From animal testing to in vitro systems: advancing standardization in microphysiological systems.

Limitations with cell cultures and experimental animal-based studies have had the scientific and industrial communities searching for new approaches that can provide reliable human models for applications such as drug development, toxicological assessment, and in vitro pre-clinical evaluation. This has resulted in the development of microfluidic-based cultures that may better represent organs and organ systems in vivo than conventional monolayer cell cultures. Although there is considerable interest from industry and regulatory bodies in this technology, several challenges need to be addressed for it to reach its full potential. Among those is a lack of guidelines and standards. Therefore, a multidisciplinary team of stakeholders was formed, with members from the US Food and Drug Administration (FDA), the National Institute of Standards and Technology (NIST), European Union, academia, and industry, to provide a framework for future development of guidelines/standards governing engineering concepts of organ-on-a-chip models. The result of this work is presented here for interested parties, stakeholders, and other standards development organizations (SDOs) to foster further discussion and enhance the impact and benefits of these efforts.

Performance assessment and economic analysis of a human Liver-Chip for predictive toxicology.

BACKGROUND: Conventional preclinical models often miss drug toxicities, meaning the harm these drugs pose to humans is only realized in clinical trials or when they make it to market. This has caused the pharmaceutical industry to waste considerable time and resources developing drugs destined to fail. Organ-on-a-Chip technology has the potential improve success in drug development pipelines, as it can recapitulate organ-level pathophysiology and clinical responses; however, systematic and quantitative evaluations of Organ-Chips' predictive value have not yet been reported. METHODS: 870 Liver-Chips were analyzed to determine their ability to predict drug-induced liver injury caused by small molecules identified as benchmarks by the Innovation and Quality consortium, who has published guidelines defining criteria for qualifying preclinical models. An economic analysis was also performed to measure the value Liver-Chips could offer if they were broadly adopted in supporting toxicity-related decisions as part of preclinical development workflows. RESULTS: Here, we show that 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%. We also show that this level of performance could generate over $3 billion annually for the pharmaceutical industry through increased small-molecule R&D productivity. CONCLUSIONS: The results of this study show how incorporating predictive Organ-Chips into drug development workflows could substantially improve drug discovery and development, allowing manufacturers to bring safer, more effective medicines to market in less time and at lower costs.

Drug development is lengthy and costly, as it relies on laboratory models that fail to predict human reactions to potential drugs. Because of this, toxic drugs sometimes go on to harm humans when they reach clinical trials or once they are in the marketplace. Organ-on-a-Chip technology involves growing cells on small devices to mimic organs of the body, such as the liver. Organ-Chips could potentially help identify toxicities earlier, but there is limited research into how well they predict these effects compared to conventional models. In this study, we analyzed 870 Liver-Chips to determine how well they predict drug-induced liver injury, a common cause of drug failure, and found that Liver-Chips outperformed conventional models. These results suggest that widespread acceptance of Organ-Chips could decrease drug attrition, help minimize harm to patients, and generate billions in revenue for the pharmaceutical industry.

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality.

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed. Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions. Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure for the in vitro modeling of interferon gamma (IFNγ)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

O ECG NA MULHER HIPERTENSA, QUAL O MELHOR CRITÉRIO DE VOLTAGEM? / THE ECG IN HYPERTENSIVE WOMEN, WHAT IS THE BEST VOLTAGE CRITERIA?

Segundo a Diretriz Brasileira de Hipertensão Arterial 2020, hipertensão arterial (HA) é uma doença crônica não transmissível (DCNT) definida por níveis pressóricos, em que os benefícios do tratamento não medicamentoso e/ou medicamentoso superam os riscos; e a falta de tratamento evolui com lesão de órgão alvo, como a hipertrofia ventricular esquerda. Neste estudo avaliamos a literatura no tocante à acurácia dos critérios eletrocardiográficos para diagnóstico de hipertrofia ventricular esquerda (HVE) e por conta das diferenças entre homens e mulheres, isso constitui um grande desafio. Dentre os fatores que mais interferem no critério sensibilidade, destacam-se a massa cardíaca e o sexo, sendo a sensibilidade do ECG maior com o aumento da massa ventricular e no sexo masculino, segundo Colossimo e Povoa. No estudo de Gasperin, onde foram utilizados critérios de voltagem, observou-se que o critério de Cornell nas mulheres foi de maior sensibilidade, de 54,90%, com alta especificidade de 81,60%. Quando a sensibilidade do critério de Cornell foi comparada à de Sokolow-Lyon-Rappaport, o segundo em sensibilidade, foi de 41,18%; sem significância estatística entre os dois. A detecção precoce da HVE tem importância prognóstica, já discutida em vários trabalhos e seus critérios de validação, um constante desafio. Definir critérios específicos em mulheres torna-se necessário para maior acurácia diagnóstica e abordagem precoce para intervenções terapêuticas sejam medicamentosas ou não. Conclui-se que o critério eletrocardiográfico de Cornell foi o método com maior sensibilidade nas mulheres, nesta revisão. São necessários estudos com análises especificas para o sexo feminino, considerando suas diferenças anatômicas e antropométricas e possivelmente ajustadas à população brasileira. Torna-se uma limitação desta análise, a lacuna resultante do pequeno número de dados e poucos estudos publicados sobre o tema.

According to the Brazilian Guidelines on Hypertension 2020, hypertension (AH) is a chronic non-communicabledisease (NCD) defined by blood pressure levels, in which the benefits of non pharmacologic and/or pharmacologic therapy out weigh The risks; and lack of treatment evolves with target-organ damage such as left ventricular hypertrophy. In this study, we evaluated the literature regarding the accuracy of the electrocardiographic criteria for the diagnosis of left ventricular hypertrophy (LVH) and, due to the differences between men and women, this represents a great challenge. Among the factors that most interfere with the sensitivity criterion, cardiac mass and gender stand out, with ECG sensitivity being greater with the increase in ventricular mass and in males, according to Colossimo and Povoa. In the study by Gasperin, where voltage criteria were used, it was observed that the Cornell criterion in women was more sensitive, 54.90%, with a high specificity of 81.60%. When the sensitivity of the Cornell criterion was compared to theSokolow-Lyon-Rappaport criterion, the latter in sensitivity was 41.18%; with no statistical significance between the two. The early detection of LVH hás prognostic importance, already discussed in several works and its validation criteria, a Constant challenge. Defining specific criteria in women becomes necessary for greater diagnostic accuracy and a nearly approach to therapeutic interventions, whether drug-related or not. It is concluded that the Cornell electrocardiographic criterion was the method with the greatest sensitivity in women in this review. However, studies with specific analyzes for females are still needed, considering their anatomical and anthropometric differences and possibly adjusted to the Brazilian population. The gap resulting from the small amount of data and few published studies on the subject becomes a limitation of this analysis

A Novel Microphysiological Colon Platform to Decipher Mechanisms Driving Human Intestinal Permeability.

BACKGROUND & AIMS: The limited availability of organoid systems that mimic the molecular signatures and architecture of human intestinal epithelium has been an impediment to allowing them to be harnessed for the development of therapeutics as well as physiological insights. We developed a microphysiological Organ-on-Chip (Emulate, Inc, Boston, MA) platform designed to mimic properties of human intestinal epithelium leading to insights into barrier integrity. METHODS: We combined the human biopsy-derived leucine-rich repeat-containing G-protein-coupled receptor 5-positive organoids and Organ-on-Chip technologies to establish a micro-engineered human Colon Intestine-Chip (Emulate, Inc, Boston, MA). We characterized the proximity of the model to human tissue and organoids maintained in suspension by RNA sequencing analysis, and their differentiation to intestinal epithelial cells on the Colon Intestine-Chip under variable conditions. Furthermore, organoids from different donors were evaluated to understand variability in the system. Our system was applied to understanding the epithelial barrier and characterizing mechanisms driving the cytokine-induced barrier disruption. RESULTS: Our data highlight the importance of the endothelium and the in vivo tissue-relevant dynamic microenvironment in the Colon Intestine-Chip in the establishment of a tight monolayer of differentiated, polarized, organoid-derived intestinal epithelial cells. We confirmed the effect of interferon-γ on the colonic barrier and identified reorganization of apical junctional complexes, and induction of apoptosis in the intestinal epithelial cells as mediating mechanisms. We show that in the human Colon Intestine-Chip exposure to interleukin 22 induces disruption of the barrier, unlike its described protective role in experimental colitis in mice. CONCLUSIONS: We developed a human Colon Intestine-Chip platform and showed its value in the characterization of the mechanism of action of interleukin 22 in the human epithelial barrier. This system can be used to elucidate, in a time- and challenge-dependent manner, the mechanism driving the development of leaky gut in human beings and to identify associated biomarkers.

A novel organ-chip system emulates three-dimensional architecture of the human epithelia and the mechanical forces acting on it.

Successful translation of in vivo experimental data to human patients is an unmet need and a bottleneck in the development of effective therapeutics. Organ-on-Chip technology aims to address this need by leveraging recent significant advancements in microfabrication and biomaterials, which enable modeling of organs and their functionality. These microengineered chips offer researchers the possibility to recreate critical elements of native tissue architecture such as in vivo relevant tissue-tissue interface, air-liquid interface, and mechanical forces, including mechanical stretch and fluidic shear stress, which are crucial to recapitulate tissue level functions. Here, we present the development of a new, comprehensive 3D cell-culture system, where we combined our proprietary Organ-Chip technology with the advantages offered by three-dimensional organotypic culture. Leveraging microfabrication techniques, we engineered a flexible chip that consists of a chamber containing an organotypic epithelium, surrounded by two vacuum channels that can be actuated to stretch the hydrogel throughout its thickness. Furthermore, the ceiling of this chamber is a removable lid with a built-in microchannel that can be perfused with liquid or air and removed as needed for direct access to the tissue. The bottom part of this chamber is made from a porous flexible membrane which allows diffusive mass transport to and from the microfluidic channel positioned below the membrane. This additional microfluidic channel can be coated with endothelial cells to emulate a blood vessel and recapitulate endothelial interactions. Our results show that the Open-Top Chip design successfully addresses common challenges associated with the Organs-on-Chip technology, including the capability to incorporate a tissue-specific extracellular matrix gel seeded with primary stromal cells, to reproduce the architectural complexity of tissues by micropatterning the gel, and to extract the gel for H&E staining. We also provide proof-of-concept data on the feasibility of using the system with primary human skin and alveolar epithelial cells.

A Microengineered Airway Lung Chip Models Key Features of Viral-induced Exacerbation of Asthma.

Viral-induced exacerbation of asthma remains a major cause of hospitalization and mortality. New human-relevant models of the airways are urgently needed to understand how respiratory infections may trigger asthma attacks and to advance treatment development. Here, we describe a new human-relevant model of rhinovirus-induced asthma exacerbation that recapitulates viral infection of asthmatic airway epithelium and neutrophil transepithelial migration, and enables evaluation of immunomodulatory therapy. Specifically, a microengineered model of fully differentiated human mucociliary airway epithelium was stimulated with IL-13 to induce a T-helper cell type 2 asthmatic phenotype and infected with live human rhinovirus 16 (HRV16) to reproduce key features of viral-induced asthma exacerbation. We observed that the infection with HRV16 replicated key hallmarks of the cytopathology and inflammatory responses observed in human airways. Generation of a T-helper cell type 2 microenvironment through exogenous IL-13 stimulation induced features of asthmatic airways, including goblet cell hyperplasia, reduction of cilia beating frequency, and endothelial activation, but did not alter rhinovirus infectivity or replication. High-resolution kinetic analysis of secreted inflammatory markers revealed that IL-13 treatment altered IL-6, IFN-λ1, and CXCL10 secretion in response to HRV16. Neutrophil transepithelial migration was greatest when viral infection was combined with IL-13 treatment, whereas treatment with MK-7123, a CXCR2 antagonist, reduced neutrophil diapedesis in all conditions. In conclusion, our microengineered Airway Lung-Chip provides a novel human-relevant platform for exploring the complex mechanisms underlying viral-induced asthma exacerbation. Our data suggest that IL-13 may impair the hosts' ability to mount an appropriate and coordinated immune response to rhinovirus infection. We also show that the Airway Lung-Chip can be used to assess the efficacy of modulators of the immune response.

Duodenum Intestine-Chip for preclinical drug assessment in a human relevant model.

Induction of intestinal drug metabolizing enzymes can complicate the development of new drugs, owing to the potential to cause drug-drug interactions (DDIs) leading to changes in pharmacokinetics, safety and efficacy. The development of a human-relevant model of the adult intestine that accurately predicts CYP450 induction could help address this challenge as species differences preclude extrapolation from animals. Here, we combined organoids and Organs-on-Chips technology to create a human Duodenum Intestine-Chip that emulates intestinal tissue architecture and functions, that are relevant for the study of drug transport, metabolism, and DDI. Duodenum Intestine-Chip demonstrates the polarized cell architecture, intestinal barrier function, presence of specialized cell subpopulations, and in vivo relevant expression, localization, and function of major intestinal drug transporters. Notably, in comparison to Caco-2, it displays improved CYP3A4 expression and induction capability. This model could enable improved in vitro to in vivo extrapolation for better predictions of human pharmacokinetics and risk of DDIs.

Human iPSC-Derived Blood-Brain Barrier Chips Enable Disease Modeling and Personalized Medicine Applications.

The blood-brain barrier (BBB) tightly regulates the entry of solutes from blood into the brain and is disrupted in several neurological diseases. Using Organ-Chip technology, we created an entirely human BBB-Chip with induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial-like cells (iBMECs), astrocytes, and neurons. The iBMECs formed a tight monolayer that expressed markers specific to brain vasculature. The BBB-Chip exhibited physiologically relevant transendothelial electrical resistance and accurately predicted blood-to-brain permeability of pharmacologics. Upon perfusing the vascular lumen with whole blood, the microengineered capillary wall protected neural cells from plasma-induced toxicity. Patient-derived iPSCs from individuals with neurological diseases predicted disease-specific lack of transporters and disruption of barrier integrity. By combining Organ-Chip technology and human iPSC-derived tissue, we have created a neurovascular unit that recapitulates complex BBB functions, provides a platform for modeling inheritable neurological disorders, and advances drug screening, as well as personalized medicine.

Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro.

Here we describe the development of a human lung 'small airway-on-a-chip' containing a differentiated, mucociliary bronchiolar epithelium and an underlying microvascular endothelium that experiences fluid flow, which allows for analysis of organ-level lung pathophysiology in vitro. Exposure of the epithelium to interleukin-13 (IL-13) reconstituted the goblet cell hyperplasia, cytokine hypersecretion and decreased ciliary function of asthmatics. Small airway chips lined with epithelial cells from individuals with chronic obstructive pulmonary disease recapitulated features of the disease such as selective cytokine hypersecretion, increased neutrophil recruitment and clinical exacerbation by exposure to viral and bacterial infections. With this robust in vitro method for modeling human lung inflammatory disorders, it is possible to detect synergistic effects of lung endothelium and epithelium on cytokine secretion, identify new biomarkers of disease exacerbation and measure responses to anti-inflammatory compounds that inhibit cytokine-induced recruitment of circulating neutrophils under flow.

Cell adhesion to plasma-coated PVC.

To produce environments suitable for cell culture, thin polymer films were deposited onto commercial PVC plates from radiofrequency acetylene-argon plasmas. The proportion of argon in the plasmas, P(Ar), was varied from 5.3 to 65.8%. The adhesion and growth of Vero cells on the coated surfaces were examined for different incubation times. Cytotoxicity tests were performed using spectroscopic methods. Carbon, O, and N were detected in all the samples using XPS. Roughness remained almost unchanged in the samples prepared with 5.3 and 28.9% but tended to increase for the films deposited with P(Ar) between 28.9 and 55.3%. Surface free energy increased with increasing P(Ar), except for the sample prepared at 28.9% of Ar, which presented the least reactive surface. Cells proliferated on all the samples, including the bare PVC. Independently of the deposition condition there was no evidence of cytotoxicity, indicating the viability of such coatings for designing biocompatible devices.

On the design of clone-based haplotyping.

BACKGROUND: Haplotypes are important for assessing genealogy and disease susceptibility of individual genomes,but are difficult to obtain with routine sequencing approaches. Experimental haplotype reconstruction based on assembling fragments of individual chromosomes is promising, but with variable yields due to incompletely understood parameter choices. RESULTS: We parameterize the clone-based haplotyping problem in order to provide theoretical and empirical assessments of the impact of different parameters on haplotype assembly. We confirm the intuition that long clones help link together heterozygous variants and thus improve haplotype length. Furthermore, given the length of the clones, we address how to choose the other parameters, including number of pools, clone coverage and sequencing coverage, so as to maximize haplotype length. We model the problem theoretically and show empirically the benefits of using larger clones with moderate number of pools and sequencing coverage. In particular, using 140 kb BAC clones, we construct haplotypes for a personal genome and assemble haplotypes with N50 values greater than 2.6 Mb. These assembled haplotypes are longer and at least as accurate as haplotypes of existing clone-based strategies, whether in vivo or in vitro. CONCLUSIONS: Our results provide practical guidelines for the development and design of clone-based methods to achieve long range, high-resolution and accurate haplotypes.

Increased response of Vero cells to PHBV matrices treated by plasma.

The copolymers poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) are being intensely studied as a tissue engineering substrate. It is known that poly 3-hydroxybutyric acids (PHBs) and their copolymers are quite hydrophobic polyesters. Plasma-surface modification is an effective and economical surface treatment technique for many materials and of growing interest in biomedical engineering. In this study we investigate the advantages of oxygen and nitrogen plasma treatment to modify the PHBV surface to enable the acceleration of Vero cell adhesion and proliferation. PHBV was dissolved in methylene chloride at room temperature. The PHBV membranes were modified by oxygen or nitrogen-plasma treatments using a plasma generator. The membranes were sterilized by UV irradiation for 30 min and placed in 96-well plates. Vero cells were seeded onto the membranes and their proliferation onto the matrices was also determined by cytotoxicity and cell adhesion assay. After 2, 24, 48 and 120 h of incubation, growth of fibroblasts on matrices was observed by scanning electron microscopy (SEM). The analyses of the membranes indicated that the plasma treatment decreased the contact angle and increased the surface roughness; it also changed surface morphology, and consequently, enhanced the hydrophilic behavior of PHBV polymers. SEM analysis of Vero cells adhered to PHBV treated by plasma showed that the modified surface had allowed better cell attachment, spreading and growth than the untreated membrane. This combination of surface treatment and polymer chemistry is a valuable guide to prepare an appropriate surface for tissue engineering application.