The drug development process is expensive and time consuming. This is largely a result of the vast majority of candidate drugs failing in clinical trials due to adverse effects in vivo. Improving the ability to predict the effectiveness and toxicity of candidate drugs in humans would significantly lower the failure rate of these drugs during clinical trials. The integration of ADME (absorption, distribution, metabolism, and excretion) profiling into the drug discovery stage has enabled better prediction of human pharmacokinetics and potential adverse side effects. However, predicting the in vivo clearance of a drug based on in vitro cell model systems remains challenging, and is even more difficult for slowly metabolized, "low-clearance" drugs. A common goal in the pharmaceutical industry is to develop low-clearance drugs that can be administered in single, once-daily doses, but with existing in vitro models, the activities of these drugs are often significantly underestimated. Therefore, the ability to accurately predict the in vivo clearance of a drug, especially a low-clearance drug, is essential for drug development and safe, accurate dosing.

Over the past decade, human primary hepatocytes (hphep cells) have become the gold standard for in vitro evaluation of drug metabolism, drug-drug interactions, safety assessment of drug candidates, and disease modeling. Primary hepatocytes mimic the functionalities of the liver, such as the expression of drug-metabolizing enzymes and transporters, which enables the prediction of in vivo drug metabolism and clearance. However, a significant limitation of hphep cells is their rapid loss of function when cultured in vitro (Richert et al. 2006). This fundamental limitation restricts the applications for which hphep cells can be used, such as clearance studies of slowly metabolized drugs, which require long incubation times. To address this problem, 3D sandwich cultures with matrix overlays (Liu et al. 1999), bioreactors (Hoffman et al. 2012), 3D spheroid cultures (Proctor et al. 2017), and coculture systems (Hultman et al. 2016) have been developed. Although these approaches can maintain some hepatocyte functions for several weeks in vitro, they do not entirely overcome the limitations of hphep cells because these culture systems restrict the types of assays that can be performed. Furthermore, they require advanced and expensive lab equipment, are not easy to use, or are not generally applicable to hphep cells from different donors.

To enable long-term cultures of primary hepatocytes in user-friendly 2D-culture formats, we developed Cellartis Power Primary HEP Medium (Power HEP medium), a new medium that maintains healthy, functional human primary hepatocytes for up to four weeks in conventional 2D cultures—without the need of overlays or sandwich cultures—overcoming a key limitation of hphep cells. Furthermore, hphep cells remain healthy and functional in Power HEP medium for several days without medium changes, and the cells can be used to predict the in vivo clearance of low clearance compounds.

Hphep cells are viable and show typical hepatocyte morphology for four weeks in 2D culture

The first basic requirement for successful long-term culture is maintenance of cell viability. Therefore, we measured the ATP content of the cultures at multiple timepoints post-thaw as an indicator of cell viability and overall hepatocyte health. To this end, we thawed and plated primary hepatocytes from six donors (purchased from four commercial vendors) according to the recommendations from each manufacturer and then changed the medium to Power HEP medium four hours post-thaw. The ATP content was measured four hours after plating (Day 0) and again on Days 1, 7, 14, 21, and 28. We found that ATP levels were stable, showing little variation between Days 7 and 28 post-thaw. These results indicate that the hepatocytes stayed viable for four weeks post-thaw (Figure 1, Panel A). Interestingly, during the first week of culture, we observed an increase in ATP content between four hours and seven days post-thaw, with some variations between donors. To further investigate the cause of this increase in ATP, cells from three different donors were dissociated and counted at Days 1, 3, 5, 7, 14, 21, and 28 post-thaw. The results indicated that the hepatocytes were proliferating during the first week of culture, with approximately one population doubling, and confirmed that the increase in ATP content was due to an increase in cell number (Figure 1, Panel B).

Figure 1. Primary hepatocytes remain viable and proliferate initially when cultured in Cellartis Power Primary HEP Medium. Panel A. Human primary hepatocytes (six donors from four vendors) were thawed and plated according to each vendor's instructions. Four hours post-thaw, the medium was changed to Power HEP medium. Cell viability was assessed four hours post-thaw (Day 0) and again at Days 1, 7, 14, 21, and 28 in culture by measuring ATP content using the CellTiter-Glo assay. Data are presented as mean values for each donor (three technical triplicates). Panel B. Number of hphep cells per well of a 96-well plate during four weeks of culturing in Cellartis Power Primary HEP Medium. Human primary hepatocytes (three donors from one vendor; B1, B2, and B3) were thawed and plated according to the vendor's instructions. Four hours post-thaw, the medium was changed to Power HEP medium. The cell number per well was assessed in the cell suspension at plating (Day 0) and on Days 1, 7, 14, 21, and 28 post-thaw by dissociating the cells using Trypsin-EDTA (4 min at 37°C) and counting them in a hemocytometer. Data are presented as mean values +/– standard deviation for n = 3 donors (two wells counted per donor).

When culturing hphep cells for an extended period of time, the biggest challenge is to prevent dedifferentiation of the cells. Therefore, we next monitored cell morphology, which is a strong indicator of the differentiation status of hphep cells. Hphep cells were cultured for four weeks in Power HEP medium or the maintenance media from three different vendors, and the morphology of the cells was monitored. The phase contrast images show that the hphep cells cultured in Power HEP medium displayed typical hepatocyte morphology after 28 days in culture: the cells were polygonal; had distinct, white cell-to-cell borders; and were occasionally bi-nucleated (Figure 2, Panel A). In contrast, hphep cells cultured in the competitors' media rapidly lost hepatocyte morphology or viability (Figure 2, Panel B).

Figure 2. Morphologies of primary hepatocytes cultured in Cellartis Power Primary HEP Medium and three commercially available hepatocyte maintenance media at Day 28 post-thaw. Panel A. Human primary hepatocytes were thawed and plated according to the manufacturer's instructions. Four hours post-thaw, the medium was changed to Power HEP medium (shown for three donors). Panel B. Cells from one donor were cultured in InVitroGRO HI Medium (BioIVT), Hepatocyte Culture Medium (Lonza), or Hepatocyte Culture Medium (Lonza) with HepExtend Supplement (50X) (Thermo Fisher Scientific). For all panels, the morphology of the hepatocytes was monitored during a 28-day culture period. Representative phase contrast images of hphep cells cultured after 28 days in the various media are shown. For all images, the scale bar is 100 µm.

Stable albumin secretion from hphep cells for four weeks

The model of metabolic zonation proposes a functional specialization of hepatocytes depending on the cells' location in the liver lobe (Jungermann and Kietzmann 2000). According to this model, albumin secretion is a predominant function of hepatocytes in the periportal zone, while hepatocytes performing drug metabolism via cytochrome P450 (CYP) enzymes are in the perivenous zone.

Albumin is the main protein of human blood plasma, and albumin secretion is a commonly used characteristic to evaluate the quality of in vitro hepatocyte models, particularly for periportal hepatocytes. Overall, we show that not only was albumin already secreted after one day in culture, but secretion was also stably maintained throughout the 28-day culture period (Figure 3). Interestingly, we also observed that while total albumin secretion stayed stable for all 28 days (Figure 3, Panel A), total protein content increased from Day 1 to Day 7; therefore, once albumin is normalized to protein content, we saw an initial drop in the normalized albumin content before it stabilized to a constant level that was maintained out to 28 days. The observed initial rise of protein content can be partly explained by the increase in hphep cell numbers that we observed during the first week in culture (Figure 1, Panel B).

Taken together, these data demonstrate that when cultured in Power HEP medium, hphep cells secrete albumin—an important indicator of high-quality, functional hepatocytes—for up to 28 days.

Figure 3. Albumin (alb) secretion in primary hepatocytes cultured in Cellartis Power Primary HEP Medium. Human primary hepatocytes (six donors from four vendors) were thawed and plated according to each manufacturer's instructions. Four hours post-plating, the media was changed to Power HEP medium. Panel A. Albumin secreted into the medium during a 24-hr period was measured by ELISA on Days 0–1, 6–7, 13–14, and 27–28 post-thaw. Panel B. Protein content per well was quantified using the Pierce BCA Protein Assay kit. Panel C. Normalization of albumin secretion to total protein content. For all graphs, data are presented as mean values +/– standard deviation for n = 6 donors.

Stable CYP activity in hphep cells for four weeks

Another major function of hepatocytes is the expression and activity of drug-metabolizing CYP enzymes, which are important for the detoxification of drug compounds. This function occurs in hepatocytes from the perivenous zone and is often used to evaluate the functionality of in vitro hepatocyte models. We investigated the activities of five key CYP enzymes in primary hepatocytes from six donors (from four commercial vendors) cultured in Power HEP medium during a four-week period. CYP activities were measured at four hours (Day 0) and 1, 7, 14, 21, and 28 days post-thaw, and levels were normalized to total protein content. CYP activities were sustained in all six hphep donors for the entire four-week culture period, and levels were remarkably stable between Days 7 and 28 post-thaw (Figure 4). CYP activities were more variable during the first seven days of culturing, likely due to the recovery period after thawing. We observed the expected interindividual variation found in the general population, as demonstrated by variation in activity levels of different CYPs depending on the donor. This variation was also maintained over the 28-day culture period.

Figure 4. CYP activities in primary hepatocytes cultured in Cellartis Power Primary HEP Medium for 28 days. Panels A–F. Human primary hepatocytes (six donors from four vendors) were thawed and plated according to each manufacturer's instructions. Four hours post-thaw, the medium was changed to Power HEP medium. CYP activity assays were performed 4 hr (Day 0) and 1, 7, 14, 21, and 28 days post-thaw. The formation of specific metabolites (CYP1A = paracetamol, CYP3A = OH-midazolam, CYP2C9 = OH-diclofenac, CYP2B6 = OH-bupropion, and CYP2D6 = OH-bufuralol) was measured by liquid chromatography/mass spectrometry (LC/MS). Metabolite concentrations were normalized to protein content and activity assay duration. Data are presented as mean values for each CYP enzyme tested (three technical triplicates).

Due to the observed variation in CYP activity between Day 0 and Day 7, we wanted to perform a more detailed investigation of the CYP activity during the first week of culture. For this experiment, we cultured primary hepatocytes from three donors in Power HEP medium for four weeks. CYP activities were measured directly after thawing in suspension (0 hr), at four hours (Day 0), and at Days 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28 post-thaw, and the levels were normalized to total protein content. During the first week post-thaw, CYP activities initially dropped compared to the 0-hr time point (in suspension), but most activities increased during the subsequent days, except for CYP2C9 activity in all three donors and CYP2D6 activity in one donor (Figure 5, Panel A). Most of the variation was observed during the first three days post-thaw. When comparing CYP activity levels after seven days to those directly after thawing (0 hr, in suspension), CYP3A, CYP2B6, and CYP2D6 were at comparable levels, whereas CYP2C9 activity was ~10 times lower and CYP1A activity ~9 times higher than at 0 hr. Consistent with the data shown in Figure 4, CYP activity levels were remarkably stable between Days 7 and 28 post-thaw (Figure 5, Panel B).

Figure 5. CYP activities in primary hepatocytes cultured in Cellartis Power Primary HEP Medium during the first week post-thaw. Human primary hepatocytes (three donors from one vendor) were thawed and plated according to the vendor's instructions. Four hours post-thaw, the medium was changed to Power HEP medium. CYP activity assays were performed directly after thawing, in suspension culture (0 hr) or 4 hr (Day 0) and Days 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28 post-thaw. The formation of specific metabolites (CYP1A = paracetamol, CYP3A = OH-midazolam, CYP2C9 = OH-diclofenac, CYP2B6 = OH-bupropion, and CYP2D6 = OH-bufuralol) was measured by liquid chromatography/mass spectrometry (LC/MS). Metabolite concentrations were normalized to protein content and activity assay duration. Data are presented as mean values for each CYP enzyme tested (technical duplicates per donor and time point).

To investigate how the performance of hphep cells cultured in Power HEP medium compares to other commercially available hepatocyte maintenance media, we cultured primary hepatocytes from two different donors either in Power HEP medium or in hepatocyte media from three vendors. Again, hphep cells showed sustained CYP activities for 28 days when cultured in Power HEP medium (Figure 6, Panel A), which is in sharp contrast to hphep cells cultured in other commercially available media where the activities rapidly dropped off (Figure 6, Panels B–D). This clearly demonstrates that in comparison to other media, Power HEP medium preserves CYP activity levels in plated hphep cells, as it is the only medium that stably maintains CYP enzyme function for four weeks in culture.

Figure 6. CYP activities in primary hepatocytes cultured in Cellartis Power Primary HEP Medium and three commercially available hepatocyte maintenance media. Human primary hepatocytes from two donors were thawed and plated according to the manufacturer's instructions. Four hours post-thaw, the medium was changed to Power HEP medium or one of the other three commercially available media. The hphep cells were cultured in Power HEP medium (Panel A), InVitroGRO HI medium (BioIVT) (Panel B), Hepatocyte Culture Medium (Lonza) (Panel C), or Hepatocyte Culture Medium (Lonza) with HepExtend Supplement (50X) (Thermo Fisher Scientific) (Panel D). CYP activity assays were performed 4 hr (Day 0) and 7, 14, 21, and 28 days post-thaw. The formation of specific metabolites (CYP1A = paracetamol, CYP3A = OH-midazolam, CYP2C9 = OH-diclofenac, CYP2B6 = OH-bupropion, CYP2D6 = OH-bufuralol) was measured by LC/MS. Metabolite concentrations were normalized to protein content and activity assay duration. Data is presented as mean values +/– standard deviation (n = 2 donors).

CYP expression remains inducible for four weeks

Drugs can increase CYP enzyme levels by inducing their mRNA expression, which can cause a change in the effects of coadministered drugs, leading to serious problems for patients taking multiple medications. Consequently, the assessment of potential drug-drug interactions is important in drug development, and hphep cells are the gold standard for assessing potential CYP inductions by a drug candidate.

To evaluate whether this important hepatocyte feature is preserved in hphep cells cultured with Power HEP medium, we exposed hphep cells to typical inducers (Table I) for 48 hr after they had reached 26 days in culture. We then analyzed CYP1A2, 2B6, 2C9, and 3A4 mRNA expression levels on Day 28 and compared them to levels in DMSO-treated control cells.

Interestingly, we found that even after 26 days in culture prior to induction, all four CYPs tested were induced at high levels (Figure 7), comparable to the fold induction shown for hphep cells induced 2–4 days post-thaw (Yajima et al. 2014). The only exception to the pattern of high levels of CYP inducibility across donors is that CYP2C9 induction by Rif was observed in only two out of three donors (Figure 7, Panel D); however, this is consistent with reports that CYP2C9 is not inducible in some donors (Yajima et al. 2014).

Figure 7. Induction of CYP mRNA expression in primary hepatocytes cultured in Cellartis Power Primary HEP Medium. Human primary hepatocytes from three donors were cultured in Power HEP medium for 26 days and subsequently treated for 48 hr with 25 µM omeprazole (Ome), 1 mM phenobarbital (PB), 50 µM rifampicin (Rif), and/or 0.2% DMSO (vehicle control). On Day 28, cells were harvested, and CYP1A2 expression in response to Ome (Panel A), CYP2B6 to PB and Rif (Panel B), CYP3A4 to PB and Rif (Panel C), and CYP2C9 to PB and Rif (Panel D) were measured by RT-qPCR and compared to the DMSO control. CYP levels are presented as fold induction over DMSO control (n = 1 for each donor).

Since hphep cells maintained their CYP inducibility for the entire culture period, we wanted to determine if they could be used for repeated CYP induction studies. This would allow hphep cells to be used for multiple CYP induction experiments, as well as enable more sophisticated tests for repeat dosing studies. To test this, we cultured primary hepatocytes from three donors in Power HEP medium. On Day 7, the hphep cells were incubated with phenobarbital for 48 hr to induce CYP2B6. CYP activity was measured directly after incubation. The cells were allowed to recover before repeating the induction at three additional time points: Days 14, 21, and 28. At all four time points, phenobarbital induced CYP2B6 to a similar extent, even though the cells had been previously exposed to the inducer (Figure 8). This demonstrates that primary hepatocytes cultured in Power HEP medium can be used for repeated CYP induction studies.

Figure 8. Repeated induction of CYP2B6 in primary hepatocytes cultured in Cellartis Power Primary HEP Medium. Human primary hepatocytes from three donors were cultured in Power HEP medium and incubated for 48 hr with 1 mM phenobarbital or 0.2% DMSO (vehicle control) Days 7–9 post-thaw, and again on Days 14–16, 21–23, and 28–30. CYP activity assays were performed immediately following each induction by washing the cells and incubating them in 10 µM bupropion, and then measuring the formation of the metabolite OH-bupropion by LC/MS. CYP levels are presented as fold induction over DMSO control. Data are presented as mean values (technical triplicates per donor and time point).

Proof-of-concept intrinsic clearance prediction study

For clearance studies on slowly metabolized compounds, hphep cells need to be incubated with the compounds for 3–10 days without medium changes. Therefore, we wanted to see how hphep cells would cope with incubation for up to 10 days without any medium changes. For this experiment, we cultured hphep cells from three different donors in Power HEP medium. The test was initiated on Day 7 post-thaw (when the initial recovery period is over) by removing the old media and adding 230 µl of fresh Power HEP medium to each well of a 96-well plate. Then, the hphep cells were cultured for 10 days without any medium changes. We evaluated morphology, ATP content, and CYP activities on Days 0, 3, 5, 6, 7, 8, and 10 after the start of incubation. For all three donors, cultures after 8 or 10 days without medium change looked healthy and similar to those at the start of incubation (Figure 9, Panel A), and no dedifferentiation or major cell loss could be observed. In agreement with this, ATP content and CYP activities were stable for 7–8 days without medium changes, with a slight decrease in ATP content and CYP activities after 7–8 days (Figure 9, Panels B and C).

Figure 9. Human primary hepatocytes cultured for 10 days in Cellartis Power Primary HEP Medium without medium changes. Human primary hepatocytes from three donors were thawed and plated according to the manufacturer's instructions. Four hours later, the medium was changed to Power HEP medium. The cells were cultured according to the user manual for six days. On Day 7, a 10-day incubation without any medium change was initiated by adding 230 µl of fresh Power HEP medium per well of a 96-well plate (after removing the old media). The morphology of the hepatocytes was monitored during the 10-day incubation period. Representative phase contrast images of hphep cells at start of incubation (Day 0), and after 8 and 10 days of incubation are shown (Panel A). For all images, the scale bar is 100 µm. Cell viability was assessed at the start of the incubation (Day 0) and again 3, 5, 6, 7, 8, and 10 days after start of the incubation by measuring ATP content using the CellTiter-Glo assay. Data are presented as mean values for each donor (two technical replicates per donor) (Panel B). CYP activity assays were performed at the start of the incubation (Day 0) and again 3, 5, 6, 7, 8, and 10 days after the start of the incubation. The formation of specific metabolites (CYP1A = paracetamol, CYP3A = OH-midazolam, CYP2C9 = OH-diclofenac, CYP2B6 = OH-bupropion, and CYP2D6 = OH-bufuralol) was measured by liquid chromatography/mass spectrometry (LC/MS). Metabolite concentrations were normalized to protein content and activity assay duration. Data are presented as mean values +/– standard deviation for each CYP enzyme tested (duplicates per donor) (Panel C).

In order to evaluate the possibility of using hphep cells cultured in Power HEP medium for intrinsic clearance (CL_int) prediction, a collaborator at AstraZeneca performed a proof-of-concept experiment using quinidine, a CYP3A4 substrate with low intrinsic clearance. The study was initiated on Day 7 post-thaw by performing a medium change in which 230 µl of fresh Power HEP medium supplemented with 1 µM quinidine was added to each well of a 96-well plate. The incubation was continued for 10 days without medium change, and quinidine content was analyzed by LC/MS at the following time points: 0, 1, 3, and 5 hr and then 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 days after the start of incubation. The disappearance of quinidine was monitored by Multiple Reaction Monitoring (MRM) and integration of the chromatographic peaks. In this experiment, the in vitro CL_int value was calculated to be 3.12 μl/min/million cells based on five timepoints (Figure 10, Panel A). Scaling in vitro CL_int to the liver and using the correction factor 3 approach (Winiwater et al. 2018) gave a predicted in vivo CL_int that is in agreement with the observed in vivo free CL_int (Figure 10, Panel B). These data indicate that hphep cells cultured in Power HEP medium can be used to study the clearance of compounds metabolized by CYP3A4. For clearance prediction of drugs mediated by other P450 enzymes and UGTs, the set of test compounds needs to be expanded. 

Figure 10. Prediction of in vivo intrinsic clearance of quinidine using primary hepatocytes cultured in Power HEP medium. Human primary hepatocytes from a single donor were thawed and plated according to the manufacturer's instructions. On Day 7 post-thaw, 230 µl of fresh Power HEP medium supplemented with 1 µM quinidine was added to each well of a 96-well plate (after removal of old media). The incubation was continued for 10 days without medium change. Concentration of quinidine at start of incubation (0 hr), 1 hr, 3 hr, 5 hr, and 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 days post-start of incubation was monitored using LC-MS (Xevo QT-S, Waters), and disappearance of quinidine was determined using Multiple Reaction Monitoring (MRM) and integration of chromatographic peaks. By plotting the natural log of the percentage of drug remaining against time for five timepoints (1 hr, 3 hr, 5 hr, 1 day, and 2 days) (Panel A), the disappearance rate of quinidine was determined. The disappearance rate was then used to calculate the half-life and in vitro CL_int for quinidine (see "CL_int calculation" in Materials and Methods). In vitro CL_int was scaled to the liver by multiplying it by scaling factors (120 million cells per g liver x 1,680 g liver weight/70 kg body weight). A regression correction method was used to predict in vivo CL_int by multiplying the scaled in vitro CL_int by a correction factor of 3. Observed in vivo CL_int was calculated by applying the well-stirred model to published human clearance data. Plotting Log(Observed in vivo CL_int) vs. Log(Predicted in vivo CL_int) shows that the predicted in vivo CL_int value based on the calculated in vitro CL_int value strongly correlated with the observed in vivo CL_int value (Panel B). (Data kindly provided by AstraZeneca.)

Primary hepatocytes are the gold standard for liver research but, to date, their utility has been strongly limited by their short functional lifespan in user-friendly 2D cultures. This short lifespan prevents hphep cells from being used for assays requiring longer times in culture such as chronic toxicity or drug clearance experiments. Our novel Cellartis Power Primary HEP Medium enables maintenance of viable and functional hepatocytes for four weeks in conventional 2D cultures. Importantly, key hepatocyte functions such as albumin secretion, CYP activities, and CYP inducibility are maintained for the four-week culture period. Notably, both periportal and perivenous features, typical for the different hepatocyte phenotypes present in the different zones of the liver lobe, are preserved.

Power HEP medium also enables long-term intrinsic clearance studies of low-clearance compounds. Primary hepatocytes cultured in Power Primary HEP medium remain viable, healthy, and functional for up to 7–8 days without medium change. Furthermore, this system provides an accurate model for predicting in vivo intrinsic clearance of slowly metabolized compounds, as demonstrated with quinidine.

The extended lifespan of primary hepatocytes cultured with Power HEP medium in user-friendly 2D cultures and with a weekend-free feeding schedule significantly enhances the utility of primary hepatocytes in multiple applications, such as drug discovery and safety toxicology studies.

Primary hepatocyte culture

Cryoplateable human primary hepatocytes were purchased from four vendors (BioIVT, Lonza, Corning, and Thermo Fisher Scientific), thawed, and plated according to each vendor's instructions. four hr post-thaw, plating media was carefully removed and replaced with prewarmed Cellartis Power Primary HEP Medium or media from other vendors. Cells were maintained for up to 28 days post-thaw. Cells cultured in Power HEP medium were cultured according to the Cellartis Power Primary HEP Medium User Manual, with media changes every second or third day. Media from other vendors were prepared according to the manufacturer's instructions, and the media was changed every second or third day.

ATP quantification

The viability of the hphep cells was determined at multiple timepoints after thawing using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. Luminescence was measured, and the values were blank corrected.

Albumin secretion

Albumin secretion from primary hepatocytes was analyzed at multiple timepoints after thawing. The culture medium was collected after 24 hr of conditioning, and albumin content was analyzed using the Albuwell kit (Exocell) according to the manufacturer's instructions, then normalized to the total protein amount per well (determined using the Pierce BCA Protein Assay Kit; see below).

Dissociation of cells for cell counting

For dissociation, cells were washed twice with warm DPBS without magnesium and calcium (150 µl per well of a 96-well plate). Next, warm 0.25% Trypsin-EDTA (Thermo Fisher Scientific) was added (75 µl per well) and incubated for 4 min at 37°C. Then, warm DPBS with magnesium and calcium + 20% FBS was added (75 µl per well) and the cell suspension mixed by carefully pipetting 5–10 times. Cells from two wells were pooled into a tube, mixed again by carefully pipetting a few times, and then counted in a hemocytometer.

CYP induction

CYP induction was assessed between Days 26 and 28 post-thaw. Hphep cells were incubated with the inducers omeprazole (25 µM), phenobarbital (1 mM), and rifampicin (50 µM) in William's medium E containing 0.1% Penicillin-Streptomycin (PEST) and HCM SingleQuots (w/o GA1000 and hydrocortisone). DMSO (0.2%) was used as a vehicle control. Medium with inducers was refreshed after 24 hr of induction, and after 48 hr cells were harvested in RNA protect reagent (Thermo Fisher Scientific). Total RNA was extracted using the MagMAX-96 Total RNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. cDNA was synthesized, and qRT-PCR amplification reactions were performed using an ABI 7500 Real-Time PCR System (Thermo Fisher Scientific). Gene expression was analyzed using TaqMan Gene Expression Assays (Thermo Fisher Scientific) according to the manufacturer's recommendations. Each sample was analyzed in duplicate.

The following assays (Thermo Fisher Scientific) were used: CEBPα (Assay ID Hs00269972_s1), CYP1A2 (Assay ID Hs 01070374_m1), CYP2B6 (Assay ID Hs04183483_g1), CYP3A4 (Assay ID Hs00604506_m1), and CYP2C9 (Assay ID Hs004260376_m1). Expression levels were calculated using the ΔΔCt method and normalized to a calibrator mix consisting of cDNA from human pluripotent stem cells (hPSC), hPSC-derived embryoid bodies, hPSC-derived definitive endoderm cells, hPSC-derived cardiomyocytes, hphep cells, HepG2 cells, and HEK293 cells. Expression was normalized to CEBPα expression and presented as relative quantification. ΔΔCt was transformed into fold change by the formula: fold change = 2–ΔΔCt.

For the repeated CYP induction experiment, hphep cells were incubated with the inducer phenobarbital (1 mM) or 0.2% DMSO (vehicle control) in phenol-red-free William's medium E containing 0.1% PEST, 25 mM HEPES, and 2 mM L-Glutamine for 48 hr on Day 7–9 post-thaw, and again on Days 14–16, 21–23, and 28–30 post-thaw. Medium with phenobarbital or DMSO was refreshed after 24 hr of induction. CYP2B6 activity was measured following 48 hr incubations as described below. Before and in between incubation with phenobarbital or DMSO, hphep cells were cultured in Power HEP medium. 

CYP activity assay

The CYP activities of primary hepatocytes were analyzed at multiple timepoints after thawing by measuring the formation of specific metabolites: paracetamol/acetaminophen (CYP1A), OH-bupropion (CYP2B6), 4-OH-diclofenac (CYP2C9), OH-bufuralol (CYP2D6), and 1-OH-midazolam (CYP3A) using LC/MS analysis (performed at Pharmacelsus GmbH, Germany). First, cells were carefully washed twice with phenol-red-free prewarmed William's medium E containing 0.1% PEST. Then, the activity assay was started by adding 110 µl/cm² culture area of prewarmed phenol-red-free William's medium E containing 0.1% PEST, 25 mM HEPES, 2 mM L-Glutamine, and the probe substrate cocktail (see Table II, below). After a 2-hr incubation at 37°C, 100 µl of the supernatant was collected and kept at –80°C until LC/MS analysis. For the CYP activity assay on hepatocytes in suspension directly after thawing, 50,000 cells in thawing medium were transferred to a vial and centrifuged for 2 min at 300g. Next, the supernatant was removed and 100 µl phenol-red-free prewarmed William's medium E containing 0.1% PEST was added to the cells. After centrifuging for 2 min at 300g and removing the supernatant, 50 µl of prewarmed phenol-red-free William's medium E containing 0.1% PEST, 25 mM HEPES, and 2 mM L-glutamine was added, and the cells were preincubated for 5 min at 37°C. Then, 50 µl of prewarmed phenol-red-free William's medium E containing 0.1% PEST, 25 mM HEPES, and 2 mM L-glutamine supplemented with a double concentration of the probe substrate cocktail was added to the cells. After mixing carefully by swirling the vial, cells were incubated for 15 min at 37°C. Next, the vial was centrifuged for 2 min at 300g and 50 µl of the supernatant was collected for LC/MS analysis. The metabolite concentrations measured by LC/MS were normalized to the amount of protein per well (determined using the Pierce BCA Protein Assay Kit; see below) and the assay duration (120 min).

Protein quantification

Cells were washed once with D-PBS with calcium and magnesium, lysed in 0.02 mM NaOH overnight at 4°C, and stored at –20°C until analysis. Protein amount was quantified using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

LC/MS method for measuring quinidine content in CLint study

Samples were analyzed on an Acquity UPLC I-Class system (Waters Corp., USA) consisting of a sample manager, pump, injector, and column manager connected to a mass spectrometer Xevo TQ-S (Waters Corp., USA). Separation was performed using a Waters Acquity UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 μm); the mobile phase consisted of Solvent A (0.1% formic acid in mQ-water) and Solvent B (0.1% formic acid in acetonitrile), and the flow rate was 1 ml/min. The elution gradient was initiated at 0.2% B for 0.3 min followed by a linear gradient up to 95% B to 1.3 min. 95% B was kept to 1.8 min, after which the elution gradient was reverted to 0.2% B. (Protocol provided by AstraZeneca.)

CL_int calculation

CL_int is based on substrate disappearance rate.

• The percent of the remaining quinidine is calculated by comparing with the 1-hr timepoint.
• CL_int is calculated from LN (LC/MS peak area) vs. time (see equation below, from Hultman et al. 2016) per million cells. For determining cell number per well on Day 0 when the incubation with quinidine was started, cells from four wells were counted as described above.
• Values where <10% of quinidine was remaining have been excluded from the CLint calculation. 

T_1/2 = Ln₂ / –slope

CL_int (µl/min/million cells) = (Ln₂ * 1,000) / [T_1/2(min) * cell conc. (million cells/ml)]

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