The protocol describes how to assess drug-induced liver injury in vitro using a microphysiological system that can maintain highly functional and metabolically active liver micro tissues for up to four weeks. This approach is a human specific cell culture model to accurately detect drug-induced liver injury liability for novel compounds, which is more predictive than simpler 2D cultures or even more complex 3D cultures. This technique can be used as part of a battery of preclinical safety tests to determine if a compound is safe to start clinical development.
A wide variety of modalities can be tested. Begin setting up the liver microphysiological system by connecting the controller to the docking station house in a cell culture incubator. Ensure fresh desiccant is added into the desiccant jar located at the back of the controller.
After switching the controller on by pressing the boat rocker switch, wait for five minutes for the system to stabilize and reach pressure. Remove each plate from the packaging. Next, prime each well by adding 500 microliters of pre-warmed seeding advanced DMEM medium to the reservoir side.
Place the driver onto the docking station in the incubator. When done, select the prime program on the controller screen until the fluid comes through the filter supports. To cover the surface channel, fill all the wells with 1.1 milliliters of seeding advanced DMEM medium.
After placing the drivers with plates in an incubator at 37 degrees Celsius and 5%carbon dioxide, connect the docking station, and run the incubate program. Thaw vials of PHHs and HJCs by holding the vials in a 37 degrees Celsius water bath until only a small sliver of ice remains. Once thawed, gently pipette a maximum of two vials of PHHs directly into a tube of pre-warmed cryopreserved hepatocyte recovery medium, CHRM media.
Then use one milliliter of CHRM to wash the remaining cells from the cryotube. Pipette the HKCs gently from the cryotube into 10 milliliters of ice cold seeding advanced DMEM medium in a 50 milliliter centrifuge tube. Later, centrifuge both the cell types separately at room temperature at 100 times G for 10 minutes.
After centrifugation, remove the supernatant and re-suspend the PHHs in warm seeding advanced DMEM medium and HKCs in ice cold seeding advanced DMEM medium using one milliliter per vial of the cells added to the tube. Re-suspend the cells with a gentle rocking action, and then place on ice. When suspended, count the cells and record the viability.
Cell viability must be above 85%Next, disconnect the driver from the docking station, and place the driver in the microbiological safety cabinet or MBSC. Then aspirate the media from above the scaffold to the stopping point, channel, and reservoir, leaving a dead volume of 0.2 milliliters in the culture well, reaching just above the scaffold. Add 400 microliters of seeding advanced DMEM medium into the well chamber before returning the driver onto the docking station in the incubator, and running the media change program for three minutes.
After three minutes, disconnect the driver from the docking station, and place the driver back in the MBSC. Aspirate the media from the above scaffold down to the stopping point and at the reservoir end of each well, followed by re-suspending the PHHs by gently rocking the tube, and adding the required volume of the cell suspension to each culture well. Carefully pipette the cell suspension, ensuring even dispersion of the cells across the plate scaffold.
Carefully re-suspend HKCs, and add the cell suspensions to each culture well. Once all the wells contain both the cell types, place the microphysiological system or MPS driver onto the docking station in the incubator without physically connecting to stand for one hour. Once one hour has passed, connect the driver to the docking station and run the seed program.
When the program automatically pauses at two minutes, remove the driver from the incubator, and slowly add 1, 000 microliters of the seeding advanced DMEM medium to the channel to achieve a total volume of 1.4 milliliters. Later, move the plates to the incubator, and run the rest of the seed program for eight hours. On day four, pause the program on the controller, and disconnect the driver and the plate from the docking station.
Transfer them to an MBSC. Use a pipette to manually collect approximately one milliliter of media from each well for soluble biomarker analysis without disturbing the cell culture by touching the scaffold. Label the collected media as day four samples, and run lactate dehydrogenase and urea assays as quality control check to ensure that seeding has been successful.
Next, dose each well according to the plate plan by performing the media change. Once complete, return the driver onto the docking station in the incubator, and run the incubate program. On day six, disconnect the driver and the plate from the docking station, and transfer to an MBSC.
Use a pipette to collect approximately one milliliter of media from each well, and label as 48 hours post dose samples and store the samples at minus 80 degrees Celsius for later assays. On day eight, remove the scaffolds from the plates using a pair of tweezers, and place the scaffolds in a 24 well plate containing 500 microliters of DPBS without calcium and magnesium in each well without disturbing the micro tissue. Take the snapshots of each scaffold under an inverted light microscope at 10 times magnification.
On day four, prior to the drug dosing, a quality control check of formed liver micro tissues was performed with LDH release and urea synthesis. On day eight, multiple health and hepatic metrics such as albumin, urea, CYP three A four, ATP were assessed to confirm high levels of hepatic functionality and reproducibility in the microtissues. Contrast phase microscopy and IF staining in the liver microtissues revealed the even distribution of HKCs in the PHH microtissues.
The acute exposure of the liver microtissues to troglitazone caused toxicity, which was detected by alanine aminotransferase or ALT, and LDH release and a rapid reduction in albumin and urea production. The ATP content and CYP three A four activity confirmed the toxicity caused by troglitazone, and EC50 values were highly comparable to other endpoints. The bright-field microscopy images taken after eight day culture in the MPS reveal a healthy liver microtissue, uniformly seeded throughout the scaffold in the vehicle control.
Tissue death or degradation was seen in the replicates treated with positive control and troglitazone. Furthermore, liver toxicity following exposure to pioglitazone was also investigated. No LDH or ALT release was detected, however, a mild reduction in albumin and urea production was observed after 48 hours.
A minor reduction in ATP content was observed at high pioglitazone concentrations. EC50 values were generated from the dose response curves. Microscopy revealed slight microtissue alteration following 96 hours of exposure to pioglitazone at the two highest tested concentrations.
In the study, the use of cells having good quality and viability above 85%is essential for generating highly functional and healthy 3D microtissues in the microphysiological system. Following this procedure, we can assess adenine and drug-drug interactions and drug-induced liver injury on disease models such as non-alcoholic Delta hepatitis or non-alcoholic fatty liver disease model that uses triple culture of liver parenchymal and non-parenchymal cells.
