CN Bio Innovations Ltd. funded this study.

All the work was conducted in the laboratory following strict Health &#38; Safety procedures and in accordance with its own laboratory risk assessments and SOPs. All equipment used is serviced according to the manufacture&#39;s guidelines. Microbiological safety cabinets (MBSCs) are serviced annually, and Ki-Discus (potassium iodide) tested to British standards. The protocol follows the United Kingdom Human Tissue Authority (HTA) Code of Practice and directives and uses ethically-sourced primary human cells supplied by vendors that fully comply with the general requirements for informed consent (45 CFR &#167;46.116 and &#167;46.117) and Good Clinical Practice (GLP), (ICH E6), and regulatory and ethics committees.
1. Preparing cell culture media
NOTE: Prepare Seeding Advanced DMEM medium on Day -1 and store at 4 &#176;C. Prepare Maintenance Advanced DMEM medium on Day 1 and store at 4 &#176;C for up to 1 week.
<ol>
	<li>Seeding Advanced DMEM medium for PHHs and HKCs coculture: Supplement one bottle of 500 mL Advanced DMEM medium (Table of Materials) with 18 mL of Cocktail A (final concentration of 3.6%) and with 25 mL of FBS (final concentration 5%).</li>
	<li>Maintenance Advanced DMEM medium for PHHs and HKCs coculture: Supplement one bottle of 500 mL Advanced DMEM medium (Table of Materials) with 20 mL of Cocktail B (final concentration of 4%) and 500 nM hydrocortisone. 
	NOTE: Hydrocortisone will be made fresh on the day of use, and the steps on how to prepare the stock solution and required dilutions are mentioned below.</li>
	<li>Preparation of 500 nM hydrocortisone in Maintenance Advanced DMEM Medium
	<ol>
		<li>Preparation of the starting stock solution (20 mM): Weigh 7.24 mg of hydrocortisone (Table of Materials) into a 1 mL glass vial. Record the exact amount of hydrocortisone weighed out and determine the volume of dimethyl sulfoxide (DMSO) using the following calculation: 
		<img alt="Equation 1" src="/files/ftp_upload/63389/63389eq01.jpg" /></li>
		<li>Preparation of the working 100 &#956;M hydrocortisone stock solution: Add 5 &#181;L of the starting 20 mM stock solution to 995 &#956;L of Advanced DMEM. 
		NOTE: Diluting 25 &#181;L of the DMSO solution from step 1 in water or media results in 0.5% DMSO concentration. In the final solution, the DMSO concentration will be 0.0025%. In this case, the additional volume of 5 &#181;L results in an insignificant change in the total volume.</li>
		<li>Preparation of the working 500 nM hydrocortisone solution in Advanced DMEM: To prepare 1 mL of 500 nM hydrocortisone solution in Advanced DMEM, add 5 &#956;L of the stock solution of 100 &#956;M hydrocortisone to 995 &#956;L of the Maintenance Advanced DMEM medium.</li>
	</ol>
	</li>
</ol>
2. MPS set-up and priming (Day -1)
<ol>
	<li>Connect the controller to its docking station house in a cell culture incubator and ensure fresh desiccant (Table of Materials) is added into the desiccant jar located at the back of the controller. 
	NOTE: The controller unit draws moisture from the incubator over time and is kept dry using fresh desiccant.</li>
	<li>Switch the controller ON by pressing the boat rocker switch located behind it and wait for 5 min for the system to stabilize and reach pressure. Then check the screen for the pneumatic report to ensure: (i) The Pressure Reservoir Output reached ~2000 mBar and (ii) The Vacuum Reservoir Output reached ~850 mBar.</li>
	<li>Remove each plate from packaging and visually inspect every well to check for possible defects (missing scaffolds, cracks etc.).</li>
	<li>Insert a driver (with a plate on) onto the docking station to check that the driver is recognized by the docking station and controller. Check the Pressure Reservoir Output has dropped by less than 100 mBar, and the Vacuum Reservoir Output has increased by less than 500 mBar.</li>
	<li>Prime each well by adding 500 &#181;L of Seeding Advanced DMEM medium (pre-warmed to 37 &#176;C) to the reservoir side.</li>
	<li>Select the Prime program on the controller screen (up flow for 3 min at 2.5 &#181;L/s) until the fluid comes through the filter supports.NOTE: &#39;Up flow&#39; is a setting on the controller that allows media to flow from the reservoir upwards through the scaffolds in the LC12 plate.</li>
	<li>Fill all wells with a further 1.1 mL of Seeding Advanced DMEM medium to cover the surface channel. All wells will then be at their full working volume of 1.6 mL.</li>
	<li>Place the drivers with plates in a 37 &#176;C and 5% CO2 incubator, connect to the docking station and run the Incubate program. 
	NOTE: All programs used in the experiment (Prime, Incubate, Seed, Media Change) are pre-set in the MPS system. Prime the plates in the incubator until ready to seed.</li>
</ol>
3. Seeding liver cells into MPS (Day 0)
<ol>
	<li>Prevalidate all PHHS and HKCs. All PHHs and HKCs Lots are pre-validated in-house prior to performing the cell culture experiment (see Supplementary Material).</li>
	<li>Thaw vials of PHHs and HKCs cells (Table of Materials) by holding the vials steadily in a 37 &#176;C water bath until only a small sliver of ice remains.</li>
	<li>Pipette PHHs directly into a tube of pre-warmed (37 &#176;C) Cryopreserved Hepatocyte Recovery Medium CHRM media (two vials max per tube).</li>
	<li>Pipette the cells gently, then use 1 mL of CHRM to wash any remaining cells from the cryotube. Be very gentle with cells when thawing and transferring into a conical tube. 
	CAUTION Do not agitate the vials during thawing, and do not pipette their content up and down.</li>
	<li>Pipette HKCs cells gently from the cryotube into 10 mL of ice-cold Seeding Advanced DMEM medium in a 15 mL centrifuge tube. 
	NOTE: Up to 2 vials of HKCs can be combined.</li>
	<li>Centrifuge both cell types at room temperature (RT) at 100 x g for 10 min. Remove the supernatant.</li>
	<li>Resuspend the PHHs in warm Seeding Advanced DMEM medium and HKCs in ice-cold Seeding Advanced DMEM medium (to help reduce cell clumping), using 1 mL per vial of cells added to the tube and place the cells on ice. Use a gentle rocking action to resuspend the cells. 
	CAUTION: Do not resuspend PHHs by pipette action, as it can lead to cell death.</li>
	<li>Combine the cell suspensions from multiple tubes (if applicable - i.e., if all PHHs are from the same donor), but do not mix cell types.</li>
	<li>Count cells. Record the viability (must be above 85% for both cell types, PHHs and HKCs) and the total number of cells. If cell viability falls below 85%, thaw a new vial of cells, and reassess cell viability.</li>
	<li>Calculate cell viability using the following formula: 
	<img alt="Equation 2" src="/files/ftp_upload/63389/63389eq02.jpg" /></li>
	<li>Calculate the desired volume of the cell suspension to seed into each well and additional volume of Seeding Advanced DMEM medium needed to take total seeding volume to 400 &#181;L. Cell numbers per well: 0.4 x 106 PHHs and 0.04 x 106 HKCs per well, and cell density of 0.25 x 106 PHHs/mL, respectively 0.025 x 106 HKCs/mL.</li>
	<li>Disconnect the driver from the docking station and place it in the MBSC.</li>
	<li>Aspirate the media from the above scaffold to the stopping point (going down the deep notch on the retaining ring), channel and reservoir. Leave a &#34;dead volume&#34; of 0.2 mL in the culture well, reaching just above the scaffold. Care must be taken not to remove the total medium from above the scaffold to avoid air bubbles forming.</li>
	<li>Add 400 &#181;L of Seeding Advanced DMEM medium into the well chamber, return the driver onto the docking station in the incubator and run the Media Change program for 3 min. The program will automatically pause after 3 min.</li>
	<li>Once complete, disconnect the driver from the docking station and place it back in the MBSC.</li>
	<li>Aspirate the media from the above scaffold down to the stopping point and at the reservoir end of each well.</li>
	<li>Carefully resuspend the PHHs by gently rocking the tube, then add the required volume of the cell suspension to each culture well. Carefully pipette the cell suspension, ensure tha the cells disperse evenly across the plate&#39;s scaffold. 
	NOTE: To ensure good coverage throughout scaffold, use a slow swirling motion to pipette cells down onto the scaffold.</li>
	<li>Similarly, carefully resuspend HKCs and add the cell suspensions to each culture well. 
	NOTE: Use a slow swirling motion to seed HKCs to ensure good coverage throughout the scaffold. The two seeding sub-steps can be separated, or the two cell types can be pre-mixed at an appropriate density and seeded concomitantly.</li>
	<li>Once all wells contain both cell types, place the MPS driver onto the docking station in the incubator without physically connecting it and leave it to stand for 1 h.</li>
	<li>After 1 h, fill up each well with the required volume of additional Seeding Advanced DMEM medium to reach 400 &#956;L and run the Seed program.</li>
	<li>After 2 min, the program will automatically pause, remove the driver from the incubator and slowly add 1000 &#181;L of the Seeding Advanced DMEM medium to the channel (closer to the reservoir end than the well chamber) to achieve a total volume of 1.4 mL (with a further 200 &#181;L dead volume in the channels).</li>
	<li>Move the plates to the incubator and run the rest of the Seed program for 8 h. 
	NOTE: Flow will automatically switch to Incubate program after 8 h.</li>
</ol>
4. Media change (Day 1)
<ol>
	<li>Disconnect the driver from the docking station and place it in the MBSC.</li>
	<li>Perform Media Change by removing the Seeding Advanced DMEM medium in the well chamber down to the stopping point.</li>
	<li>Add 400 &#181;L of the Maintenance Advanced DMEM medium into the well chamber, return the driver onto the docking station in the incubator and run the Media Change program for 3 min. The program will automatically pause after 3 min.</li>
	<li>Disconnect the driver from the docking station and, in the MBSC, aspirate away media from the reservoir chamber, channel, and stopping point above the scaffold. At this point, the culture well will be returned to the dead volume.</li>
	<li>Top up the reservoir chamber with 1.4 mL of fresh pre-warmed (37 &#176;C) Maintenance Advanced DMEM medium.</li>
	<li>Return the driver onto the docking station in the incubator and run the Incubate program.</li>
</ol>
5. Liver microtissue quality control (QC), media collection, media change, and drug dosing (Day 4)
<ol>
	<li>On day 4 perform a Media Change using the Maintenance Advanced DMEM medium and QC checks to ensure seeding has been successful. 
	NOTE: QC is a process used to check the health of the formed microtissues by measuring Lactate dehydrogenase (LDH) and urea.</li>
	<li>Prior to running the QC, prepare fresh stock solutions for each compound to test (either in Maintenance Advanced DMEM medium or Maintenance Advanced DMEM medium containing 0.1% DMSO, depending on the solubility of each compound). Prepare dilutions accordingly to yield test concentrations for each compound.</li>
	<li>Disconnect the driver and plate from the docking station and transfer to a MBSC.</li>
	<li>Transfer 50 &#181;L of media from each well using a pipette to a 96 well plate to perform an LDH assay (Table of Materials) before sampling and 25 &#181;L for a Urea assay (Table of Materials). 
	NOTE: LDH and urea assays will be performed following the manufacturer&#39;s instructions.</li>
	<li>Continue the experiment after QC if LDH readings are lower than 2 AU/106 cells and urea is above 40 &#181;g/day/106 cells. 
	NOTE: Albumin is not used as QC because it is a lengthy assay to run on the day and will be assayed later once the experiment is complete from the samples withdrawn on Day 4.</li>
	<li>If any wells fail QC, remove them from the experimental design.</li>
	<li>Once the experimental layout is confirmed, sample the remaining media from each well, making sure not to disturb the cell culture by touching the scaffold. Store the collected media (labeled as pre-dose samples) at -80 &#176;C for later assays.</li>
	<li>Perform Media Change in a MBSC following steps 4.3-4.5. Change the wells to Maintenance Advanced DMEM medium with the right drug concentration according to the experimental design.</li>
	<li>Once complete, return the driver onto the docking station in the incubator and run the Incubate program.</li>
</ol>
6. Media collection, media change and drug dosing (Day 6)
<ol>
	<li>Prepare fresh stock solutions for each compound to test (either in Maintenance Advanced DMEM medium or Maintenance Advanced DMEM medium containing 0.1% DMSO, depending on the solubility of each compound). Prepare dilutions accordingly to yield test concentrations for each compound according to the plate plan.</li>
	<li>Disconnect the driver and the plate from the docking station and transfer to a MBSC.</li>
	<li>Collect media from each well (~1 mL) manually with a pipette making sure not to disturb the cell culture by touching the scaffold, assay for LDH and Urea. Store the rest of the collected media at -80 &#176;C for later assays, and label them 48 h post-dose samples.</li>
	<li>Re-dose each well with the same drug concentration as on Day 4 and according to the plate plan by performing Media Change following steps 4.3-4.5.</li>
	<li>Once complete, return the driver onto the docking station in the incubator and run the Incubate program.</li>
</ol>
7. Ending the experiment (Day 8)
<ol>
	<li>Disconnect the driver and the plate from the docking station and transfer to a MBSC.</li>
	<li>Sample media from each well manually using a pipette, making sure not to disturb the cell culture by touching the scaffold.</li>
	<li>Assay the withdrawn media for LDH and Urea and store the rest of the collected media at -80 &#176;C for later assays.</li>
	<li>Running CYP3A4-glo assay.
	<ol>
		<li>Measure the effects of the drugs tested on cytochrome P450 CYP3A4 activity in PHHs at the end of the experiment using this assay.</li>
		<li>Reconstitute the detection reagent (for CYP3A4 assay, see Table of Materials) following the manufacturer&#39;s instructions. If the detection reagent has previously been reconstituted and frozen, remove it from the -20 &#176;C freezer and allow it to thaw at RT.</li>
		<li>Prepare 20 mM stock D-luciferin standard following the manufacturer&#39;s instructions.</li>
		<li>Prepare working luminogenic substrate medium with a 1:1000 dilution of Luciferin IPA in Maintenance Advanced DMEM medium (2 mL of luminogenic substrate medium per well).</li>
		<li>Perform a Media Change as described in steps 4.3-4.5 with luminogenic substrate medium. Save 500 &#181;L of the luminogenic substrate medium in a 1.5 mL glass vial (Table of Materials) as input material.</li>
		<li>Return the driver onto the docking station in the incubator and run the Incubate program for 1.5 h.</li>
		<li>Prepare D-luciferin standard curve in culture medium in 1.5 mL tubes following the manufacturer&#39;s instructions and pipette 50 &#181;L of each standard in duplicate on a white opaque 96-well plate (see Table of Materials), using culture medium as blank or 0 &#181;M.</li>
		<li>Once the time has elapsed, remove the driver from the docking station and sample media for CYP3A4 assay following steps 7.4.9-7.4.13.</li>
		<li>After incubation, transfer 50 &#181;L of the sample medium from each well and input material to the 96-well opaque white luminometer plate containing standards. Take care to leave at least two empty rows on the opaque plate between the standards and samples to avoid light carryover between the top standards and the samples readings.</li>
		<li>Add 50 &#181;L of Luciferin Detection Reagent to each well to initiate a luminescent reaction.</li>
		<li>Incubate the plate at RT on a plate shaker for 20 min in the dark to stabilize the luminescent signal.</li>
		<li>Record the luminescence using a luminometer or CCD camera.</li>
		<li>Plot the standard curve by taking the average of each point and then subtracting the average of the blanks. Use the equation of the line to calculate the metabolic rate (pmol/min/106 cells) in the rest of the samples, remembering to include any dilutions done.</li>
	</ol>
	</li>
	<li>Remove the scaffolds from the plates using a pair of tweezers and place them in a 24 well plate containing 500 &#181;L of D-PBS (Without Ca++ and Mg++) in each well, taking care not to disturb the microtissue.</li>
	<li>Take snapshots of each scaffold using an inverted light microscope at magnification 10x.</li>
	<li>Running the ATP assay (see Table of Materials) following the manufacturer&#39;s instructions:
	<ol>
		<li>Thaw the reagent at RT.</li>
		<li>Wash the scaffolds twice with 500 &#181;L of D-PBS (Without Ca++ and Mg++) for each wash step.</li>
		<li>Add 120 &#956;L of reagent and 120 &#956;L of PBS to each scaffold and the same volumes to an empty well (this will serve as blank). Place the plate covered in aluminum foil on a shaker and shake vigorously (500 rpm) for 5 min followed by 30 min of incubation to stabilize the luminescence signal.</li>
		<li>Transfer 100 &#956;L of the lysed samples in duplicate to a clear flat-bottom 96-well assay plate for measurement. Ensure that the blank wells are not placed next to the other measuring wells of high luminescence.</li>
		<li>Record the luminescence using a microplate reader.</li>
		<li>Compare the luminescence of the samples to the luminescence of the standards to determine ATP detected by the reagent in the samples.</li>
	</ol>
	</li>
</ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+microfluidic+liver+cultures+as+a+physiological+pre-clinical+tool+for+hepatitis+B+virus+infection.">3D microfluidic liver cultures as a physiological pre-clinical tool for hepatitis B virus infection.</a> Nature Communications. 9 (1), 682 (2018).</li><li>Kostrzewski, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+perfused+human+in+vitro+model+of+non-alcoholic+fatty+liver+disease.">Three-dimensional perfused human in vitro model of non-alcoholic fatty liver disease.</a> World Journal of Gastroenterology. 23 (2), 204-215 (2017).</li><li>Kostrzewski, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microphysiological+system+for+studying+nonalcoholic+steatohepatitis.">A microphysiological system for studying nonalcoholic steatohepatitis.</a> Hepatology Communications. 4 (1), 77-91 (2020).</li><li>Vacca, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+morphogenetic+protein+8B+promotes+the+progression+of+non-alcoholic+steatohepatitis.">Bone morphogenetic protein 8B promotes the progression of non-alcoholic steatohepatitis.</a> Nature Metabolism. 2 (6), 514-531 (2020).</li><li>Long, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+therapeutic+antibody-small+molecule+drug-drug+interactions+using+a+three-dimensional+perfusable+human+liver+co-culture+platforms.">Modeling therapeutic antibody-small molecule drug-drug interactions using a three-dimensional perfusable human liver co-culture platforms.</a> Drug Metabolism and Disposition. 44, 1940-1948 (2016).</li><li>Bai, J., Wang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+and+microphysiological+systems:+New+tools+for+ophthalmic+drug+discovery.">Organoids and microphysiological systems: New tools for ophthalmic drug discovery.</a> Frontiers in Pharmacology. 11, 407 (2020).</li><li>Ribeiro, A. J. S., Yang, X., Patel, V., Madabushi, R., Strauss, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+microphysiological+systems+for+predicting+and+evaluating+drug+effects.">Liver microphysiological systems for predicting and evaluating drug effects.</a> Clinical Pharmacology &amp; Therapeutics. 106 (1), 139-147 (2019).</li><li>Clark, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microphysiological+system+model+of+therapy+for+liver+micrometastases+hhs+public+access.">A microphysiological system model of therapy for liver micrometastases hhs public access.</a> Experimental Biology and Medicine (Maywood). 239 (9), 1170-1179 (2014).</li><li>Qosa, H., Ribeiro, A. J. S., Hartman, N. R., Volpe, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+commercially+available+line+of+iPSC+hepatocytes+as+models+of+hepatocyte+function+and+toxicity+for+regulatory+purposes.">Characterization of a commercially available line of iPSC hepatocytes as models of hepatocyte function and toxicity for regulatory purposes.</a> Journal of Pharmacological and Toxicological Methods. 110, 107083 (2021).</li></ol>

All authors are employees of CN Bio Innovations Limited.

MPS are designed to recapitulate functional units of human organs in vitro and have been developed to address the limitations of conventional 3D cell culture models27. The liver is one of the most modeled organs using MPS, and a wide variety of systems have been developed. The human liver is responsible for drug metabolism and generation of toxic drug metabolites, and its function is a key element to model for drug development, including the assessment of DILI liability of compounds28. Here we have introduced a new method for assessing DILI using a liver MPS; the protocol enables mechanistic insights to be sought for each compound assayed to determine how it may cause DILI as well as being a highly sensitive and robust assay. Liver microtissues are formed in the MPS plates, which are a coculture of PHH and HKCs and are highly functional with high levels of albumin and urea production as well as high CYP3A4 activity compared to standard in vitro liver models20.
Although the DILI model described here can serve as a useful tool in later stages of preclinical testing in the drug development process, it also has several limitations. As the majority of MPS currently available on the market, it is a low-throughput platform and, therefore, more difficult to use for large-scale drug screening activities. Consisting of microtissues formed by coculturing PHHs and HKCs the DILI model also cannot entirely capture the complexity of the human liver, and further optimization by incorporating different types of cells (e.g., immune cells) would be beneficial to add value to the existing model. This single-organ MPS could also be combined with other organ platforms that can share a common medium and allow organ crosstalk at the cellular or endocrine level, and that can help to better understand the mechanistic insights of toxicity not limited only to the liver itself. Furthermore, as any relatively new technology, it might be considered costly and hence of limited accessibility.
MPS is a platform used to develop organotypic models of single or multi- human tissues. The system is composed of a controller, umbilical cable, and MPS driver into which the plate is inserted (Figure 5A). Each liver MPS plate has 12 independent open wells for culturing primary liver cells in 3D on engineered scaffolds. In summary, the system is QC checked, and the plates are primed at Day -1, the PHHs and HKCs are seeded on the plates at Day zero (Figure 5B, see 1). Embedded micropumps facilitate the circulation of cell culture media through the scaffolds to facilitate the formation of 3D microtissues (Figure 5B, see 2). Formed microtissues are QC&#39;d at Day 4, dosed with different concentrations of each compound every 48 h for 4 days, and assayed for endpoint biomarkers at Day 8 (Figure 5C). The experimental timeline of the DILI assay in the MPS plate is depicted in Figure 5D.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63389/63389fig05.jpg" /> 
Figure 5: The microphysiological systemand experimental timeline of a standard DILI assay. (A) The microphysiological system with its components: controller (1), umbilical cable (2), docking station (3), MPS driver (4) and LC12 plate (5). (B) Seeding of PHHs and HKCs on LC12 plate at Day 1 (1) and embedded micropumps facilitate circulation of cell culture media with tuneable flow rates through the 3D microtissues seeded on the scaffolds (2). (C) Taking down the scaffolds at the end of the study. (D) Experimental timeline. <a href="https://www.jove.com/files/ftp_upload/63389/63389fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
When performing the protocol, it is important that a robust system QC check is performed prior to starting, checking the system is functioning pneumatically correctly and the consumable plates are visually inspected and primed efficiently to ensure even functionality across all wells. Having high-quality primary human cells is essential for this protocol, with hepatocytes known to adhere consistently in cell culture experiments and form 3D interactions. Thawing these cells is also a critical step, as primary hepatocytes should not be resuspended by pipetting action as this can rapidly lead to cell death. Having cell viability above 85% is critical for successful seeding, as large amounts of cellular debris will interfere with 3D microtissue formation. The QC check of formed liver microtissues at Day 4 is also important, and the user needs to ensure that acceptable levels of LDH and Urea are measured, as out-of-range parameters might be indicative of poor-quality tissue formation and allow straightforward troubleshooting. Finally, the hydrocortisone used in the cell culture media must be prepared fresh on the day of use to prevent any unwanted degradation that might impact cell culture functionality, as it is required to maintain the metabolic functionality of the hepatocytes.
Despite having significant complexity, the liver MPS does not contain all the cell types of the human liver. It is possible to add further cells types to the model24,29 to increase physiological relevance, but these should only be added with a clear justification for the context of use. For studying DILI PHH are the key cell type, and the incorporation of HKCs in this model allows some immunological responses to be determined. It should also be noted that PHHs isolated from human livers and commercially available cryopreserved PHHs tend to demonstrate some variations from lot to lot. We have demonstrated here that this protocol produces reproducible results when used with high-quality preparations of cells. However, some lot-lot variation would be expected, and this could be further overcome by using pooled lots of multiple donors. These limitations could be overcome by using hepatocyte-like cells differentiated from iPSC that recapitulate many functional properties of PHHs and that have been used in the drug development process30. HKCs also show a lot to lot variability and a high level of activation upon thaw; therefore, HKCs donors are pre-validated in-house prior to use in experimental cell culture (coculture with validated PHHs) and must have low levels of post-thaw activation; this is assessed by measuring biomarkers IL-6 and TNF-alpha (see Supplementary Material).
The data presented here confirm that the assay can detect DILI accurately, helping to identify hepatotoxicants that might not be detected by 2D10,11&#160;and even some 3D models. Data generated from MPS are still not used as a standard by the pharmaceutical industry for regulatory submissions or drug screening purposes due to the lack of process standardization and harmonization, including inter-site reproducibility20. The data and experimental approaches demonstrated here address this, showing that the liver model can be used routinely and robustly in DILI screens to accurately predict the liability of novel compounds.
By measuring a range of endpoints to produce a &#34;signature of hepatotoxicity&#34;, helping to identify compounds with different levels of DILI concern (including compounds not detectable by other in vitro methods) and their mechanisms of toxicity revealed. This technology can close the gap between traditional cell culture and animal models on one side and human clinical trials, advancing towards the simulation of human biological conditions for the preclinical assessment of liver toxicity as part of the drug development process.

DILI remains the most common cause for acute liver failure in the USA and Europe and is a leading cause of attrition of compounds in the drug development process1. Nearly all classes of medications can cause hepatotoxicity, with central nervous system agents and antibiotics being by far the most common treatments that cause DILI in patients2. Drug-induced hepatotoxicity is caused by a complex interaction of genetic, non-genetic, and environmental factors, leading to the death of hepatocytes and other liver cell types, including cholangiocytes and endothelial cells1,3.
DILI causing agents can be classified in two ways: those which cause predictable dose-dependent liver damage or those which cause idiosyncratic DILI that is rare and develops independently of drug dose, or route, or duration of administration, but is responsible for up to a sixth of all acute liver failures in the USA only4. Unfortunately, DILI is often not detected until drugs have reached the clinical stages of the drug development process. Drug-induced liver injury rank (or DILIrank) consists of more than a thousand FDA-approved medicines that are divided into four classes according to their potential for causing DILI, and their use in patients must be closely monitored5.
Studying mechanisms of drug hepatotoxicity remains very challenging, and therefore, many preclinical models have been developed to explore mechanisms of DILI. Current in vitro and in vivo models used to predict DILI in preclinical development have several limitations to providing insights into the complex, multifaceted interactions in a living human body. Cancerous hepatic cell lines (i.e., HepG2, HepaRG) cultured in 2D are still used in the early stages of drug development for evaluating the toxicity of candidate compounds6. Even so, these cell lines come from single donors and show abnormal levels of liver function, and do not always exhibit high sensitivity for detection of DILI7,8. As an alternative to cancerous hepatic cell lines, PHHs better represent human liver physiology if cultured appropriately in vitro, although several limitations exist with their culture, like short incubation time with drugs, relatively short life span, loss of hepatic gene expression, and changes of drug metabolic functions9,10,11. PHHs can be cultured on extracellular matrix proteins in standard 2D cell culture plates, but as a downside, the rapid decline in their function means they have low sensitivity (&#60;50%) for DILI prediction12.
On the other hand, testing on animal models is slow, expensive, and needs a cross-species translation to extrapolate prediction to humans. Most newly developed drugs fail to gain approval making this process costly and risky5. Furthermore, for testing new human-specific modalities, animal models are less suitable due to gene sequence or immunological response differences versus humans13.
Consequently, interest in more advanced three-dimensional (3D) in vitro liver models has exponentially grown. Culturing PHHs as spheroidal structures generated by gravitational aggregation in hanging drops or on ultralow attachment surfaces represents a high-throughput method for assessing compound liabilities14. PHH spheroids have been used to assess DILI in a diseased background (e.g., steatosis and cholestasis)15. A wide variety of models have been developed to include plated micro-patterned cocultures of hepatocytes with stromal fibroblasts16, 3D bio-printed liver tissues17, 3D spheroid cultures with or without hepatic non-parenchymal cells15. However, all these methods still have drawbacks, and culturing PHHs in a more physiologically relevant microenvironment could provide them with higher levels of functionality for extended periods of time to enable the investigation of prolonged exposure to potential hepatotoxicants. Additionally, to improve the translational relevance of any advanced in vitro PHH culture, clinically relevant functional endpoints or toxicity output biomarkers must be utilized to allow data to be compared in vivo or clinical scenarios18.
In this study, we assessed whether a MPS, also known as an Organ-on-a-Chip (OOC), in vitro liver model could be used to understand the detailed mechanistic aspects of liver toxicity. The MPS has previously been shown to maintain highly functional 3D liver microtissues, under flow, for up to 4 weeks19. The system has been recently tested by FDA and shown to have high reproducibility when performing drug toxicity, metabolism, and intracellular accumulation20. Moreover, when compared with spheroids and sandwich cultures, the system had a more stable function and higher sensitivity in detecting the toxicity of several drugs20. To date, the MPS has been used in a wide range of applications that cover ADME21, disease modeling (HBV22, NAFLD23,24,25), and drug-drug interactions26, potentially making it highly suited to assessing acute and chronic DILI. The technology presented here offers an alternative to close the gap between more traditional cell cultures and animal models and human clinical trials, advancing towards the simulation of human biological conditions to support the assessment of candidate compounds&#39; liver toxicity in preclinical stages of the drug development process.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24 well cell culture cluster plates flat bottom</td>
			<td>Corning</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well clear assay plates, flat bottom clear plastic</td>
			<td>Greiner</td>
			<td>655101</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates black flat bottom</td>
			<td>Corning</td>
			<td>3915</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well White/Clear Bottom Plate, TC Surface</td>
			<td>ThermoScientific</td>
			<td>165306</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM (1x)</td>
			<td>Gibco</td>
			<td>12491015</td>
			<td>Cell culture media.</td>
		</tr>
		<tr>
			<td>AssayMax Albumin ELISA Kit</td>
			<td>AssayPro</td>
			<td>EA3201-1</td>
			<td>Dilution 1:250. Time point Day 4, 6, and 8.</td>
		</tr>
		<tr>
			<td>Cell Maintenance Cocktail B, (Primary Hepatocyte Maintenance Supplements)</td>
			<td>Gibco</td>
			<td>CM4000</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D Cell Viability Assay</td>
			<td>Promega</td>
			<td>G9682</td>
			<td>Dilution 1:1. Time point Day 8.</td>
		</tr>
		<tr>
			<td>Chlorpromazine HCl</td>
			<td>Sigma Aldrich</td>
			<td>C8138</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromacol blue lids, 9 mm Autosampler Vial Screw Thread Caps</td>
			<td>ThermoScientific</td>
			<td>9-SCK(B)-ST1</td>
			<td>glass vial</td>
		</tr>
		<tr>
			<td>Chromacol vials, 9 mm Clear Glass Screw Thread Vials</td>
			<td>ThermoScientific</td>
			<td>2-SVW</td>
			<td></td>
		</tr>
		<tr>
			<td>Class 2 Microbiological Safety Cabinets - Trimat2 1500 exhaust</td>
			<td>Contained Air Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes 50 mL</td>
			<td>Greiner</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryopreserved Hepatocyte Recovery Medium (CHRM)</td>
			<td>ThermoFisher Scientific</td>
			<td>Gibco CM7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryopreserved primary human hepatocytes</td>
			<td>BioIVT Europe</td>
			<td>Lot. RAS</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoTox 96 Cytotoxicty (LDH) Assay Kit</td>
			<td>Promega</td>
			<td>G1781</td>
			<td>Dilution - none. Time point Day 4, 6 and 8</td>
		</tr>
		<tr>
			<td>&#62;Data analysis model used to generate the graph and EC:50 curves was nonlinear regression (curve fit) asymmetric sigmoidal, 5PL, where X is log(concentration</td>
			<td>GraphPad Prism 9</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable PES Filter Units 500mL</td>
			<td>Fisher Scientific</td>
			<td>15913307</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Pipette Basins 50ml</td>
			<td>Fisher Scientific</td>
			<td>12369175</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO (Dimethyl sulfoxide)</td>
			<td>Sigma-Aldrich</td>
			<td>Sigma D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbeco&#8217;s Phosphate Buffered Saline without Ca2+ and Mg2+ (D-PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy Reader Conical Polypropylene Centrifuge Tubes 15 mL</td>
			<td>Fisher Scientific</td>
			<td>11889640</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum</td>
			<td>Gibco</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Human ALT ELISA Kit</td>
			<td>Abcam</td>
			<td>&#160;ab 234578</td>
			<td>Dilution 1:5. Time point Day 6 and 8.</td>
		</tr>
		<tr>
			<td>Human Cryopreserved Kupffer Cells</td>
			<td>Lonza Europe</td>
			<td>Lot. 190088KC</td>
			<td></td>
		</tr>
		<tr>
			<td>hydrocortisone</td>
			<td>Merck</td>
			<td>H0888-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubators models: New Brunswick&#160; Galaxy 170 S, New Brunswick&#160; Galaxy 170 R and CellXpert&#174; C170.</td>
			<td>Eppendorf</td>
			<td></td>
			<td>All serviced yearly; paperwork available upon request.</td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Leica DMIL LED</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MPS know as Organ-on-a-Chip (OOC)</td>
			<td>CN Bio Innovations Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MPS LC-12 plate</td>
			<td>CN Bio Innovations Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer Improved C-Chip Disposable Haemocytometer (2 channel)</td>
			<td>Cambridge Bioscience</td>
			<td>DHC-N01-50</td>
			<td></td>
		</tr>
		<tr>
			<td>P450-Glo CYP3A4 Assay and Screening System</td>
			<td>Promega</td>
			<td>V9002</td>
			<td>Dilution - none. Time point Day 8</td>
		</tr>
		<tr>
			<td>PhysioMimix MPS platform</td>
			<td>CN Bio Innovations Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pioglitazone</td>
			<td>MedChemExpress Tocris</td>
			<td>HY-13956/CS-1700</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantichrom Urea Assay Kit &#8211; Bioassay systems</td>
			<td>Bioassay Systems</td>
			<td>DY970-05</td>
			<td>Dilution 1:2 if initial reading is too high. Time point Day 4, 6 and 8.</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Sigma-Aldrich</td>
			<td>S7625</td>
			<td></td>
		</tr>
		<tr>
			<td>Software used to analyse and generate all the graphs was</td>
			<td>GraphPad Prism 9</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stripettes 10 mL</td>
			<td>Fisher Scientific</td>
			<td>11839660</td>
			<td></td>
		</tr>
		<tr>
			<td>Stripettes 25 mL</td>
			<td>Fisher Scientific</td>
			<td>11839181</td>
			<td></td>
		</tr>
		<tr>
			<td>Thawing plate Cocktail A, (Primary Hepatocyte Thawing and Plating Supplements)</td>
			<td>Gibco</td>
			<td>CM3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Troglitazone</td>
			<td>MedChemExpress Tocris</td>
			<td>97322-87-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubes 1.5 mL</td>
			<td>Greiner</td>
			<td>616201</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing balance - model PA214C and AV213C</td>
			<td>Ohaus Corp</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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).

The manuscript describes a liver MPS model used for assessing DILI. The MPS facilitates the generation of 3D liver microtissues that are maintained highly functional under flow for up to 4 weeks. PHHs/HKCs are seeded onto collagen-coated scaffolds to form liver microtissues which are perfused with a growth medium and, after passing the QC check, are dosed with compounds. Here, we show data for troglitazone and pioglitazone, two structurally similar compounds but with different DILI severities.
At Day 4, prior to drug dosing, a QC check of formed liver microtissues is assessed and consists of LDH release and urea synthesis (Figure 1A). The QC aims to confirm that the liver MPS produces highly consistent and functioning liver microtissues. The data presented here are generated from three experiments and shows good levels of reproducibility with low intra- and inter-study variability. After an 8-day culture, multiple health and hepatic metrics (albumin, urea, CYP3A4, ATP) are assessed and control microtissues show high levels of hepatic functionality and reproducibility (Figure 1B,C). Contrast phase microscopy and IF staining of the liver microtissues (See Supplementary Material) shows high seeding consistency throughout the scaffold&#39;s microchannels and reveal the distribution of HKCs in the PHH microtissues (Figure 1D)
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63389/63389fig01.jpg" /> 
Figure 1: Liver MPS produces highly reproducible data and consistent microtissues. (A) 3D Liver microtissue QC metrics at Day 4, and functionality assessment at the end of the study at Day 8 -(B) Albumin and Urea, (C) CY3A4 and ATP). Data is collected from 3 experiments; in each experiment, there were 3 vehicle control replicates. Data shown are Mean &#177; SD, N = 9. (D) Phase contrast microscopy (10x and 20x) and IF of 3D liver microtissues generated by coculturing PHHs and HKCs in liver MPS platform for assessing DILI. To visualize the HKCs, prior to seeding HKCs were transduced with an adenoviral vector expressing eGFP (see Supplementary Material). Representative photomicrographs are shown. The transduction and imaging were performed as a standalone experiment to demonstrate cell localization and not done with the DILI protocol described. HKCs cells are pre-validated in-house prior to use in experimental cell culture and must have low levels of post-thaw activation; this is assessed by measuring biomarkers IL-6 and TNF-alpha. <a href="https://www.jove.com/files/ftp_upload/63389/63389fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Troglitazone is known to cause severe DILI; following its license for the treatment of type 2 diabetes, it was withdrawn by the FDA after 3 years on the market because of the frequency of liver injury associated with its use. To date, published animal studies failed to predict troglitazone&#39;s potential to cause severe liver injury. The toxicity of this compound was also not detected in standard in vitro 2D hepatic assays14.
Liver microtissues in the MPS were dosed with troglitazone for 96 h, and it caused an acute toxic response, Cmax driven, which was detected by ALT and LDH release and a rapid reduction in albumin and urea production, at circa 15 x Cmax, following acute exposure to troglitazone (Figure 2A). Cellular endpoint (ATP content) and CYP3A4 activity (for assessing metabolic biotransformation), sampled after 96 h exposure, further confirmed toxicity caused by troglitazone and EC:50 values were highly comparable to other endpoints (Figure 2B). Brightfield microscopy images taken after 8-day culture in the MPS reveal a healthy liver microtissue, uniformly seeded throughout the scaffold (vehicle control) in contrast to generalized tissue death/degradation as seen in the replicates treated with positive control and troglitazone at the top two test concentrations (Figure 2C).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63389/63389fig02.jpg" /> 
Figure 2: Determining DILI risk of troglitazone using multiple hepatotoxic endpoints. Liver microtissues were exposed to seven test concentrations of troglitazone for 96 h and compared for (A) LDH release, ALT release, Albumin production, Urea synthesis, CYP3A4 activity, and ATP content. Blue lines - 48 h exposure (media endpoints only), red lines - 96 h exposure. Positive control was 100 &#956;M chlorpromazine. All endpoints are measured from the same liver MPS cultures. Data shown are mean &#177; SD, N = 3. (B) Summary of E:50 numbers generated from data. N.D. = data not plottable. Line = not assayed. (C) Representative brightfield microscopy of liver microtissues after 8-day culture (magnification 10x). <a href="https://www.jove.com/files/ftp_upload/63389/63389fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Liver toxicity following exposure to pioglitazone was also investigated. Pioglitazone is a compound known to be of low-DILI concern4 and did not exert hepatotoxicity in classic 2D primary hepatocytes cultures and even in some more advanced 3D models10, 11. Mild hepatotoxic effects were observed at both tested time points (Figure 3). No LDH or ALT release was detected; however, after 48 h, a mild reduction in albumin and urea production was observed, at approx. 25x Cmax (Figure 3A). Very minor reduction in ATP content was also observed at high pioglitazone concentrations, but this was not significant. EC:50 values generated from dose-response curves are presented in Figure 3B. Microscopy revealed slight microtissue alteration following 96 h exposure to pioglitazone at the two highest tested concentrations (Figure 3C). The results demonstrate the ability of the liver MPS to detect the toxicity of compounds with mild DILI concern.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63389/63389fig03.jpg" /> 
Figure 3: Determining DILI risk of pioglitazone using multiple hepatotoxic endpoints. Liver microtissues were exposed to seven test concentrations of pioglitazone for 96 h and compared for (A) LDH release, ALT release, Albumin production, Urea synthesis, CYP3A4 activity, and ATP content. Blue lines - 48 h exposure (media endpoints only), red lines - 96 h exposure. Positive control was 100 &#956;M chlorpromazine. All endpoints are measured from the same liver MPS cultures. Data shown are mean &#177; SD, N = 3. (B) Summary of EC:50 numbers generated from data. N.D. = data not plottable. Line = not assayed. (C) Representative brightfield microscopy of liver microtissues after 8-day culture (magnification 10x). <a href="https://www.jove.com/files/ftp_upload/63389/63389fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
By assessing all functional endpoints and toxicity output biomarkers that might represent in vivo or clinical scenarios (LDH release, Urea synthesis, Albumin production, CYP3A4 activity, ATP content, ALT release) and corroborating the data generated for both tested compounds dosed at a seven-point dose range for 48 h and 96 h, a heatmap has been generated to yield a &#34;signature of hepatotoxicity&#34;, helping to identify compounds with varying level of DILI concern (Figure 4).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63389/63389fig04.jpg" /> 
Figure 4: Determining &#34;signature of toxicity&#34; with Liver MPS. Heatmap showing of troglitazone and pioglitazone from six functional liver-specific endpoints (LDH release, Urea synthesis, Albumin production, ALT release, CYP3A4 activity, and ATP content) following 48 h and 96 h exposure to seven-point dose range. Each value is generated as Mean, N = 3, and normalized to control samples. The values on the color bars represent a fold increase over baseline controls. <a href="https://www.jove.com/files/ftp_upload/63389/63389fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Material: Fluorescent microscope imaging of microtissues and pre-qualification assessment of cells. <a href="https://www.jove.com/files/ftp_upload/63389/Supplementary Material-63389R1.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro at JoVE.com

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.

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 ...

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4.1K Views.  CN Bio Innovations Ltd. 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.

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

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on host and/or pathogen chromatin. Chromatin preparation from tissues creates additional challenges that need to be overcome to obtain reproducible and reliable protocols comparable to those used for cell culture. Tissue disruption and fixation are critical steps to achieve efficient shearing of chromatin. Coexistence of different cell types and clusters may also require different shearing times to reach optimal fragment size and hinders shearing reproducibility. The purpose of this method is to achieve reliable and reproducible host chromatin preparations from frozen tissue (liver) suitable for both ChIP-qPCR and next generation sequencing (NGS) applications. We observed that the combination of liquid nitrogen tissue pulverization followed by homogenization leads to increased reproducibility compared to homogenization only, since it provides a suspension consisting mostly of dissociated single cells that can be efficiently sheared. Moreover, the fixation step should be performed under mild rotation to provide homogeneous crosslinking. The fixed material is then suitable for buffer-based nuclei isolation, to reduce contamination of cytoplasmic protein and pathogen DNAs and RNAs (when applicable), avoiding time-consuming centrifugation gradients. Subsequent sonication will complete nuclear lysis and shear the chromatin, producing a specific size range according to the chosen shearing conditions. The size range should fall between 100 and 300 nt for NGS applications, while it could be higher (300-700 nt) for ChIP-qPCR analysis. Such protocol adaptations can greatly improve chromatin analyses from frozen tissue specimens.

This protocol focuses on chromatin preparation from snap frozen tissues and it is suitable for Crosslinking Chromatin Immunoprecipitation (X-ChIP) followed by either quantitative PCR analysis (X-ChIP-qPCR) or next generation sequencing approaches (X-ChIP-seq).

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on ...

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective treatments. Considerable limiting factors hindering the timely and accurate diagnosis (including grading) and monitoring of NAFLD, as well as the development of potential therapies, are the current inadequacies in the characterization of the hepatic microenvironment structure and the scoring of the disease stage in a spatiotemporal and non-invasive manner. Using a diet-induced NAFLD mouse model, we investigated the use of in vivo micro-computed tomography (CT) imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation. This imaging methodology allows for longitudinal analysis of liver steatosis and functional tissue uptake, as well as the evaluation of the relative blood volume, portal vein diameter, and density of the vascular network. Understanding the adaptations of the hepatic vascular network during NAFLD progression and correlating this with other ways of characterizing the disease progression (steatosis, inflammation, fibrosis) using the proposed method can pave the way toward the establishment of new, more efficient, and reproducible approaches for NAFLD research in mice. This protocol is also expected to upgrade the value of preclinical animal models for investigating&#160;the development of novel therapies against disease progression.

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Using a diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we describe the use of novel in vivo micro-computed tomography imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation.