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

Summary

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.

Abstract

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 µM and 2 µM 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.

Introduction

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 relevance^2,3,4

Protocol

1. Production of tissue equivalents for cultivation in the 2-OC

1. Small intestine barrier equivalent production
   1. 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 “DMEM S” in this manuscript.
   2. 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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
1x DPBS	Thermo Fisher Scientific	14190235	No calcium, no magnesium
2-OC	TissUse GmbH		Two-organ chip
384-well Spheroid Microplate	Corning	3830	Black/Clear, Round Bottom, Ultra-Low Attachment
4% Paraformaldehyde			Use to fix cell
Acetaminophen	Sigma Aldrich	A7085	Use to MPS assays
Acetonitrile	Tedia		Used to perform HPLC
Alexa Fluor 647 phalloidin	Thermo Fisher Scientific		confocal experiment
Ammonium acetate	Sigma Aldrich		Used to perform HPLC
Caco-2 cells	Sigma Aldrich	86010202
Cacodylate buffer
Cell culture flasks	Sarstedt
Confocal Fluorescence microscope	Leica	DMI6000
Cryostat	Leica	CM1950
DMEM high glucose	Thermo Fisher Scientific	12800017	Add supplements: 10% fetal bovine serum, 100 units per mL penicillin, 100 µg/mL streptomycin, and 1% non-essential amino acids
DMSO	Sigma Aldrich	D4540	Add 2% to HepaRG media
Ethanol	Synth
Fetal Bovine Serum	Thermo Fisher Scientific	12657029
Freezing medium OCT	Tissue-Tek		Tissue-Tek® O.C.T.™ Compound is a formulation of watersoluble glycols and resins, providing a convenient specimen matrix for cryostat sectioning at temperatures of -10°C and below.
Hematoxylin & Eosin
HepaRG cells	Biopredic International	HPR101	Undifferentiated cells
HHSTeC	ScienCell Research Laboratories	5300	Cells and all culture supplements
Hoechst 33342			HCA experiments
HT-29 cells	Sigma Aldrich	85061109
Human Insulin	Invitrogen - Thermo Fisher Scientific	12585014
Hydrocortisone	Sigma Aldrich	H0888
Isopropanol	Merck	278475
Karnovsky’s fixative
L-glutamine	Thermo Fisher Scientific	A2916801
Luna C18 guard column SS	Phenomenex		Used to perform HPLC
Microscope	Leica	DMi4000
Microtome	Leica	RM2245
Millicell 0.4 µm pore size inserts	Merck	PIHP01250
Millicell ERS-2 meter	Merck	MERS00002	Used to TEER measurement
MitoTracker Deep Red			HCA experiments
MTT	Thermo Fisher Scientific	M6494
MX3000P system	Agilent Technologies
Neubauer chamber			Counting cells
Operetta High Content Imaging System	Perkin Elmer		Used to perform HCA
P450-Glo CYP3A4 Assay with Luciferin-IPA	Promega	Cat.# V9001
Penicillin/Streptomycin	Thermo Fisher Scientific	15070063	Cell culture
Permount	Thermo Fisher Scientific		Histology
Primers			RT-qPCR
PVDF membrane	BioRad
PVDF Syringe filter			0.22 μm pore size
Reversed-phase Luna C18 column	Phenomenex		Used to perform HPLC
Shaker (IKA VXR Basic Vibrax)	IKA Works GmbH & Co	2819000	Used for spheroids to improve MTT assay
Stellate Cell Media (STeC CM)	ScienCell	5301	Add STeC CM supplements
SuperScriptIITM Reverse Transcriptase	Thermo Fisher Scientific
SYBR Green PCR Master Mix	Thermo Fisher Scientific
TRizol TM reagent	Thermo Fisher Scientific		Trizol is a monophasic solution of phenol and guanidine isothiocyanate.
Trypsin/EDTA solution	Thermo Fisher Scientific	R001100
Ultra-low-attachment plates	Corning	CLS3471-24EA	6 wells
Vectashield plus DAPI mounting media
White Opaque 96-well Microplate	PerkinHelmer
Wide-bore tips
Williams E	Pan Biotech	P04-29510	Add supplements: 10% fetal bovine serum, 2 mM L-glutamine, 100 units per ml penicillin, 100 µg/mL streptomycin and 5 µg/mL human insulin