Publications by authors named "Donald E Ingber"

243 Publications

Is it Time for Reviewer 3 to Request Human Organ Chip Experiments Instead of Animal Validation Studies?

Authors:
Donald E Ingber

Adv Sci (Weinh) 2020 Nov 12;7(22):2002030. Epub 2020 Oct 12.

Wyss Institute for Biologically Inspired Engineering at Harvard University Boston MA 02115 USA.

For the past century, experimental data obtained from animal studies have been required by reviewers of scientific articles and grant applications to validate the physiological relevance of in vitro results. At the same time, pharmaceutical researchers and regulatory agencies recognize that results from preclinical animal models frequently fail to predict drug responses in humans. This reviews recent advances in human organ-on-a-chip (Organ Chip) microfluidic culture technology, both with single Organ Chips and fluidically coupled human "Body-on-Chips" platforms, which demonstrate their ability to recapitulate human physiology and disease states, as well as human patient responses to clinically relevant drug pharmacokinetic exposures, with higher fidelity than other in vitro models or animal studies. These findings raise the question of whether continuing to require results of animal testing for publication or grant funding still makes scientific or ethical sense, and if more physiologically relevant human Organ Chip models might better serve this purpose. This issue is addressed in this article in context of the history of the field, and advantages and disadvantages of Organ Chip approaches versus animal models are discussed that should be considered by the wider research community.
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http://dx.doi.org/10.1002/advs.202002030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7675190PMC
November 2020

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

J Cell Sci 2020 Nov 2;133(20). Epub 2020 Nov 2.

Vascular Biology Program, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA

One of the most rapid (less than 4 ms) transmembrane cellular mechanotransduction events involves activation of transient receptor potential vanilloid 4 (TRPV4) ion channels by mechanical forces transmitted across cell surface β1 integrin receptors on endothelial cells, and the transmembrane solute carrier family 3 member 2 (herein denoted CD98hc, also known as SLC3A2) protein has been implicated in this response. Here, we show that β1 integrin, CD98hc and TRPV4 all tightly associate and colocalize in focal adhesions where mechanochemical conversion takes place. CD98hc knockdown inhibits TRPV4-mediated calcium influx induced by mechanical forces, but not by chemical activators, thus confirming the mechanospecificity of this signaling response. Molecular analysis reveals that forces applied to β1 integrin must be transmitted from its cytoplasmic C terminus via the CD98hc cytoplasmic tail to the ankyrin repeat domain of TRPV4 in order to produce ultrarapid, force-induced channel activation within the focal adhesion.
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http://dx.doi.org/10.1242/jcs.248823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657480PMC
November 2020

Treatment of psoriasis with NFKBIZ siRNA using topical ionic liquid formulations.

Sci Adv 2020 Jul 22;6(30):eabb6049. Epub 2020 Jul 22.

John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.

Systemic antibodies targeting tumor necrosis factor-α (TNF-α) and interleukin-17A (IL-17A) are effective in plaque psoriasis. Despite their popularity, safety concerns pose a challenge for systemic biologics. While anti-TNF-α and anti-IL-17A antibodies effectively inhibit respective proteins, we hypothesize that an approach based on local silencing of an upstream target such as NFKBIZ can be advantageous for treating psoriasis. However, effective delivery of small interfering RNA (siRNA) into the skin is a substantial hurdle due to skin's barrier function and poor stability of siRNA. Using ionic liquids as an enabling technology, we report on the effective delivery of NFKBIZ siRNA into the skin and its therapeutic efficacy in a psoriasis model. Treatment with IL-siRNA suppressed aberrant gene expression and resulted in down-regulation of psoriasis-related signals including TNF-α and IL-17A. These results provide a framework for a topical delivery platform for siRNA.
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http://dx.doi.org/10.1126/sciadv.abb6049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439648PMC
July 2020

Proteomic and Metabolomic Characterization of Human Neurovascular Unit Cells in Response to Methamphetamine.

Adv Biosyst 2020 09 3;4(9):e1900230. Epub 2020 Aug 3.

Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA, 02115, USA.

The functional state of the neurovascular unit (NVU), composed of the blood-brain barrier and the perivasculature that forms a dynamic interface between the blood and the central nervous system (CNS), plays a central role in the control of brain homeostasis and is strongly affected by CNS drugs. Human primary brain microvascular endothelium, astrocyte, pericyte, and neural cell cultures are often used to study NVU barrier functions as well as drug transport and efficacy; however, the proteomic and metabolomic responses of these different cell types are not well characterized. Culturing each cell type separately, using deep coverage proteomic analysis and characterization of the secreted metabolome, as well as measurements of mitochondrial activity, the responses of these cells under baseline conditions and when exposed to the NVU-impairing stimulant methamphetamine (Meth) are analyzed. These studies define the previously unknown metabolic and proteomic profiles of human brain pericytes and lead to improved characterization of the phenotype of each of the NVU cell types as well as cell-specific metabolic and proteomic responses to Meth.
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http://dx.doi.org/10.1002/adbi.201900230DOI Listing
September 2020

Human Organs-on-Chips for Virology.

Trends Microbiol 2020 11 13;28(11):934-946. Epub 2020 Jul 13.

Medical Systems Biophysics and Bioengineering, Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands. Electronic address:

While conventional in vitro culture systems and animal models have been used to study the pathogenesis of viral infections and to facilitate development of vaccines and therapeutics for viral diseases, models that can accurately recapitulate human responses to infection are still lacking. Human organ-on-a-chip (Organ Chip) microfluidic culture devices that recapitulate tissue-tissue interfaces, fluid flows, mechanical cues, and organ-level physiology have been developed to narrow the gap between in vitro experimental models and human pathophysiology. Here, we describe how recent developments in Organ Chips have enabled re-creation of complex pathophysiological features of human viral infections in vitro.
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http://dx.doi.org/10.1016/j.tim.2020.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357975PMC
November 2020

Phosphorylation of ACTN4 Leads to Podocyte Vulnerability and Proteinuric Glomerulosclerosis.

J Am Soc Nephrol 2020 07 15;31(7):1479-1495. Epub 2020 Jun 15.

Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts

Background: Genetic mutations in -actinin-4 (ACTN4)-an important actin crosslinking cytoskeletal protein that provides structural support for kidney podocytes-have been linked to proteinuric glomerulosclerosis in humans. However, the effect of post-translational modifications of ACTN4 on podocyte integrity and kidney function is not known.

Methods: Using mass spectrometry, we found that ACTN4 is phosphorylated at serine (S) 159 in human podocytes. We used phosphomimetic and nonphosphorylatable ACTN4 to comprehensively study the effects of this phosphorylation and . We conducted x-ray crystallography, F-actin binding and bundling assays, and immunofluorescence staining to evaluate F-actin alignment. Microfluidic organ-on-a-chip technology was used to assess for detachment of podocytes simultaneously exposed to fluid flow and cyclic strain. We then used CRISPR/Cas9 to generate mouse models and assessed for renal injury by measuring albuminuria and examining kidney histology. We also performed targeted mass spectrometry to determine whether high extracellular glucose or TGF- levels increase phosphorylation of ACTN4.

Results: Compared with the wild type ACTN4, phosphomimetic ACTN4 demonstrated increased binding and bundling activity with F-actin . Phosphomimetic Actn4 mouse podocytes exhibited more spatially correlated F-actin alignment and a higher rate of detachment under mechanical stress. Phosphomimetic Actn4 mice developed proteinuria and glomerulosclerosis after subtotal nephrectomy. Moreover, we found that exposure to high extracellular glucose or TGF- stimulates phosphorylation of ACTN4 at S159 in podocytes.

Conclusions: These findings suggest that increased phosphorylation of ACTN4 at S159 leads to biochemical, cellular, and renal pathology that is similar to pathology resulting from human disease-causing mutations in ACTN4. ACTN4 may mediate podocyte injury as a consequence of both genetic mutations and signaling events that modulate phosphorylation.
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http://dx.doi.org/10.1681/ASN.2019101032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351002PMC
July 2020

Biology-inspired microphysiological systems to advance patient benefit and animal welfare in drug development

ALTEX 2020 28;37(3):365-394. Epub 2020 Feb 28.

F. Hoffmann-La Roche Ltd, Roche Innovation Center Basel, Switzerland.

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.
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http://dx.doi.org/10.14573/altex.2001241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7863570PMC
February 2020

YAP Regulates Hematopoietic Stem Cell Formation in Response to the Biomechanical Forces of Blood Flow.

Dev Cell 2020 02 6;52(4):446-460.e5. Epub 2020 Feb 6.

Stem Cell Program, Division of Pediatric Hematology and Oncology, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address:

Hematopoietic stem and progenitor cells (HSPCs), first specified from hemogenic endothelium (HE) in the ventral dorsal aorta (VDA), support lifelong hematopoiesis. Their de novo production promises significant therapeutic value; however, current in vitro approaches cannot efficiently generate multipotent long-lived HSPCs. Presuming this reflects a lack of extrinsic cues normally impacting the VDA, we devised a human dorsal aorta-on-a-chip platform that identified Yes-activated protein (YAP) as a cyclic stretch-induced regulator of HSPC formation. In the zebrafish VDA, inducible Yap overexpression significantly increased runx1 expression in vivo and the number of CD41 HSPCs downstream of HE specification. Endogenous Yap activation by lats1/2 knockdown or Rho-GTPase stimulation mimicked Yap overexpression and induced HSPCs in embryos lacking blood flow. Notably, in static human induced pluripotent stem cell (iPSC)-derived HE culture, compound-mediated YAP activation enhanced RUNX1 levels and hematopoietic colony-forming potential. Together, our findings reveal a potent impact of hemodynamic Rho-YAP mechanotransduction on HE fate, relevant to de novo human HSPC production.
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http://dx.doi.org/10.1016/j.devcel.2020.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398148PMC
February 2020

Quantitative prediction of human pharmacokinetic responses to drugs via fluidically coupled vascularized organ chips.

Nat Biomed Eng 2020 04 27;4(4):421-436. Epub 2020 Jan 27.

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.

Analyses of drug pharmacokinetics (PKs) and pharmacodynamics (PDs) performed in animals are often not predictive of drug PKs and PDs in humans, and in vitro PK and PD modelling does not provide quantitative PK parameters. Here, we show that physiological PK modelling of first-pass drug absorption, metabolism and excretion in humans-using computationally scaled data from multiple fluidically linked two-channel organ chips-predicts PK parameters for orally administered nicotine (using gut, liver and kidney chips) and for intravenously injected cisplatin (using coupled bone marrow, liver and kidney chips). The chips are linked through sequential robotic liquid transfers of a common blood substitute by their endothelium-lined channels (as reported by Novak et al. in an associated Article) and share an arteriovenous fluid-mixing reservoir. We also show that predictions of cisplatin PDs match previously reported patient data. The quantitative in-vitro-to-in-vivo translation of PK and PD parameters and the prediction of drug absorption, distribution, metabolism, excretion and toxicity through fluidically coupled organ chips may improve the design of drug-administration regimens for phase-I clinical trials.
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http://dx.doi.org/10.1038/s41551-019-0498-9DOI Listing
April 2020

Robotic fluidic coupling and interrogation of multiple vascularized organ chips.

Nat Biomed Eng 2020 04 27;4(4):407-420. Epub 2020 Jan 27.

Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA, USA.

Organ chips can recapitulate organ-level (patho)physiology, yet pharmacokinetic and pharmacodynamic analyses require multi-organ systems linked by vascular perfusion. Here, we describe an 'interrogator' that employs liquid-handling robotics, custom software and an integrated mobile microscope for the automated culture, perfusion, medium addition, fluidic linking, sample collection and in situ microscopy imaging of up to ten organ chips inside a standard tissue-culture incubator. The robotic interrogator maintained the viability and organ-specific functions of eight vascularized, two-channel organ chips (intestine, liver, kidney, heart, lung, skin, blood-brain barrier and brain) for 3 weeks in culture when intermittently fluidically coupled via a common blood substitute through their reservoirs of medium and endothelium-lined vascular channels. We used the robotic interrogator and a physiological multicompartmental reduced-order model of the experimental system to quantitatively predict the distribution of an inulin tracer perfused through the multi-organ human-body-on-chips. The automated culture system enables the imaging of cells in the organ chips and the repeated sampling of both the vascular and interstitial compartments without compromising fluidic coupling.
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http://dx.doi.org/10.1038/s41551-019-0497-xDOI Listing
April 2020

On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.

Nat Biomed Eng 2020 04 27;4(4):394-406. Epub 2020 Jan 27.

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.

The inaccessibility of living bone marrow (BM) hampers the study of its pathophysiology under myelotoxic stress induced by drugs, radiation or genetic mutations. Here, we show that a vascularized human BM-on-a-chip (BM chip) supports the differentiation and maturation of multiple blood cell lineages over 4 weeks while improving CD34 cell maintenance, and that it recapitulates aspects of BM injury, including myeloerythroid toxicity after clinically relevant exposures to chemotherapeutic drugs and ionizing radiation, as well as BM recovery after drug-induced myelosuppression. The chip comprises a fluidic channel filled with a fibrin gel in which CD34 cells and BM-derived stromal cells are co-cultured, a parallel channel lined by human vascular endothelium and perfused with culture medium, and a porous membrane separating the two channels. We also show that BM chips containing cells from patients with the rare genetic disorder Shwachman-Diamond syndrome reproduced key haematopoietic defects and led to the discovery of a neutrophil maturation abnormality. As an in vitro model of haematopoietic dysfunction, the BM chip may serve as a human-specific alternative to animal testing for the study of BM pathophysiology.
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http://dx.doi.org/10.1038/s41551-019-0495-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160021PMC
April 2020

Biomimetic smoking robot for in vitro inhalation exposure compatible with microfluidic organ chips.

Nat Protoc 2020 02 10;15(2):183-206. Epub 2020 Jan 10.

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.

Exposure of lung tissues to cigarette smoke is a major cause of human disease and death worldwide. Unfortunately, adequate model systems that can reliably recapitulate disease biogenesis in vitro, including exposure of the human lung airway to fresh whole cigarette smoke (WCS) under physiological breathing airflow, are lacking. This protocol extension builds upon, and can be used with, our earlier protocol for microfabrication of human organs-on-chips. Here, we describe the engineering, assembly and operation of a microfluidically coupled, multi-compartment platform that bidirectionally 'breathes' WCS through microchannels of a human lung small airway microfluidic culture device, mimicking how lung cells may experience smoke in vivo. Several WCS-exposure systems have been developed, but they introduce smoke directly from above the cell cultures, rather than tangentially as naturally occurs in the lung due to lateral airflow. We detail the development of an organ chip-compatible microrespirator and a smoke machine to simulate breathing behavior and smoking topography parameters such as puff time, inter-puff interval and puffs per cigarette. Detailed design files, assembly instructions and control software are provided. This novel platform can be fabricated and assembled in days and can be used repeatedly. Moderate to advanced engineering and programming skills are required to successfully implement this protocol. When coupled with the small airway chip, this protocol can enable prediction of patient-specific biological responses in a matched-comparative manner. We also demonstrate how to adapt the protocol to expose living ciliated airway epithelial cells to smoke generated by electronic cigarettes (e-cigarettes) on-chip.
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http://dx.doi.org/10.1038/s41596-019-0230-yDOI Listing
February 2020

AAV-mediated gene therapy targeting TRPV4 mechanotransduction for inhibition of pulmonary vascular leakage.

APL Bioeng 2019 Dec 2;3(4):046103. Epub 2019 Dec 2.

Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts 02115, USA.

Enhanced vascular permeability in the lungs can lead to pulmonary edema, impaired gas exchange, and ultimately respiratory failure. While oxygen delivery, mechanical ventilation, and pressure-reducing medications help alleviate these symptoms, they do not treat the underlying disease. Mechanical activation of transient receptor potential vanilloid 4 (TRPV4) ion channels contributes to the development of pulmonary vascular disease, and overexpression of the high homology (HH) domain of the TRPV4-associated transmembrane protein CD98 has been shown to inhibit this pathway. Here, we describe the development of an adeno-associated virus (AAV) vector encoding the CD98 HH domain in which the AAV serotypes and promoters have been optimized for efficient and specific delivery to pulmonary cells. AAV-mediated gene delivery of the CD98 HH domain inhibited TRPV4 mechanotransduction in a specific manner and protected against pulmonary vascular leakage in a human lung Alveolus-on-a-Chip model. As AAV has been used clinically to deliver other gene therapies, these data raise the possibility of using this type of targeted approach to develop mechanotherapeutics that target the TRPV4 pathway for treatment of pulmonary edema in the future.
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http://dx.doi.org/10.1063/1.5122967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887658PMC
December 2019

Human Colon-on-a-Chip Enables Continuous In Vitro Analysis of Colon Mucus Layer Accumulation and Physiology.

Cell Mol Gastroenterol Hepatol 2020 26;9(3):507-526. Epub 2019 Nov 26.

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts; Department of Surgery, Harvard Medical School, Boston Children's Hospital, Boston, Massachusetts; Vascular Biology Program and Department Surgery, Harvard Medical School, Boston Children's Hospital, Boston, Massachusetts; Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University, Boston, Massachusetts. Electronic address:

Background & Aims: The 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.

Methods: A 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 noninvasively.

Results: The 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 the human colon, while maintaining a subpopulation of proliferative epithelial cells. Live imaging of the mucus layer formation on-chip showed that stimulation of the colonic epithelium with prostaglandin E2, which is increased during inflammation, causes rapid mucus volume expansion via an Na-K-Cl cotransporter 1 ion channel-dependent increase in its hydration state, but no increase in de novo mucus secretion.

Conclusions: This study shows the production of colonic mucus with a physiologically relevant bilayer structure in vitro, which can be analyzed in real time noninvasively. 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.
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http://dx.doi.org/10.1016/j.jcmgh.2019.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036549PMC
November 2019

An antifouling coating that enables affinity-based electrochemical biosensing in complex biological fluids.

Nat Nanotechnol 2019 12 11;14(12):1143-1149. Epub 2019 Nov 11.

Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA, USA.

Affinity-based electrochemical detection in complex biological fluids could enable multiplexed point-of-care diagnostics for home healthcare; however, commercialization of point-of-care devices has been limited by the rapid loss of sensitivity caused by electrode surface inactivation and biofouling. Here, we describe a simple and robust antifouling coating for electrodes consisting of a three-dimensional porous matrix of cross-linked bovine serum albumin supported by a network of conductive nanomaterials composed of either gold nanowires, gold nanoparticles or carbon nanotubes. These nanocomposites prevent non-specific interactions while enhancing electron transfer to the electrode surface, preserving 88% of the original signal after 1 month of exposure to unprocessed human plasma, and functionalization with specific antibodies enables quantification of anti-interleukin 6 in plasma with high sensitivity. The easy preparation, stability and simplicity of this nanocomposite allow the generation of electrochemical biosensors that can operate in complex biological fluids such as blood plasma or serum.
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http://dx.doi.org/10.1038/s41565-019-0566-zDOI Listing
December 2019

Reproducing human and cross-species drug toxicities using a Liver-Chip.

Sci Transl Med 2019 11;11(517)

Emulate Inc., 27 Drydock Avenue, Boston, MA 02210, USA.

Nonclinical rodent and nonrodent toxicity models used to support clinical trials of candidate drugs may produce discordant results or fail to predict complications in humans, contributing to drug failures in the clinic. Here, we applied microengineered Organs-on-Chips technology to design a rat, dog, and human Liver-Chip containing species-specific primary hepatocytes interfaced with liver sinusoidal endothelial cells, with or without Kupffer cells and hepatic stellate cells, cultured under physiological fluid flow. The Liver-Chip detected diverse phenotypes of liver toxicity, including hepatocellular injury, steatosis, cholestasis, and fibrosis, and species-specific toxicities when treated with tool compounds. A multispecies Liver-Chip may provide a useful platform for prediction of liver toxicity and inform human relevance of liver toxicities detected in animal studies to better determine safety and human risk.
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http://dx.doi.org/10.1126/scitranslmed.aax5516DOI Listing
November 2019

Tumor-Derived Extracellular Vesicles Breach the Intact Blood-Brain Barrier Transcytosis.

ACS Nano 2019 12 10;13(12):13853-13865. Epub 2019 Sep 10.

Vascular Biology Program , Boston Children's Hospital , Boston , Massachusetts 02115 , United States.

The restrictive nature of the blood-brain barrier (BBB) creates a major challenge for brain drug delivery with current nanomedicines lacking the ability to cross the BBB. Extracellular vesicles (EVs) have been shown to contribute to the progression of a variety of brain diseases including metastatic brain cancer and have been suggested as promising therapeutics and drug delivery vehicles. However, the ability of native tumor-derived EVs to breach the BBB and the mechanism(s) involved in this process remain unknown. Here, we demonstrate that tumor-derived EVs can breach the intact BBB , and by using state-of-the-art and models of the BBB, we have identified transcytosis as the mechanism underlying this process. Moreover, high spatiotemporal resolution microscopy demonstrated that the endothelial recycling endocytic pathway is involved in this transcellular transport. We further identify and characterize the mechanism by which tumor-derived EVs circumvent the low physiologic rate of transcytosis in the BBB by decreasing the brain endothelial expression of rab7 and increasing the efficiency of their transport. These findings identify previously unknown mechanisms by which tumor-derived EVs breach an intact BBB during the course of brain metastasis and can be leveraged to guide and inform the development of drug delivery approaches to deliver therapeutic cargoes across the BBB for treatment of a variety of brain diseases including, but not limited to, brain malignancies.
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http://dx.doi.org/10.1021/acsnano.9b04397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7169949PMC
December 2019

Controllable Fabrication of Inhomogeneous Microcapsules for Triggered Release by Osmotic Pressure.

Small 2019 10 25;15(42):e1903087. Epub 2019 Aug 25.

John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA.

Inhomogeneous microcapsules that can encapsulate various cargo for controlled release triggered by osmotic shock are designed and reported. The microcapsules are fabricated using a microfluidic approach and the inhomogeneity of shell thickness in the microcapsules can be controlled by tuning the flow rate ratio of the middle phase to the inner phase. This study demonstrates the swelling of these inhomogeneous microcapsules begins at the thinnest part of shell and eventually leads to rupture at the weak spot with a low osmotic pressure. Systematic studies indicate the rupture fraction of these microcapsules increases with increasing inhomogeneity, while the rupture osmotic pressure decreases linearly with increasing inhomogeneity. The inhomogeneous microcapsules are demonstrated to be impermeable to small probe molecules, which enables long-term storage. Thus, these microcapsules can be used for long-term storage of enzymes, which can be controllably released through osmotic shock without impairing their biological activity. The study provides a new approach to design effective carriers to encapsulate biomolecules and release them on-demand upon applying osmotic shock.
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http://dx.doi.org/10.1002/smll.201903087DOI Listing
October 2019

Broad-spectrum capture of clinical pathogens using engineered Fc-mannose-binding lectin enhanced by antibiotic treatment.

F1000Res 2019 25;8:108. Epub 2019 Jan 25.

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, 02115, USA.

Fc-mannose-binding lectin (FcMBL), an engineered version of the blood opsonin 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 has also been used to create an enzyme-linked lectin sorbent assay (ELLecSA) for use as a rapid (<1 h) diagnostic of bloodstream infections. Here we extended this work by using the ELLecSA to test FcMBL's ability to bind to more than 190 different isolates from over 95 different pathogen species. FcMBL bound to 85% of the isolates and 97 of the 112 (87%) different pathogen species tested, including bacteria, fungi, viral antigens and parasites. FcMBL 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 (PIM ) from . The efficiency of pathogen detection and variation between binding of different strains of the same species could be improved by treating the bacteria with antibiotics, or mechanical disruption using a bead mill, 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.
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http://dx.doi.org/10.12688/f1000research.17447.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544136PMC
June 2020

Author Correction: A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip.

Nat Biomed Eng 2019 Jul;3(7):583

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.

In the version of this Article originally published, the authors mistakenly cited Fig. 5d in the sentence beginning 'Importantly, the microbiome cultured in these primary Intestine Chips...'; the correct citation is Supplementary Table 2. This has now been amended.
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http://dx.doi.org/10.1038/s41551-019-0428-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7735410PMC
July 2019

Cellular nanoscale stiffness patterns governed by intracellular forces.

Nat Mater 2019 10 17;18(10):1071-1077. Epub 2019 Jun 17.

Department of Biological Sciences, Columbia University, New York, NY, USA.

Cell stiffness measurements have led to insights into various physiological and pathological processes. Although many cellular behaviours are influenced by intracellular mechanical forces that also alter the material properties of the cell, the precise mechanistic relationship between intracellular forces and cell stiffness remains unclear. Here we develop a cell mechanical imaging platform with high spatial resolution that reveals the existence of nanoscale stiffness patterns governed by intracellular forces. On the basis of these findings, we develop and validate a cellular mechanical model that quantitatively relates cell stiffness to intracellular forces. This allows us to determine the magnitude of tension within actin bundles, cell cortex and plasma membrane from the cell stiffness patterns across individual cells. These results expand our knowledge on the mechanical interaction between cells and their environments, and offer an alternative approach to determine physiologically relevant intracellular forces from high-resolution cell stiffness images.
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http://dx.doi.org/10.1038/s41563-019-0391-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754298PMC
October 2019

Hypoxia-enhanced Blood-Brain Barrier Chip recapitulates human barrier function and shuttling of drugs and antibodies.

Nat Commun 2019 06 13;10(1):2621. Epub 2019 Jun 13.

Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA, 02115, USA.

The high selectivity of the human blood-brain barrier (BBB) restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system. Here, we describe an in vitro microfluidic organ-on-a-chip BBB model lined by induced pluripotent stem cell-derived human brain microvascular endothelium 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 and functional efflux pumps, and it displays selective transcytosis of peptides and antibodies previously observed in vivo. Increased 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.
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http://dx.doi.org/10.1038/s41467-019-10588-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565686PMC
June 2019

Human Intestinal Morphogenesis Controlled by Transepithelial Morphogen Gradient and Flow-Dependent Physical Cues in a Microengineered Gut-on-a-Chip.

iScience 2019 May 3;15:391-406. Epub 2019 May 3.

Department of Biomedical Engineering, Cockrell School of Engineering, The University of Texas at Austin, Austin, TX 78712, USA; Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, TX 78712, USA; Department of Medical Engineering, Yonsei University College of Medicine, Seoul 03722, Republic of Korea. Electronic address:

We leveraged a human gut-on-a-chip (Gut Chip) microdevice that enables independent control of fluid flow and mechanical deformations to explore how physical cues and morphogen gradients influence intestinal morphogenesis. Both human intestinal Caco-2 and intestinal organoid-derived primary epithelial cells formed three-dimensional (3D) villi-like microarchitecture when exposed to apical and basal fluid flow; however, 3D morphogenesis did not occur and preformed villi-like structure involuted when basal flow was ceased. When cells were cultured in static Transwells, similar morphogenesis could be induced by removing or diluting the basal medium. Computational simulations and experimental studies revealed that the establishment of a transepithelial gradient of the Wnt antagonist Dickkopf-1 and flow-induced regulation of the Frizzled-9 receptor mediate the histogenesis. Computational simulations also predicted spatial growth patterns of 3D epithelial morphology observed experimentally in the Gut Chip. A microengineered Gut Chip may be useful for studies analyzing stem cell biology and tissue development.
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http://dx.doi.org/10.1016/j.isci.2019.04.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6526295PMC
May 2019

A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip.

Nat Biomed Eng 2019 07 13;3(7):520-531. Epub 2019 May 13.

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.

The diverse bacterial populations that comprise the commensal microbiome of the human intestine play a central role in health and disease. A method that sustains complex microbial communities in direct contact with living human intestinal cells and their overlying mucus layer in vitro would thus enable the investigation of host-microbiome interactions. Here, we show the extended coculture of living human intestinal epithelium with stable communities of aerobic and anaerobic human gut microbiota, using a microfluidic intestine-on-a-chip that permits the control and real-time assessment of physiologically relevant oxygen gradients. When compared to aerobic coculture conditions, the establishment of a transluminal hypoxia gradient in the chip increased intestinal barrier function and sustained a physiologically relevant level of microbial diversity, consisting of over 200 unique operational taxonomic units from 11 different genera and an abundance of obligate anaerobic bacteria, with ratios of Firmicutes and Bacteroidetes similar to those observed in human faeces. The intestine-on-a-chip may serve as a discovery tool for the development of microbiome-related therapeutics, probiotics and nutraceuticals.
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http://dx.doi.org/10.1038/s41551-019-0397-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658209PMC
July 2019

Seeing Your Way to New Insights in Biology.

J Mol Biol 2019 06 26;431(14):2485-2486. Epub 2019 Apr 26.

Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA 02115, USA; Vascular Biology Program, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; "aHarvard John A. Paulson School of Engineering and Applied Sciences, Cambridge, MA 02139, USA.

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http://dx.doi.org/10.1016/j.jmb.2019.04.033DOI Listing
June 2019

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

Microbiome 2019 03 20;7(1):43. Epub 2019 Mar 20.

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, 02115, USA.

Background: Species-specific differences in tolerance to infection are exemplified by the high susceptibility of humans to enterohemorrhagic Escherichia 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.

Results: We 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.

Conclusion: Organ-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.
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http://dx.doi.org/10.1186/s40168-019-0650-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425591PMC
March 2019

Platelet decoys inhibit thrombosis and prevent metastatic tumor formation in preclinical models.

Sci Transl Med 2019 02;11(479)

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA.

Platelets are crucial for normal hemostasis; however, their hyperactivation also contributes to many potentially lethal pathologies including myocardial infarction, stroke, and cancer. We hypothesized that modified platelets lacking their aggregation and activation capacity could act as reversible inhibitors of platelet activation cascades. Here, we describe the development of detergent-extracted human modified platelets (platelet decoys) that retained platelet binding functions but were incapable of functional activation and aggregation. Platelet decoys inhibited aggregation and adhesion of platelets on thrombogenic surfaces in vitro, which could be immediately reversed by the addition of normal platelets; in vivo in a rabbit model, pretreatment with platelet decoys inhibited arterial injury-induced thromboembolism. Decoys also interfered with platelet-mediated human breast cancer cell aggregation, and their presence decreased cancer cell arrest and extravasation in a microfluidic human microvasculature on a chip. In a mouse model of metastasis, simultaneous injection of the platelet decoys with tumor cells inhibited metastatic tumor growth. Thus, our results suggest that platelet decoys might represent an effective strategy for obtaining antithrombotic and antimetastatic effects.
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http://dx.doi.org/10.1126/scitranslmed.aau5898DOI Listing
February 2019

Rapid Coating Process Generates Omniphobic Dentures in Minutes to Reduce Biofouling.

ACS Biomater Sci Eng 2019 Feb 27;5(2):420-424. Epub 2018 Dec 27.

Department of Dentistry, Boston Children's Hospital, Boston, Massachusetts 02115, United States.

Localized infections caused by biofilm formation on dentures pose a serious health risk for patients, especially the elderly, as they can lead to complications such as pneumonia. Daily enzymatic denture cleaners do not fully prevent biofilm formation on dentures. Here we developed a rapid coating process to apply a liquid repellent surface to dentures in ∼5 min and demonstrated a significant 225-fold reduction of adhesion over 6 days, compared to uncoated dentures. This rapid coating process could be applied to dentures and other dental devices chair-side and allow the research community to quickly and easily generate ominphobic surfaces.
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http://dx.doi.org/10.1021/acsbiomaterials.8b01214DOI Listing
February 2019