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

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.