Novel Screening System for Biliary Excretion of Drugs Using Human Cholangiocyte Organoid Monolayers with Directional Drug Transport

Abstract

It has recently been reported that cholangiocyte organoids can be established from primary human hepatocytes. The purpose of this study was to culture the organoids in monolayers on inserts to investigate the biliary excretory capacity of drugs. Cholangiocyte organoids prepared from hepatocytes had significantly higher mRNA expression of CK19, a bile duct epithelial marker, compared to hepatocytes. The organoids also expressed mRNA for efflux transporters involved in biliary excretion of drugs, P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP). The subcellular localization of each protein was observed. These results suggest that the membrane-cultured cholangiocyte organoids are oriented with the upper side being the apical membrane side (A side, bile duct lumen side) and the lower side being the basolateral membrane side (B side, hepatocyte side), and that each efflux transporter is localized to the apical membrane side. Transport studies showed that the permeation rate from the B side to the A side was faster than from the A side to the B side for the substrates of each efflux transporter, but this directionality disappeared in the presence of inhibitor of each transporter. In conclusion, the cholangiocyte organoid monolayer system has the potential to quantitatively evaluate the biliary excretion of drugs. The results of the present study represent an unprecedented system using human cholangiocyte organoids, which may be useful as a screening model to directly quantify the contribution of biliary excretion to the clearance of drugs.

INTRODUCTION

In screening and optimization studies for new drugs, in vitro test systems and animal studies are important in evaluating kinetic characteristics such as absorption, distribution, metabolism, and excretion. Drug elimination from the body can be broadly classified into metabolism in the liver or excretion into bile and urine via the kidneys. Among these, it is possible to accurately predict the presence or absence of urinary excretion of a drug in clinical practice by the identification of transporters involved in the urinary excretion of the drug, animal studies and physico-chemical properties. Various methods have been proposed for hepatic metabolism using human hepatocytes as a resource.^1–4) The combination of these methods is a very useful tool for predicting pharmacokinetics in clinical trials, and the hepatic metabolism and urinary excretion of drugs can be verified in early trials. In contrast, during the drug discovery phase, animal studies are relied upon to estimate the biliary excretion of drugs. However, the transporters involved in drug biliary excretion vary among species.⁵⁾ In addition, results from laboratory animals, in which metabolic processes are qualitatively and/or quantitatively different from those of humans, do not provide much useful information regarding biliary excretion of drugs and their metabolites. Furthermore, drugs excreted via bile could be reabsorbed via enterohepatic circulation, which makes it difficult to determine the profile of biliary excretion of drugs.^6–8) Furthermore, biliary excretion is extremely difficult to verify in clinical practice.

Several in vitro test systems have been proposed to estimate the biliary excretion of drugs.^9,10) In many cases, liver-derived cells have been cultured in two-dimensional (2D) or three-dimensional (3D) and used known fluorescent substrates excreted in bile to qualitatively demonstrate the localization of the substrates, indicating the formation of a bile duct-like network.^11–15) However, these evaluation systems are not applicable to general substrates that do not fluoresce. Therefore, attempts have been made to quantitatively measure biliary excretion of drugs by quantifying drugs in the presence or absence of calcium and calculating the difference.^16,17) This is based on the fact that the bile duct morphology between cells is disrupted in the absence of calcium, and the drug accumulated in the bile duct-like network leaks out. Although this is a reasonable method, it is complicated as a test system and has several problems such as the inability to directly quantify the drug excreted in bile.

We previously reported that human liver cell lines HepG2 and Huh-7 were successfully cultured on a membrane insert in a monolayer with the upper side of the insert oriented toward the bile duct and the lower side toward the blood.¹⁸⁾ When a substrate of multidrug resistance-associated protein 2 (MRP2) was added to this system, the secretion of the drug known to be excreted in the bile from the lower side to the upper side (apparent permeability coefficient: P_{app, B to A}) exceeded that from the upper side to the lower side (P_{app, A to B}). The efflux ratio (ER) value (P_{app, B to A}/P_{app, A to B}) was 1.38. This system can directly quantify drugs and is considered to be an in vivo method for observing the biliary excretion of drugs. However, the fundamental question remains as to whether hepatocytes, which are mesenchymal cells, can be cultured in membranes in the same way as epithelial cells. Moreover, there are still several areas to be improved, such as the type of insert, culture method, membrane resistance, and ER value.

It has recently been reported that cholangiocyte organoids can be established from primary human hepatocytes (PHH) that can proliferate over the long term. Organoids are 3D constructions of living tissue in vitro by embedding cells of biological origin in an extracellular matrix and using a special culture medium.^19–23) This organoid was reported to express P-glycoprotein (P-gp) and MRP2 at the apical surface of the bile duct region.²⁴⁾ Moreover, examples using rhodamine123 (Rho123), a substrate of P-gp, has been reported.^22,25,26) However, there are concerns that evaluation in 3D culture is not reproducible and that it is difficult to remove the drug from the system and directly quantify it.^27,28) The objective of this study was to establish cholangiocyte organoids from available PHHs^20,29) and then culture them in monolayers on inserts to investigate the biliary excretory capacity of drugs. Drugs that are substrates for P-gp, MRP2, and breast cancer resistance protein (BCRP), transporters involved in drug biliary excretion, were used. In addition, these cholangiocyte organoids can be cultured indefinitely, as can the strain cells. The organoid which was used in this study is in the experimental availability stage.

MATERIALS AND METHODS

Materials

Human cryopreserved hepatocytes (Lot: HEP187216) were purchased from BIOPREDIC International (Saint Grégoire, France). This lot was from a 39-year-old female human adult of unknown race. The liver condition was metastases from carcinoma of the colon. Cryopreserved intra-hepatic biliary epithelial cells (Lot: IHBEC103118D) were purchased from Zen-Bio, Inc. (NC, U.S.A.). Matrigel^® was obtained from Corning (NY, U.S.A.). Vitrigel membranes (ad-MED Vitrigel^® 2) were purchased from Kanto Chemical (Tokyo, Japan). Adherent cell culture plates 24-well plates and suspension cell culture 6-well plates were purchased from Sumitomo Bakelite (Tokyo, Japan). William’s E medium, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), TrypLE™ Express Enzyme, Goat anti Mouse immunoglobulin G (IgG) (H + L) Secondary Antibody/Alexa Fluor^® 555, Goat anti Rabbit IgG (H + L) Secondary Antibody/Alexa Fluor^® 555, and Phalloidin/Alexa Fluor™ 488 were purchased from Thermo Fisher Scientific (MA, U.S.A.). MK-571, and Ko143 were purchased from Cayman Chemical (MI, U.S.A.). Rho123, and bisbenzimide H 33342 trihydrochloride (H33342) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Verapamil was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). 5-(and-6)-Carboxy-2′,7′-dichlorofiuoresein diacetate (CDCFDA) was purchased from PromoKine (Heidelberg, Germany). RNA extraction kit (Direct-zol RNA Kit micro) was purchased from Zymo Research Corporation (CA, U.S.A.). cDNA synthesis kit (PrimeScript RT Master Mix) and qPCR reagent (TB Green Premix Ex Taq) were purchased from TaKaRa Bio (Shiga, Japan). Antibody against P-gp (ab170904), MRP2 (ab3373), BCRP (ab3380), and CK19 (ab7755) were purchased from Abcam (Cambridge, U.K.). 4′,6-Diamidino-2-phenylindole (DAPI) was purchased from DOJINDO (Kumamoto, Japan). All other reagents were commercially available special grade reagents.

Culture of Cholangiocyte Organoids

Cholangiocyte organoids were established from PHHs by modifying the reported method.^20,29) In brief, PHHs were seeded in Matrigel^® 40 µL/well in 24-well plates at a density of 1.0 × 10⁴ cells/well and incubated with the reported medium at 37 °C, 5% CO₂, 95% air for 7–10 d³⁰⁾ (Supplementary Table 1, Supplementary Fig. 1A). The established organoids were picked using a pipette tip (Supplementary Fig. 1B). Only cholangiocyte organoids were picked for differences in organoid morphology and single-cell dispersal passages were performed using TrypLE™ Express Enzyme. For expansion cultures, cholangiocyte organoids (50–500 cells/µL) were cultured in suspension in Matrigel^® medium at a final concentration of 10%. In addition, when cholangiocyte-like organoids isolated by picking were passaged, only the organoids increased, and the remaining cells died without increasing.

Determination of mRNA Expression Levels

The mRNA expression levels of CK19, P-gp, MRP2, and BCRP in established cholangiocyte organoid cells were determined. Total RNA was extracted from organoid cells, PHHs, and intra-hepatic biliary epithelial cells using an RNA extraction kit and reverse transcribed using a cDNA synthesis kit. Quantitative real-time RT-PCR was performed using a LightCycler^® 480 System (F. Hoffmann-La Roche, Ltd., Basel, Switzerland). PCR primer sequences are listed in Supplementary Table 2. β-Glucuronidase (GUSB) mRNA was used as a control.³¹⁾

Monolayer Cell Preparation

Cholangiocyte organoids were seeded onto 5% Matrigel^®-coated Vitrigel membranes after dissociation into single cells at a density of 1.0 × 10⁴ cells/well and replaced with fresh medium every 3 to 4 d to form the monolayer of cholangiocyte organoids.

Immunofluorescence Microscopy

Cells cultured on vitrigel membranes for 14 d were fixed in 4%-para-formaldehyde/phosphate buffered saline (PBS) for 10 min at room temperature. The cells were then washed with PBS and permeabilised with 0.5% TritonX100/PBS for 2 min at room temperature. One percent bovine serum albumin (BSA)/PBS was added and blocking was carried out for 1 h at 4 °C. Only in the case of CK19 staining, the cells were fixed with ice-cold methanol for 3 min at room temperature. The samples were incubated with antibodies against P-gp, MRP2, BCRP, or CK19 (1 : 200 dilution) overnight at 4 °C. The samples were washed with PBS, and then incubated with secondary antibody (1 : 1000 dilution) in 1% BSA/PBS as the secondary antibody. After washing with PBS, Alexa Fluor™ 488 Phalloidin, and 1 µg/mL DAPI/PBS were added and incubated for 30 min at room temperature. After washing with PBS, fluorescence intensity was observed using a CellVoyager CQ1 (Yokogawa Electric Corporation, Tokyo, Japan). To assess the localization of the transporters, the sum of the fluorescence intensities was measured from the images using ImageJ/FIJI image processing software version 1.53q.³²⁾

Transport Study

Cholangiocyte organoids were cultured on Vitrigel membranes for 10–14 d, and the transepithelial electrical resistance difference (ΔTEER) of their cell monolayers was measured using Millicell^® ERS-2 (Millipore, MA, U.S.A.). On the donor side, culture medium containing 10 µM Rho123 (1% dimethyl sulfoxide, DMSO), 10 µM CDCFDA (0.1% DMSO), or 10 µM H33342 (0.1% ethanol) was added at 37 °C. The receiver side was incubated with culture medium containing equal concentrations of DMSO or ethanol. For transport tests in the presence of inhibitors, culture medium containing 30 µM verapamil, 100 µM MK-571, or 20 µM Ko143 was added to both the donor and receiver sides. Samples were taken up to 120 min after the start of the test and replaced with an equal volume of culture medium. It was confirmed that 1% DMSO did not affect the assay. The fluorescence intensities of Rho123 and CDCF (a metabolite of CDCFDA) (excitation 485 nm/emission 535 nm) and H33342 (excitation 365 nm/emission 430 nm) were measured using an ARVO MX (PerkinElmer, Inc., MA, U.S.A.) or an Infinite 200 PRO M Nano⁺ (Tecan Group Ltd., Männedorf, Switzerland).

Data Analysis

All experimental data are expressed as mean ± standard deviation (S.D.). P_app (pmol/well/min) was determined from the slope of the initial linear portion of the permeability (pmol/well) versus time (min) curves by linear regression analysis. Statistical analysis was performed by Student's t-test or Holm test using Pharmaco Basic software (Scientist Press, Inc., Tokyo, Japan). Values of p < 0.05 were considered significant.

RESULTS

Expression of mRNA for Proteins in Cholangiocyte Organoids

The mRNA expression level of the bile duct marker CK19 was significantly higher than that of PHHs in cholangiocyte organoids before and after membranization. The mRNA levels of transports in cholangiocyte organoids before membranization were higher or comparable to those in PHHs. Post-membranization mRNA levels were decreased, and approached expression levels in normal cholangiocytes (Fig. 1).

Fig. 1. mRNA Expression in Cholangiocyte Organoids

Black, gray, white, and shaded bars indicate PHHs, intra-hepatic biliary epithelial cells, and cholangiocyte organoids before and after membranization, respectively. Values represent mean ± S.D. (n = 3). **, ††, ‡‡ Significant difference (p < 0.01) from PHHs, intra-hepatic biliary epithelial cells, cholangiocyte organoids before membranization, respectively (Holm test).

Localization of Efflux Transporters in Cholangiocyte Organoids Monolayers

Localization of each protein expressed on the membranized cholangiocyte organoids was evaluated (Fig. 2). The signal ratio of CK19 from the upper side to lower side was 0.85 ± 0.07, indicating no localization. The ratio of P-gp, MRP2, and BCRP were 3.56 ± 0.95, 3.99 ± 1.09, and 2.12 ± 0.96, respectively. The signal ratio of DAPI was 0.47 ± 0.16, whereas that of phalloidin was 1.40 ± 0.39.

Fig. 2. Localization of Various Proteins in the Cholangiocyte Organoid Monolayers

(A) These images indicated z–x axis. Targeted CK19 and transporters (P-gp, MRP2 and BCRP) are shown in red, DAPI in grey, and phalloidin in green, respectively. (B) The upper side is indicated as the A side and the lower side as the B side. Scale bar indicates 50 µm. The DAPI and phalloidin images are based on the BCRP-targeted image shown in Fig. 2B. (C) Summed values of fluorescent intensity indicating the amount of expression and fold change from side A to side B. Values represent mean ± S.D. (n = 3). ** p < 0.01 (Holm test).

Evaluation of Efflux Transporters

The time course of substrate transport was assessed using a monolayer of cholangiocyte organoids (Fig. 3, Table 1). In both studies, A to B and B to A permeation increased with time. P_{app, A to B} of Rho123, a substrate of P-gp, was 0.11 ± 0.01 pmol/well/min, and P_{app, B to A} was 0.57 ± 0.02 pmol/well/min. The ER value was 5.31 ± 0.15. When P-gp inhibitor verapamil was added, P_{app, A to B} was 0.10 ± 0.02 pmol/well/min and P_{app, B to A} was 0.09 ± 0.01 pmol/well/min. The addition of the inhibitor did not change P_{app, A to B} but decreased P_{app, B to A}, resulting in an ER of 0.97 ± 0.13 (Fig. 4A), indicating the directionality of Rho123 permeation was abolished. P_{app, A to B} of CDCF, a substrate of MRP2, was 45.3 ± 6.3 (×10⁻³) pmol/well/min and P_{app, B to A} was 54.7 ± 7.9 (×10⁻³) pmol/well/min. The addition of MK-571, an inhibitor of MRP2, resulted in P_{app, A to B} of 46.8 ± 7.7 (×10⁻³) pmol/well/min and P_{app, B to A} of 40.1 ± 1.9 (×10⁻³) pmol/well/min. The addition of the inhibitor did not change the P_{app, A to B}, but decreased P_{app, B to A}. The ER value of CDCF was 1.21 ± 0.17, and the addition of the MRP2 inhibitor resulted in an ER of 0.86 ± 0.04 (Fig. 4B). The P_{app, A to B} and P_{app, B to A} of H33342, a substrate of BCRP, were 1.11 ± 0.22 pmol/well/min and 2.64 ± 0.36 pmol/well/min, respectively. With the addition of Ko143, an inhibitor of BCRP, P_{app, A to B} and P_{app, B to A} of H33342, was 1.49 ± 0.34 pmol/well/min and 1.99 ± 0.17 pmol/well/min. With the addition of the BCRP inhibitor, the decrease in B to A permeability was accompanied by an increase in A to B permeability. The ER was 2.36 ± 0.33 without inhibitors and the addition of inhibitors resulted in an ER of 1.33 ± 0.11 (Fig. 4C).

Fig. 3. Time Course of Drug Permeation through the Cholangiocyte Organoid Monolayers

(A) Permeation of Rho123 as P-gp substrate. (B) Permeation of CDCF as MRP2 substrate (CDCFDA is hydrolyzed during permeation through the monolayers and is detected as CDCF). (C) Permeation of H33342 as a BCRP substrate. White circles indicate permeation from the A to B side, black circles from the B to A side. The left panel shows the absence of inhibitors of each transporter, while the right panel shows the presence of 30 µM verapamil, 100 µM MK-571, and 20 µM Ko143 as inhibitors of P-gp (A), MRP2 (B), and BCRP (C), respectively. Values represent mean ± S.D. (n = 3–4).

Table 1. Summarized P_app Values of Rho123, CDCF, and H33342

		Papp, A to B (pmol/well/min)	Papp, B to A (pmol/well/min)
Rho123	Control	0.11 ± 0.01	0.57 ± 0.02 **
	with verapamil	0.10 ± 0.02	0.09 ± 0.01
CDCF	Control	45.3 ± 6.3 (×10−3)	54.7 ± 7.9 (×10−3)
	with MK-571	46.8 ± 7.7 (×10−3)	40.1 ± 1.9 (×10−3)
H33342	Control	1.11 ± 0.22	2.64 ± 0.36 **
	with Ko143	1.49 ± 0.34	1.99 ± 0.17

Values represent mean ± S.D. (n = 3–4). ** p < 0.01 (Student’s t-test).

Fig. 4. Effect of Inhibitors on the Efflux Ratio of Drug Permeation through the Cholangiocyte Organoid Monolayers

(A), (B), and (C) show efflux ratio of Rho123 as P-gp substrate, CDCF as MRP2 substrate, and H33342 as BCRP substrate, respectively. Values represent mean ± S.D. (n = 3–4). * p < 0.05 or ** p < 0.01 (Student’s t-test).

DISCUSSION

The mRNA expression level of the bile duct marker CK19 was significantly higher than that of PHHs in cholangiocyte organoids before and after membranization. Since a previous report³³⁾ showed that the expression of CK19 differs more than 100-fold between hepatocytes and cholangiocyte cells, this organoid was considered to be a cholangiocyte epithelial-like organoid. The mRNA levels of efflux transports in cholangiocyte organoids before membranization were higher or comparable to those in PHHs, but the levels after membranization were lower than those prior, and tended to approach expression levels in normal cholangiocyte cells (Fig. 1). Cholangiocyte epithelial-like organoids have already been established using hepatocytes from different donors.^{20,22,29,33,34)} In our and other reported cases, the expression of key genes was comparable. In addition, we previously reported on the development of an evaluation system for biliary excretion of drugs using the monolayer of human liver cell lines HepG2 and Huh-7.¹⁸⁾ We investigated the mRNA expression levels of P-gp, MRP2, and BCRP, efflux transporters involved in biliary excretion of drugs in that system. Our results showed that this system expressed only MRP2. Therefore, that evaluation system limited the assessment of drug excretion into bile to MRP2 substrates. In contrast, our current system has the potential to evaluate drugs excreted in bile via various transporters. The subcellular localization of each protein was observed in our current system (Fig. 2). CK19 was distributed evenly in the cytoplasm.^35,36) P-gp, MRP2, and BCRP were localized on the upper side rather than the lower side; phalloidin, a marker of the cytoskeleton localized mainly at the apical side of the epithelial cells,^37,38) was localized on the upper side. DAPI, a marker of the nucleus localized at the basolateral side,^39,40) was localized on the lower side. These results suggest that the membrane-cultured cholangiocyte organoids are oriented with the upper side being the apical membrane side (A side, bile duct lumen side) and the lower side being the basolateral membrane side (B side, hepatocyte side), and that each efflux transporter is localized to the apical membrane. Epithelial-derived cells seeded on membranes, such as Caco-2 cells from the gastrointestinal tract and LLC-PK1 cells from the renal tubules, also have an orientation of the upper side toward the apical membrane and the lower side toward the basolateral membraneand are used for transport studies to estimate gastrointestinal absorption and urinary excretion of drugs.^41–44) The direction of drug transport in the present study system was shown to be consistent with these reports.

At the transport study via P-gp and BCRP, P_{app, B to A} of each substrate was significantly higher than P_{app, A to B} (Table 1). In the presence of inhibitor, the difference between P_{app, B to A} and P_{app, A to B} of each substrate disappeared. ER values of Rho123 and H33342 were all above 1.0 in the absence of inhibitor, while each ER value was significantly decreased in the presence of inhibitor (Fig. 4). In several cell monolayer systems, compounds were evaluated as efflux transported when the ER value was greater than 1.0.^45–47) Since the similar result was observed in our evaluation system, we thought that each substrate was transported in the efflux direction. Therefore, the substrate drugs for P-gp and BCRP were shown to permeate towards the bile duct with a directional effect, which were completely abolished by the addition of the inhibitor of each transporter. These results indicate this system is capable of directly, quantitatively and directionally assessing the biliary excretion of the drugs that are substrates of these transporters.

The ER value of CDCF, a substrate of MRP2, exceeded 1.0, and its ER value decreased in the presence of inhibitors (Table 1, Fig. 4). This suggests that at least MRP2 is involved in the permeation of cholangiocyte organoids of CDCF. In addition, the ER value was less than 1.0 in the presence of the inhibitor, suggesting that uptake transporter(s) may also be involved in the membrane permeation of CDCF.

For P-gp and MRP2, the addition of inhibitors decreased the B to A permeability, resulting in a loss of directionality. For BCRP, the decrease in B to A permeability was accompanied by an increase in A to B permeability. The cause of this is not clear, but it is thought to be due to differences in membrane permeation properties caused by differences in the transporters. Furthermore, the substrates and inhibitors used in this study might change the membrane permeation properties of the transporters. Further studies are needed.

In conclusion, the cholangiocyte organoid monolayer system examined in this study has the potential to quantitatively assess drug biliary excretion. The results of this study represent the first system in the world that can assess biliary excretion of drugs using human cholangiocyte organoids and may be useful as a screening model to directly quantify the contribution of biliary excretion to drug clearance.

Acknowledgments

This work was supported by JSPS KAKENHI Grant No. JP23K14392, and by the Japan Agency for Medical Research and Development (AMED) Grant No. 23ak0101187 for Yamazaki group, and No. 16nk0101051h0201 for Ishida group. These projects of AMED are partially supported by Suntory Holding Ltd.

Conflict of Interest

We have the following financial relationships to disclose for our study: RO and MI are employees of JSR corporation and NM is an employee of MBL. The other authors have no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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