure in vitro, which can be analyzed in real-time non-invasively. The Colon Chip may offer a new preclinical tool to analyze the role of mucus in human intestinal homeostasis as well as diseases, such as ulcerative colitis and cancer.

cell biology↗

bioRxiv2019

Molecular mapping of transmembrane mechanotransduction through the β1 integrin-CD98hc-TRPV4 axis

One of the most rapid (< 4 msec) transmembrane cellular mechanotransduction events involves activation of Transient Receptor Potential Vanilloid 4 (TRPV4) ion channels by mechanical forces transmitted across {beta}1 integrin receptors in endothelial cells, and the transmembrane Solute Carrier family 3 member 2 (CD98hc) protein has been implicated in this response. Here, we show that {beta}1 integrin, CD98hc, and TRPV4 all tightly associate and co-localize in focal adhesions where mechanochemical conversion takes place. CD98hc knock down inhibits TRPV4-mediated calcium influx induced by mechanical forces, but not by chemical activators, thus confirming the mechanospecificity of this signaling response. Molecular analysis revealed that forces applied to {beta}1 integrin must be transmitted from its cytoplasmic C-terminus to the CD98hc cytoplasmic tail, and from there to the ankyrin repeat domain of TRPV4 in order to produce ultra-rapid, force-induced, channel activation within the focal adhesion.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC=\"FIGDIR/small/617092v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (44K):\norg.highwire.dtl.DTLVardef@11bfb51org.highwire.dtl.DTLVardef@10bcdaorg.highwire.dtl.DTLVardef@debf89org.highwire.dtl.DTLVardef@1f0bee5_HPS_FORMAT_FIGEXP M_FIG Graphical abstract\n\nC_FIG SUMMARYA direct path of mechanical signal transfer between {beta}1 integrin, CD98hc, and TRPV4 channels is identified that mediates ultra-rapid transmembrane mechanochemical conversion within focal adhesions.

cell biology↗

bioRxiv2019

Species-specific enhancement of enterohemorrhagic E. Coli pathogenesis mediated by microbiome metabolites

BackgroundSpecies-specific differences in tolerance to infection are exemplified by the high susceptibility of humans to enterohemorrhagic E. coli (EHEC) infection whereas mice are relatively resistant to this pathogen. This intrinsic species-specific difference in EHEC infection limits the translation of murine research to human. Furthermore, studying the mechanisms underlying this differential susceptibility is a difficult problem due to complex in vivo interactions between the host, pathogen, and disparate commensal microbial communities.\n\nResultsWe utilize organ-on-a-chip (Organ Chip) microfluidic culture technology to model damage of the human colonic epithelium induced by EHEC infection, and show that epithelial injury is greater when exposed to metabolites derived from the human gut microbiome compared to mouse. Using a multi-omics approach, we discovered four human microbiome metabolites -- 4-methyl benzoic acid, 3,4-dimethylbenzoic acid, hexanoic acid, and heptanoic acid -- that are sufficient to mediate this effect. The active human microbiome metabolites preferentially induce expression of flagellin, a bacterial protein associated with motility of EHEC and increased epithelial injury. Thus, the decreased tolerance to infection observed in humans versus other species may be due in part to the presence of compounds produced by the human intestinal microbiome that actively promote bacterial pathogenicity.\n\nConclusionOrgan on chip technology allowed the identification of specific human microbiome metabolites modulating EHEC pathogenesis. These identified metabolites are sufficient to increase susceptibility to EHEC in our human Colon Chip model and they contribute to species-specific tolerance. This work suggests that higher concentrations of these metabolites could be the reason for higher susceptibility to EHEC infection in certain human populations, such as children. Furthermore, this research lays the foundation for therapeutic-modulation of microbe products in order to prevent and treat human bacterial infection.

bioengineering↗

bioRxiv2018

Hypoxia-enhanced Blood-Brain Barrier Chip recapitulates human barrier function, drug penetration, and antibody shuttling properties

The highly specialized human brain microvascular endothelium forms a selective blood-brain barrier (BBB) with adjacent pericytes and astrocytes that restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system. Here, we describe an in vitro microfluidic organ-on-a-chip (Organ Chip) model of the BBB lined by induced pluripotent stem cell-derived human brain microvascular endothelium (iPS-BMVEC) interfaced with primary human brain astrocytes and pericytes that recapitulates the high level of barrier function of the in vivo human BBB for at least one week in culture. The endothelium expresses high levels of tight junction proteins, multiple functional efflux pumps, and displays selective transcytosis of peptides and anti-transferrin receptor antibodies previously observed in vivo. This increased level of barrier functionality was accomplished using a developmentally-inspired induction protocol that includes a period of differentiation under hypoxic conditions. This enhanced BBB Chip may therefore represent a new in vitro tool for development and validation of delivery systems that transport drugs and therapeutic antibodies across the human BBB.\n\nThe human blood-brain barrier (BBB) is a unique and selective physiological barrier that controls transport between the blood and the central nervous system (CNS) to maintain homeostasis for optimal brain function. The BBB is composed of brain microvascular endothelial cells (BMVECs) that line the capillaries as well as surrounding extracellular matrix (ECM), pericytes, and astrocytes, which create a microenvironment that is crucial to BBB function1. The brain microvascular endothelium differs from that found in peripheral capillaries based on its complex tight junctions, which restrict paracellular transit and instead, require that transcytosis be used to transport molecules from the blood through the endothelium and into the CNS2. BMVECs also express multiple broad-spectrum efflux pumps on their luminal surface that inhibit uptake of lipophilic molecules, including many drugs, into the brain3,4. The astrocytes and pericytes provide signals that are required for differentiation of the BMVECs5,6, and all three cell types are needed to maintain BBB integrity in vivo as well as in vitro7-9. The BBB is also of major clinical relevance because dysfunction of the BBB associated is observed in many neurological diseases, and the efficacy of drugs designed to treat neurological disorders is often limited by their inability to cross the BBB10. Unfortunately, neither animal models of the BBB nor in vitro cultures of primary or immortalized human BMVECs alone effectively mimic the barrier and transporter functions of the BBB observed in humans11-14. Thus, there is a great need for a human BBB model that could be used to develop new and more effective CNS-targeting therapeutics and delivery technologies as well as advance fundamental and translational research8,9.\n\nDevelopment of human induced pluripotent stem (iPS) cell technology has enabled differentiation of brain-like microvascular endothelial cells (iPS-BMVECs) that exhibit many properties of the human BBB, including well-organized tight junctions, expression of nutrient transporters and polarized efflux transporter activity15,16. The trans-endothelial electrical resistance (TEER) values exhibited by the permeability barrier generated by these human iPS-BMVECs reach physiological levels ([~]3000-5000 {Omega}{middle dot}cm2) within 24-48 h when cultured in Transwell inserts or within a microfluidic organ-on-a-chip (Organ Chip) device15,17-19, a level that is more than an order of magnitude higher than TEER values previously reported in other in vitro human BBB models6,17,20.\n\nHowever, the usefulness of these iPS-BMVEC models for studies on targeted delivery to the CNS is limited because they can only maintain these high TEER levels for [~]2 days, and the expression of efflux pumps in these iPS-BMVECs does not fully mimic those of human brain endothelium in vivo21. Here, we describe the development of an enhanced human BBB model created with microfluidic Organ Chip culture technology22,23 that contains human iPS-BMVECs interfaced with primary human pericytes and astrocytes, and that uses a developmentally-inspired differentiation protocol24-26. The resulting human BBB Chip exhibits physiologically relevant levels of human BBB function for at least one week in vitro, including low barrier permeability and expression of multiple efflux pumps and transporter functions that are required for analysis of drug and therapeutic antibody transport.

bioengineering↗

bioRxiv2018

Human bone marrow disorders recapitulated in vitro using organ chip technology

Understanding human bone marrow (BM) pathophysiology in the context of myelotoxic stress induced by drugs, radiation, or genetic mutations is of critical importance in clinical medicine. However, study of these dynamic cellular responses is hampered by the inaccessibility of living BM in vivo. Here, we describe a vascularized human Bone Marrow-on-a-Chip (BM Chip) microfluidic culture device for modeling bone marrow function and disease states. The BM Chip is comprised of a fluidic channel filled with a fibrin gel in which patient-derived CD34+ cells and bone marrow-derived stromal cells (BMSCs) are co-cultured, which is separated by a porous membrane from a parallel fluidic channel lined by human vascular endothelium. When perfused with culture medium through the vascular channel, the BM Chip maintains human CD34+ cells and supports differentiation and maturation of multiple blood cell lineages over 1 month in culture. Moreover, it recapitulates human myeloerythroid injury responses to drugs and gamma radiation exposure, as well as key hematopoietic abnormalities found in patients with the genetic disorder, Shwachman-Diamond Syndrome (SDS). These data establish the BM Chip as a new human in vitro model with broad potential utility for studies of BM dysfunction.

bioengineering↗

bioRxiv2018

Complex human gut microbiome cultured in anaerobic human intestine chips

The diverse bacterial populations that comprise the commensal microbiota of the human intestine play a central role in health and disease, yet no method is available to sustain these complex microbial communities in direct contact with living human intestinal cells and their overlying mucus layer in vitro. Here we describe a human Organ-on-a-Chip (Organ Chip) microfluidic platform that permits control and real-time assessment of physiologically-relevant oxygen gradients, and which enables co-culture of living human intestinal epithelium with stable communities of aerobic and anaerobic human gut microbiota. When compared to aerobic co-culture conditions, establishment of a transluminal hypoxia gradient sustained higher microbial diversity with over 200 unique operational taxonomic units (OTUs) from 11 different genera, and an abundance of obligate anaerobic bacteria with ratios of Firmicutes and Bacteroidetes similar to those observed in human feces, in addition to increasing intestinal barrier function. The ability to culture human intestinal epithelium overlaid by complex human gut microbial communities within microfluidic Intestine Chips may enable investigations of host-microbiome interactions that were not possible previously, and serve as a discovery tool for development of new microbiome-related therapeutics, probiotics, and nutraceuticals.

bioengineering↗

bioRxiv2018

Broad spectrum capture of clinical pathogens using engineered Fc-Mannose-Binding Lectin (FcMBL) enhanced by antibiotic treatment

FcMBL, an engineered version of the blood opsonin mannose-binding lectin (MBL) that contains the carbohydrate recognition domain (CRD) and flexible neck regions of MBL fused to the Fc portion of human IgG1, has been shown to bind various microbes and pathogen-associated molecular patterns (PAMPs). FcMBL also has been used to create an enzyme-linked lectin sorbent assay (ELLecSA) for use as a rapid (< 1 hr) diagnostic of bloodstream infections. Here we extended this work by using the ELLecSA to test FcMBLs ability to bind to more than 200 different isolates from over 100 different pathogen species. FcMBL bound to 86% of the isolates and 110 of the 122 (90%) different pathogen species tested, including bacteria, fungi, viruses, and parasites. It also bound to PAMPs including, lipopolysaccharide endotoxin (LPS) and lipoteichoic acid (LTA) from Gram-negative and Gram-positive bacteria, as well as lipoarabinomannan (LAM) and phosphatidylinositol mannoside 6 (PIM6) from Mycobacterium tuberculosis. The efficiency of pathogen detection and variation between binding of different strains of the same species also could be improved by treating the bacteria with antibiotics prior to FcMBL capture to reveal previously concealed binding sites within the bacterial cell wall. As FcMBL can bind to pathogens and PAMPs in urine as well as blood, its broad-binding capability could be leveraged to develop a variety of clinically relevant technologies, including infectious disease diagnostics, therapeutics, and vaccines.

immunology↗