Name: an intestine/liver microphysiological system for drug pharmacokinetic and toxicological assessment
Uploaded: 2020-12-03T12:30:00
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.

1. Production of tissue equivalents for cultivation in the 2-OC
<ol>
	<li>Small intestine barrier equivalent production
	<ol>
		<li>Maintain Caco-2 and HT-29 cells using the intestine equivalent medium: DMEM supplemented with 10% FBS, 1% penicillin and streptomycin, and 1% non-essential amino acids, which is named as &#8220;DMEM S&#8221; in this manuscript.</li>
		<li>Remove the medium, wash twice with 1x DPBS and add 8 mL of 0.25% Trypsin/EDTA to dissociate Caco-2 cells grown in cell culture flasks (175 cm2...

<ol>
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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 &#181;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&#174; O.C.T.&#8482; Compound is a formulation of watersoluble glycols and resins, providing a convenient specimen matrix for cryostat sectioning at temperatures of -10&#176;C and below.</td>
		</tr>
		<tr>
			<td>Hematoxylin &#38; 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&#8217;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 &#181;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 &#956;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 &#38; 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 &#181;g/mL streptomycin and 5 &#181;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 ...

Microphysiological systems have the ability to emulate pharmacokinetic and the toxicological response of the human body to specific treatments of interests. Microphysiological systems have the potential to replace animal tests as their use can improve the predictive power of in vitro methods and reduce the cost and time of pharmacokinetical and toxicological studies. 24 hours before the test substance administration, split an 800 microliter aliquot of William ES medium between the larger and smaller compartments of the two organ chip, and aspirate the basolateral and apical medium from each intestinal barrier equivalent in the prepared 24 well plates.Using sterile forceps, integrate one insert per two organ chip circuit into the larger compartment and add 200 microliters of DMEMS to the apical side. Using wide bore tips, integrate 20 liver equivalents per circuit into the smaller compartment of the two organ chip and connect the system to the control unit. Then connect the control unit to a pressurized air supply and set the pressure to approximately plus or minus 300 bars and a pumping frequency of 0.3 Hertz.The next day dilute the stock acetaminophen solution to a 12 micromolar concentration. To replace the medium in the system, aspirate the basolateral and apical media from the intestinal barrier equivalent in the two organ chip and add 500 microliters of fresh appropriate culture medium into the large compartment at the organoid basolateral side and 300 microliters to the small compartment. After checking for bubbles, emulate oral administration with the addition of 200 microliters of a 12 micromolar acetaminophen solution on the apical side of the intestinal culture inserts and connect the microphysiological system to the control unit.Collect the total volume from both the intestinal apical and basolateral sides, as well as from the small compartment in triplicate at the indicated time points. When all of the samples have been collected, set all of the relevant parameters for the HPLC analysis as indicated in the table and filter the mobile phase through a 0.4 micron membrane filter under vacuum. Then filter the samples through a 0.22 micron pore size PVDF syringe filter and store the samples in a vial before starting the HPLC UV measurement.To assess the organoid viability, transfer all 20 spheroids from each replicate to individual wells of a 96 well plate and transfer the cell culture inserts to individual wells of a 24 well plate. Wash the tissue equivalents three times with fresh DPBS per wash and add 300 microliters of a one milligram per milliliter MTT solution to each experimental well. After a three hour incubation in the cell culture incubator, replace the MTT solution with 200 microliters of isopropanol per well for an overnight incubation at four degrees Celsius to extract the MTT formazan from the intestine and liver equivalents.The next morning, transfer 200 microliters of each supernatant to the appropriate well in the 96 well plate and read the formazan on a plate reader at 570 nanometers to allow calculation of the relative ability of the cells to reduce MTT using the average optical density of each time point compared to the negative control. For cytochemical and histological analysis of the samples, first fix the intestine and liver equivalents in 4%paraformaldehyde in 0.1 molar PBS for 25 minutes at room temperature. At the end of the incubation, wash the organoids five times in PBS for 10 minutes per wash, before staining the intestinal and liver equivalents with tetramethylrhodamine isothiocyanate phalloidin Alexa Fluor 647 phalloidin for one hour at room temperature.At the end of the incubation, transfer the samples to freezing medium for a few minutes before snap freezing the tissues in liquid nitrogen. Next, use a cryostat to acquire 10 to 12 micron thick liver spheroid cryo-sections and mount the tissue sections in mounting medium with DAPI for imaging by confocal fluorescence microscopy. For mitochondrial and nuclear staining, completely cover the samples with mitochondrial staining solution for a 15 to 45 minute incubation at 37 degrees Celsius in a humidified atmosphere with 5%carbon dioxide.At the end of the incubation, carefully replace the staining solution with 2 to 4%paraformaldehyde in PBS for 15 minutes at room temperature. After fixing, gently rinse the cells two times with fresh PBS for five minutes per wash before labeling the samples with nucleic acid staining working solution for 10 minutes at room temperature. At the end of the incubation, wash the samples with three, five minute washes in PBS protected from light, and use the nucleic acid stain to facilitate quantification of the number of mitochondrial stain expressing cells on a fluorescent microscope with the appropriate filter sets.Integrating the human intestinal barrier and a liver equivalent into a two organ chip microfluidic device allows assessment of the pharmacokinetic and toxicological properties of treatments of interest. After treatment, the media samples can be analyzed by HPLC. The organoids can be analyzed for their gene expression, trans-epithelial electrical resistance values, and protein expression and activity, as well as for their morphological characteristics.MTT analysis can be performed to assess organoid viability, as well as to detect very early toxic events in response to acetaminophen treatment. The media flow does not significantly affect acetaminophen absorption, whereas it significantly improves liver equivalent functionality, indicating that human intestinal acetaminophen absorption and hepatic metabolism can be emulated in this microphysiological system. Microphysiological systems have the potential to replace animal models in drug development and discovery as they better mimic human physiology and can be used to promote drug development at higher speed and lower cost to risk and complexity.

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