Name: an intestine/liver microphysiological system for drug pharmacokinetic and toxicological assessment
Uploaded: 2020-12-03T12:30:00.000+00:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment at JoVE.com

We thanks to Dr. Christie Guguen-Guillouzo, Dr. Philippe Gripon at Unit 522 INSERM and to Dr. Christian Trepo at Unit 271 INSERM for the use of the Biological Material (Hepa RG cells) and for making then available for us in order to perform the academic research.

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The authors have nothing to disclose.

The accurate and reliable assessment of the pharmacologic properties of investigative new drugs is critical for reducing the risk in the following development steps. The MPS is a relatively new technology, that aims at improving the predictive power and reducing the costs and time spent with preclinical tests. Our group is advancing in the assessment of pharmacokinetic and toxicological properties mostly needed for lead optimization. We worked with the 2-OC microfluidic device, which has two chambers, allowing the integr...

Due to genomic and proteomic differences, animal models have limited predictive value for several human outcomes. Moreover, they are time-consuming, expensive and ethically questionable1. MPS is a relatively new technology that aims at improving the predictive power and reduce the costs and time spent with pre-clinical tests. They are microfluidic devices cultivating organoids (artificial mimetics functional units of organs) under media flow that promotes organoid-organoid communication. Organoids made of human cells increase translational relevance2,3,4. MPS is expected to perform better than the animal tests because they are genetically human and recapitulate the interplay among tissues. When fully functional, the MPS will provide more meaningful results, at higher speed and lower costs and risks4. Many groups are developing MPS for several purposes, especially disease models to tests drug&#8217;s efficacy.
Exposure level is one of the most critical parameters for evaluating drug efficacy and toxicity5,6,7,8,9,10,11,12. MPS allows organoid integration that emulates systemic exposure and is expected to perform better than the traditional 2D human tissue culture. This technology can significantly improve the prediction of compound intestinal absorption and liver metabolism4.
An MPS integrating human equivalent model of intestine and liver is a good starting point, considering the central role of these two organs in drug bioavailability and systemic exposure13,14,15. APAP is an attractive drug for studying an MPS without a kidney equivalent because its metabolization is done mainly by the liver16,17.
The 2-OC is a two-chamber microfluidic device suitable for the culture of two different human equivalent tissues/organoids interconnected by microchannels16. In order to emulate an in vitro human oral/intravenous administration of a drug and assess the effects of the cross-talk between the intestine and liver equivalents on APAP pharmacokinetics, besides the organoids functionality and viability, three different MPS assemblies were performed: (1) an &#8220;Intestine 2-OC MPS&#8221; comprised of an intestine equivalent based in a culture insert containing a Caco-2 + HT-29 cells coculture, integrated into the 2-OC device; (2) a &#8220;Liver 2-OC MPS&#8221; comprised of liver spheroids made of HepaRG + HHSteC (Human Hepatic Stellate Cells) integrated in the 2-OC device; and (3) an &#8220;Intestine/Liver 2-OC MPS&#8221; comprised of the intestine equivalent in one device compartment communicating with the liver equivalent in the other by the media flow through&#160;the microfluidic channels.
All assays were performed under static (no flow) and dynamic (with flow) conditions due to the impact of the mechanical stimuli (compression, stretching, and shear) on the cell viability and functionalities18,19,20. The present article describes the protocol for APAP oral/intravenous administration emulation and the respective absorption/metabolism and toxicological analyses in the 2-OC MPS containing human intestine and liver equivalent models.

<table><tbody><tr><td>1x DPBS</td><td>Thermo Fisher Scientific</td><td>14190235</td><td>No calcium, no magnesium</td></tr><tr><td>2-OC</td><td>TissUse GmbH</td><td></td><td>Two-organ chip</td></tr><tr><td>384-well Spheroid Microplate</td><td>Corning</td><td>3830</td><td>Black/Clear, Round Bottom, Ultra-Low Attachment</td></tr><tr><td>4% Paraformaldehyde</td><td></td><td></td><td>Use to fix cell</td></tr><tr><td>Acetaminophen</td><td>Sigma Aldrich</td><td>A7085</td><td>Use to MPS assays</td></tr><tr><td>Acetonitrile</td><td>Tedia</td><td></td><td>Used to perform HPLC</td></tr><tr><td>Alexa Fluor 647 phalloidin</td><td>Thermo Fisher Scientific</td><td></td><td>confocal experiment</td></tr><tr><td>Ammonium acetate</td><td>Sigma Aldrich</td><td></td><td>Used to perform HPLC</td></tr><tr><td>Caco-2 cells</td><td>Sigma Aldrich</td><td>86010202</td><td></td></tr><tr><td>Cacodylate buffer</td><td></td><td></td><td></td></tr><tr><td>Cell culture flasks</td><td>Sarstedt</td><td></td><td></td></tr><tr><td>Confocal Fluorescence microscope</td><td>Leica</td><td>DMI6000</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM1950</td><td></td></tr><tr><td>DMEM high glucose</td><td>Thermo Fisher Scientific</td><td>12800017</td><td>Add supplements: 10% fetal bovine serum, 100 units per mL penicillin, 100 &amp;micro;g/mL streptomycin, and 1% non-essential amino acids</td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>D4540</td><td>Add 2% to HepaRG media</td></tr><tr><td>Ethanol</td><td>Synth</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>12657029</td><td></td></tr><tr><td>Freezing medium OCT</td><td>Tissue-Tek</td><td></td><td>Tissue-Tek&lt;sup&gt;&amp;reg;&lt;/sup&gt; O.C.T.&amp;trade; Compound is a formulation of watersoluble glycols and resins, providing a convenient specimen matrix for cryostat sectioning at temperatures of -10&amp;deg;C and below.</td></tr><tr><td>Hematoxylin &amp;amp; Eosin</td><td></td><td></td><td></td></tr><tr><td>HepaRG cells</td><td>Biopredic International</td><td>HPR101</td><td>Undifferentiated cells</td></tr><tr><td>HHSTeC</td><td>ScienCell Research Laboratories</td><td>5300</td><td>Cells and all culture supplements</td></tr><tr><td>Hoechst 33342</td><td></td><td></td><td>HCA experiments</td></tr><tr><td>HT-29 cells</td><td>Sigma Aldrich</td><td>85061109</td><td></td></tr><tr><td>Human Insulin</td><td>Invitrogen - Thermo Fisher Scientific</td><td>12585014</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma Aldrich</td><td>H0888</td><td></td></tr><tr><td>Isopropanol</td><td>Merck</td><td>278475</td><td></td></tr><tr><td>Karnovsky&amp;rsquo;s fixative</td><td></td><td></td><td></td></tr><tr><td>L-glutamine</td><td>Thermo Fisher Scientific</td><td>A2916801</td><td></td></tr><tr><td>Luna C18 guard column SS</td><td>Phenomenex</td><td></td><td>Used to perform HPLC</td></tr><tr><td>Microscope</td><td>Leica</td><td>DMi4000</td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>RM2245</td><td></td></tr><tr><td>Millicell 0.4 &amp;micro;m pore size inserts</td><td>Merck</td><td>PIHP01250</td><td></td></tr><tr><td>Millicell ERS-2 meter</td><td>Merck</td><td>MERS00002</td><td>Used to TEER measurement</td></tr><tr><td>MitoTracker Deep Red</td><td></td><td></td><td>HCA experiments</td></tr><tr><td>MTT</td><td>Thermo Fisher Scientific</td><td>M6494</td><td></td></tr><tr><td>MX3000P system</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>Neubauer chamber</td><td></td><td></td><td>Counting cells</td></tr><tr><td>Operetta High Content Imaging System</td><td>Perkin Elmer</td><td></td><td>Used to perform HCA</td></tr><tr><td>P450-Glo CYP3A4 Assay with Luciferin-IPA</td><td>Promega</td><td>Cat.# V9001</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>15070063</td><td>Cell culture</td></tr><tr><td>Permount</td><td>Thermo Fisher Scientific</td><td></td><td>Histology</td></tr><tr><td>Primers</td><td></td><td></td><td>RT-qPCR</td></tr><tr><td>PVDF membrane</td><td>BioRad</td><td></td><td></td></tr><tr><td>PVDF Syringe filter</td><td></td><td></td><td>0.22 &amp;mu;m pore size</td></tr><tr><td>Reversed-phase Luna C18 column</td><td>Phenomenex</td><td></td><td>Used to perform HPLC</td></tr><tr><td>Shaker (IKA VXR Basic Vibrax)</td><td>IKA Works GmbH &amp;amp; Co</td><td>2819000</td><td>Used for spheroids to improve MTT assay</td></tr><tr><td>Stellate Cell Media (STeC CM)</td><td>ScienCell</td><td>5301</td><td>Add STeC CM supplements</td></tr><tr><td>SuperScriptIITM Reverse Transcriptase</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>SYBR Green PCR Master Mix</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>TRizol TM reagent</td><td>Thermo Fisher Scientific</td><td></td><td>Trizol is a monophasic solution of phenol and guanidine isothiocyanate.</td></tr><tr><td>Trypsin/EDTA solution</td><td>Thermo Fisher Scientific</td><td>R001100</td><td></td></tr><tr><td>Ultra-low-attachment plates</td><td>Corning</td><td>CLS3471-24EA</td><td>6 wells</td></tr><tr><td>Vectashield plus DAPI mounting media</td><td></td><td></td><td></td></tr><tr><td>White Opaque 96-well Microplate</td><td>PerkinHelmer</td><td></td><td></td></tr><tr><td>Wide-bore tips</td><td></td><td></td><td></td></tr><tr><td>Williams E</td><td>Pan Biotech</td><td>P04-29510</td><td>Add supplements: 10% fetal bovine serum, 2 mM L-glutamine, 100 units per ml penicillin, 100 &amp;micro;g/mL streptomycin and 5 &amp;micro;g/mL human insulin</td></tr></tbody></table>

an intestine/liver microphysiological system for drug pharmacokinetic and toxicological assessment

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests phase of drug developing process because they are genetically human and recapitulate the interplay among tissues. In this study, the human intestinal barrier (emulated by a co-culture of Caco-2 and HT-29 cells) and the liver equivalent (emulated by spheroids made of differentiated HepaRG cells and human hepatic stellate cells) were integrated into a two-organ chip (2-OC) microfluidic device to assess some acetaminophen (APAP) pharmacokinetic (PK) and toxicological properties. The MPS had three assemblies: Intestine only 2-OC, Liver only 2-OC, and Intestine/Liver 2-OC with the same media perfusing both organoids. For PK assessments, we dosed the APAP in the media at preset timepoints after administering it either over the intestinal barrier (emulating the oral route) or in the media (emulating the intravenous route), at 12 &#181;M and 2 &#181;M respectively. The media samples were analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Organoids were analyzed for gene expression, for TEER values, for protein expression and activity, and then collected, fixed, and submitted to a set of morphological evaluations. The MTT technique performed well in assessing the organoid viability, but the high content analyses (HCA) were able to detect very early toxic events in response to APAP treatment. We verified that the media flow does not significantly affect the APAP absorption whereas it significantly improves the liver equivalent functionality. The APAP human intestinal absorption and hepatic metabolism could be emulated in the MPS. The association between MPS data and in silico modeling has great potential to improve the predictability of the in vitro methods and provide better accuracy than animal models in pharmacokinetic and toxicological studies.

To perform the PK APAP tests in the 2-OC MPS, the first step is to manufacture the human intestine and liver equivalents (organoids). They are integrated into the 2-OC microfluidic device (Figure 1A) 24 h before starting of the PK APAP assay. The next day, the medium is changed, and the model is exposed to APAP. Figure 1 illustrates the intestine and liver equivalents placed inside the 2-OC device (Figure 1B) and the APAP PK experim...

Watch this Scientific Journal Video about An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment at JoVE.com

An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment

We exposed a microphysiological system (MPS) with intestine and liver organoids to acetaminophen (APAP). This article describes the methods for organoid production and APAP pharmacokinetic and toxicological property assessments in the MPS. It also describes the tissue functionality analyses necessary to validate the results.

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests ...

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Pharmacokinetics and Pharmacodynamics: Introduction

Pharmacokinetics

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7.7K Views.  Brazilian Center for Research in Energy and Materials (CNPEM). We exposed a microphysiological system (MPS) with intestine and liver organoids to acetaminophen (APAP). This article describes the methods for organoid production and APAP pharmacokinetic and toxicological property assessments in the MPS. It also describes the tissue functionality analyses necessary to validate the results.

Video: An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

Biochemistry

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Bioengineering

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

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&#160;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&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-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.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Medicine

A Necrotizing Enterocolitis Model for Mechanistic Studies and Drug Discovery

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, which remains incompletely understood. The pathophysiology of NEC includes disruption of intestinal tight junctions, increased gut barrier permeability, epithelial cell death, microbial dysbiosis, and dysregulated inflammation. Traditional tools to study NEC include animal models, cell lines, and human or mouse intestinal organoids. While studies using those model systems have improved the field&#39;s understanding of disease pathophysiology, their ability to recapitulate the complexity of human NEC is limited. An improved in vitro model of NEC using microfluidic technology, named NEC-on-a-chip, has now been developed. The NEC-on-a-chip model consists of a microfluidic device seeded with intestinal enteroids derived from a preterm neonate, co-cultured with human endothelial cells and the microbiome from an infant with severe NEC. This model is a valuable tool for mechanistic studies into the pathophysiology of NEC and a new resource for drug discovery testing for neonatal intestinal diseases. In this manuscript, a detailed description of the NEC-on-a-chip model will be provided.

Author Spotlight: Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model 

This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC.

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, ...

Protocol for Studying Gastrointestinal Dynamics and Drug Effects on Motility

Using An In Vitro Tissue Perfusion System to Detect the Functional Activities of Isolated Intestinal Tubes in Real Time

Gastrointestinal diseases, which have a high incidence, pose considerable challenges for humans. The small intestine is integral to food and drug digestion and absorption and plays a crucial role in treating these diseases. The intestinal tube movement experiment, a common and essential in vitro method, is utilized to study gastrointestinal dynamics. This includes the preparation of the isolated intestinal tube, as well as the suspension of the prepared intestinal tube in the bath and its connection to a signal detector. This is followed by the recording and analysis of a series of parameters, such as tension, which can be used to assess intestinal motor function, as well as considerations for keeping the intestinal tube active in vitro. The standardized program from sampling to data collection greatly improves the repeatability of the experimental data and ensures the authenticity of the recording of intestinal tension after physiological, pathological, and drug intervention. Here we present the key problems in experimental operation and a valuable reference experimental protocol for studying drugs that regulate gastrointestinal motility.

Author Spotlight: Reliable and Reproducible In Vitro Assessment of Drug Impact on Rat Intestinal Tubes

We present a method for isolating rat intestinal tubes and assessing the impact of drugs on their tension, frequency, and amplitude in vitro. This method offers a valuable approach for researchers investigating intestinal tubes.

Gastrointestinal diseases, which have a high incidence, pose considerable challenges for humans. The small intestine is integral to food and drug ...

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is often not detected until drugs have reached clinical stages, risking patients' safety and leading to substantial losses for the pharma industry. Taking into account that standard 2D models have limitations in detecting DILI it is essential to develop in vitro models that are more predictive to improve data translatability. To understand causality and mechanistic aspects of DILI in detail, a human liver MPS consisting of human primary liver parenchymal and non-parenchymal cells (NPCs) and cultured in 3D microtissues on an engineered scaffold under perfusion has been developed. Cryopreserved primary human hepatocytes (PHHs) and Kupffer cells (HKCs) were cocultured as microtissues in the MPS platform for up to two weeks, and each compound of interest was repeatably dosed onto liver microtissues at seven test concentrations for up to four days. Functional liver-specific endpoints were analyzed (including clinical biomarkers such as alanine aminotransferase, ALT) to evaluate liver function. Acute and chronic exposure to compounds of various DILI severities can be assessed by comparing responses to single and multi-dosed microtissues. The methodology has been validated with a broad set of severe and mildly hepatotoxic compounds. Here we show the data for pioglitazone and troglitazone, well-known hepatotoxic compounds withdrawn from the market for causing liver failures. Overall, it has been shown that the liver MPS model can be a useful tool to assess DILI and its association with changes in hepatic function. The model can additionally be used to assess how novel compounds behave in distinct patient subsets and how toxicity profiles may be affected by liver disease states (e.g., viral hepatitis, fatty liver disease).

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

Drug-induced liver injury (DILI) is a major cause of drug failure. A protocol has been developed to accurately predict the DILI liability of a compound using a liver microphysiological system (MPS). The liver model uses the coculture of primary hepatic cells and translationally relevant endpoints to assess cellular responses to treatment.

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is ...

Biology

Construction of a 3D Intestinal Mucosa Model with Paraffin Embedding

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the pharmacological screening of potentially beneficial substances, and for toxicity studies on potentially harmful food components. Of relevance, there is a current demand for the development of cell-based in vitro models to substitute animal models. Here, a protocol for a basic, &#8220;healthy tissue&#8221; three-dimensional (3D) intestinal equivalent model using cell lines is presented with the dual benefit of providing both experimental simplicity (standardized and easily repeatable system) and physiological complexity (Caco-2 enterocytes with a supporting immune component of U937 monocytes and L929 fibroblasts). The protocol also includes paraffin embedding for light microscopic evaluation of fixed intestinal equivalents, thereby providing the advantage of analyzing multiple visual parameters from a single experiment. Hematoxylin and eosin (H&#38;E) stained sections showing the Caco-2 columnar cells forming a tight and regular monolayer in control treatments are used to verify the efficacy of the model as an experimental system. Using gluten as a pro-inflammatory food component, parameters analyzed from sections include reduced monolayer thickness, as well as disruption and detachment from the underlying matrix (H&#38;E), decreased tight junction protein expression as shown from occludin staining (quantifiable statistically), and immune-activation of migrating U937 cells as evidenced from the cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. As shown by using lipopolysaccharide to simulate intestinal inflammation, additional parameters that can be measured are increased mucus staining and cytokine expression (such as midkine) that can be extracted from the medium prior to fixation. The basic three-dimensional (3D) intestinal mucosa model and fixed sections can be recommended for inflammatory status and barrier integrity studies with the possibility of analyzing multiple visual quantifiable parameters.

Author Spotlight: Investigating the Effects of Compounds on Intestinal Tissue Using 3D Human Cell Line Models 

Here we describe constructing a basic three-dimensional (3D) intestinal cell line model system and a paraffin embedding protocol for light microscopic evaluation of fixed intestinal equivalents. Staining of selected proteins permits the analysis of multiple visual parameters from a single experiment for potential use in preclinical drug screening studies.

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the ...

A Physiologically-Relevant All-Human 2D+ Hepatic System for Drug Discovery

An All-Human Hepatic Culture System for Drug Development Applications

Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. Current in vitro model platforms are often inconvenient and complex, lack phenotypic stability over time, and do not support multiple PHH lots, lacking experimental reproducibility and flexibility. Here, we provide a detailed protocol for the thawing, plating, and maintenance of an all-human 2D+ hepatic system (TV2D+), which takes advantage of standard two-dimensional (2D) culture techniques and equipment while maintaining the longevity and phenotypic stability over time that typically accompany more complex three-dimensional (3D) systems. The results show attachment and percent plateability in TV2D+ as a function of PHH seeding density, as well as stable functionality for at least 2 weeks in culture. A range of PHH seeding densities are assessed to achieve a successful long-term culture. When established properly, the PHHs in TV2D+ organize into hepatocyte colonies, express a hepatic-specific marker, and maintain viability, architectural integrity, and physiologically relevant levels of albumin and urea. This unique combination of attributes makes the TV2D+ system a suitable hepatic model for a variety of pharmacological and toxicological applications.

Author Spotlight: Developing a Simple and Robust Hepatic Model for Pharmacological and Toxicological Applications 

Recent technical advances enabled the scaled production of an in vitro platform for drug metabolism and toxicity applications. An all-human 2D+ hepatic system (TV2D+) provides physiologically relevant results using traditional two-dimensional culture methods. This protocol will support end-users in the setup, maintenance, and application of the system.

Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. ...

An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment

Summary

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The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

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