Placental samples used for this study were collected after normal term cesarean deliveries from John Sealy hospital [University of Texas Medical Branch (UTMB)] at Galveston, Texas, United States in accordance with the relevant guidelines and regulations of approved protocols for various studies (UTMB 11-251). As discarded placentas after delivery were used for the study, no subject recruitment or consent was required.

Placental and FM (i.e., amniochorion) tissues from term, not in labor, cesarean deliveries (TNIL) (n = 5) were used for this project and prepared as previously described by our laboratory (n = 6 tissues for western blotting and n = 5 tissues for explant culture) (Ayad et al., 2018; Sheller-Miller et al., 2020). Briefly, the FM was dissected from the placenta, washed three times in normal saline, cleaned with sterile saline-moistened gauze to clear any adherent blood clots and all the decidua, and washed in Hank’s balanced solution (HBSS) with penicillin 100 U/mL and streptomycin 100 µg/ml. Using a skin biopsy punch, the membranes were cut into 6-mm disks. Sections were taken from midzone of the fetal membranes, avoiding the regions overlying the cervix or placenta. Four disks of the amniochorion membranes were placed in each well of a 24-well tissue culture plate with 0.4 ml of Dulbecco’s modified Eagle’s medium (DMEM) with Ham F12 nutrient mixture 1:1; antibiotics: penicillin (100 IU/ml), streptomycin (100 µg/ml), and amphotericin B (2.5 µg/ml); glutamine (2 mmol/L); and 15% heat-inactivated fetal bovine serum. Cultures were incubated at 37°C with 5% CO₂. The medium was changed at 24 h (hrs.) when the study reagents were added.

Placental explants were prepared as described previously (Behnia et al., 2016; Arita et al., 2018). Briefly, placental tissues were washed with sterile saline. After removal of the blood clots by gentle blotting, segments of the maternal part of the placental villi were isolated by sharp dissection, and subsequently washed and cut 3–5 times. Chopped tissues were washed in DMEM supplemented with 5% fetal bovine serum (v/v), penicillin (100 U/mL), and streptomycin (100 µg/ml). Placental tissue (0.1 gm) was placed in each well of a 24-well tissue culture plate in 0.5 ml DMEM with Ham F12 nutrient mixture 1:1; antibiotics: penicillin (100 IU/ml), streptomycin (100 µg/ml), and amphotericin B (2.5 µg/ml); glutamine (2 mmol/L); and 10% heat-inactivated fetal bovine serum. The medium was changed at 24 h when the study reagents were added.

Maternal decidual and FM cells [amnion epithelial cells (AECs); amnion mesenchymal cells (AMCs), chorionic trophoblast cells (CTCs)] were isolated from term, not-in-labor, cesarean-delivered FM and immortalized by a standard HPV16 E6E7 retrovirus protocol (Halbert et al., 1991). Retroviruses were obtained from the PA317 LXSN 16E6E7 (ATCC® CRL-2203™) cell line. Cells derived from healthy FM tissues were seeded in 6-well plates. The next day, cells were infected with 500 μL of the mixture containing 50 μL of retrovirus supernatant, 5 μg of protamine sulfate in serum-free culture medium, and incubated at 37°C for 6 h. Culture medium was replaced with fresh media, and 48 h after the viral infection, antibiotic selection was started. Five days after the antibiotic selection process, the immortalized cells were expanded and passaged several times (up to passage 8) before using them in the experiments. AMCs and maternal decidual cells were cultured and maintained in complete media consisting of Dulbecco’s modified Eagle’s medium (DMEM) with Ham F12 nutrient mixture 1:1; antibiotics: penicillin (100 IU/ml), streptomycin (100 µg/ml), and amphotericin B (2.5 µg/ml); and 5% heat-inactivated fetal bovine serum. AECs were cultured in complete keratinocyte serum-free media (KSFM), a culture highly selective for epithelial cells, supplemented with human recombinant epidermal growth factor (0.1 ng/ml), bovine pituitary extract (30 µg/ml), and primocin (0.5 mg/ml) (ant-pm-1; Invivogen). Supplements were added to the media immediately before use. CTCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with Ham F12 nutrient mixture 1:1 supplemented with the following: fetal bovine serum, penicillin, streptomycin, bovine serum albumin, ITS-X, CHIR99021, A83-01, SB431542, L-ascorbic acid, epidermal growth factor, VPA, and Y27632 (ROCK inhibitor). Cells used for all experiments were within 10 passages.

Cigarette smoke extract (CSE) was prepared as previously reported by our laboratory (Menon et al., 2013; Sheller et al., 2016; Richardson et al., 2020a). Briefly, smoke drawn from a single lit commercial cigarette (unfiltered Camel™, R.J. Reynolds Tobacco Co.; Winston Salem, NC) was bubbled through 50 ml of tissue culture medium (DMEM: F12 Ham’s mixture with antimicrobial agents and FBS). Each cigarette is reported to contain 26 mg of tar and 1.7 mg of nicotine. The stock CSE was filter-sterilized using a 0.25 µm pore Millipore filter (Millipore, Bedford, MA) to remove insoluble particles. Fetal membrane and placental explants were then stimulated with CSE (diluted 1:10 in culture media) for 0, 4, 8, 24 and 48 h. CSE was prepared freshly for each experiment. After the treatments, the explants (placental and FM) were removed and processed for western blotting for OATP2B1. Both placental and FM explants were used to better understand the expression levels of OATP2B1 in response to CSE stimulation.

Immunohistochemistry (IHC) for OATP2B1 was performed on FM tissues using methods previously described by our laboratory (Kammala et al., 2020). Briefly, FM sections were fixed in 4% paraformaldehyde for 48 h and embedded in paraffin. Tissue sections were cut to 5 μm thickness, placed on a positively charged slide, and attached by keeping them at 50°C for 45 min. Slides were deparaffinized using xylene, and then rehydrated with 100% alcohol, 95% alcohol, and normal saline (pH 7.4), followed by staining. Tissue sections were probed with the antihuman OATP2B1 antibody (1:500, ab222094, Abcam, Cambridge, United Kingdom) overnight at 4°C, and IHC anti-rabbit/anti-mouse secondary antibody (Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit, ab64264, Abcam, Cambridge, United Kingdom) was added for 45 min at room temperature, followed by adding DAB as a chromogen for 15 s, and then hematoxylin as a counterstain for color development. Bright-field microscopy images were captured using a Nikon Eclipse TS100 microscope (20x and 100x) (Nikon).

Firstly, anti-human OATP2B1 antibody (ab222094, Abcam, Cambridge, United Kingdom) was labeled using the CF® Dye and Biotin Protein Labeling Kits (Biotium, Fremont, CA) along with Rabbit IgG (AB_2532938, Invitrogen) as per the manufacturer’s protocol. The next day, the conjugated OATP2B1 antibody was used for flow cytometry analysis. Briefly, FM cells (AEC, AMC, and CTC), maternal decidua, and BeWo cells (used as a surrogate for placental cells) were harvested, washed in ice-cold PBS, 10% FCS, 1% sodium azide and centrifuged at 3,000 rpm for 10 min. Cell pellets were incubated with specific fluorophore conjugated antihuman OATP2B1 antibody (1:50) and propidium iodide dye (BioLegend, San Diego, CA) for 30 min at 4°C in dark. Cells were washed three times by centrifugation at 400 g for 5 min and resuspended in 500 µL of ice-cold PBS, 10% FCS, 1% sodium azide, and analyzed immediately on a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA).

Based on our IHC and western blotting data, we chose AECs and CTCs to further study the uptake function of cell surface OATP2B1 by flow cytometry. Previous studies have demonstrated that intracellular accumulation of zombie violet dye is increased explicitly by OATPs (Patik et al., 2015; Patik et al., 2018). OATP2B1 is known to function almost exclusively at acidic extracellular pH (Patik et al., 2018). Based on these criteria, we chose the zombie violet dye uptake assay to determine the functional capacity of OATP2B1 in FM cells. Briefly, cells were washed in uptake buffer (125 mM NaCl, 4.8 mM KCl, 1.2 mM CaCl₂, 1.2 mM KH₂PO₄, 12 mM MgSO₄, 25 mM MES, and 5.6 mM glucose, with the pH adjusted to 5.5 using 1 M HEPES and 1 N NaOH). After washing 5 × 10⁵ cells were incubated for 15 min at 37°C with Zombie violet (BioLegend®, San Diego, CA, United States; 0.4 µL zombie violet/5 × 10⁵ cell) and propidium iodide dye (BioLegend, San Diego, CA) in a final volume of 100 µL. The reaction was stopped by adding 1 ml ice-cold PBS and the fluorescence of 10,000 living cells was immediately determined on a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA). Dead cells were excluded by propidium iodide labeling.

Placental and FM explants as well as immortalized maternal DECs and FM cells (AECs, AMCs, CTCs) were lysed with radioimmunoprecipitation lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 1% Triton X-100, 1.0 mM EDTA (pH 8.0), and 0.1% SDS] supplemented with a protease and phosphatase inhibitor cocktail and also phenyl methyl sulfonyl fluoride. Human FM were homogenized as previously described (Richardson et al., 2020a). After centrifugation at 12,000 g for 20 min, the supernatant was collected and protein concentrations were determined using the Pierce bicinchoninic acid kit (Thermo Scientific, Waltham, MA, United States). The protein samples were separated using SDS-polyacrylamide gel electrophoresis on a gradient (4–15%) Mini-PROTEAN TGX Precast Gel (Bio-Rad, Hercules, CA, United States) and transferred to the membrane using a Bio-Rad Gel Transfer Device (Bio-Rad). Membranes were blocked in 5% non-fat milk in 1× Tris-buffered saline-Tween 20 for a minimum of 1 h at room temperature and then probed overnight at 4°C using primary antibody overnight. The membranes were then incubated with secondary antibody conjugated with horseradish peroxidase, and immunoreactive proteins were visualized using chemiluminescence reagents ECL WB detection system (Amersham Piscataway, NJ, United States). The following anti-human antibodies were used for western blot: OATP2B1 (1:1,000 for tissues and 1:250 for FM cells, ab245246, Abcam, United States) and β-actin (1:15,000, Sigma- Aldrich, St. Louis, MO, United States). Protein band quantification were normalized to β-actin expression in each lane, and the expression levels were densitometrically determined using the Bio-Rad Image Lab 6.0 software.

CTCs were found to have maximum levels of OATP2B1 compared to all other cell types in FMs. Therefore, we chose CTCs to further study the role of OATP2B1 in the uptake of fluorescent zombie violet dye and substrate (Rosuvastatin) propagation across the FM layers based on our immunohistochemistry western blot analyses. CTCs were cultured to 50-60% confluency in DMEM/F12 medium supplemented with 10% FBS and antimicrobial agents (penicillin, streptomycin, and amphotericin), and then transfected with 15 µL of 20 µM oligos (Dharmacon, Lafayette, CO, United States) directed against OATP2B1 or nontarget scrambled (control) siRNAs using RNAimax lipofectamine as per the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, United States). After 24 h, OATP2B1 knockdown CTCs were separately used for the fluorescent zombie violet uptake assay BeWo cells was used as a surrogate of placental cells. BeWo cells is a commonly used in vitro model for studying the placental barrier. BeWo cells emulate a term human placental barrier in culture conditions because of its ability to fuse and form a syncytium in vitro. In another set of experiments, control and knockdown CTCs were loaded into the feto-maternal interface OOC device as previously described (Radnaa et al., 2021), as further described in the following section.

The development and validation of the feto-maternal interface -OOC device have been previously reported by our laboratory (Richardson et al., 2019; Richardson et al., 2020b; Richardson et al., 2020c; Radnaa et al., 2021). In brief, the microfluidic feto-maternal interface -OOC is composed of four concentric cell culture compartments (one for maternal cells and three for fetal cells) interconnected through arrays of microfluidic channels to allow co-cultivation of four different cell types. Chamber one, the central chamber is for maternal DECs, chamber two is for fetal CTCs, chamber three is for AMCs, and the outermost chamber is for AECs. Each cell chamber is 250 μm in height, and the width of each chamber was designed to mimic the differences in thickness of each maternal and fetal layer as seen in utero. The chambers are interconnected through an array of 24 microchannels (all width: 35 μm; length: 600 µm for CTCs to DECs and AMCs to CTCs, 300 µm for AECs to AMCs; all height: 5 µm) that performed several functions, such as preventing the flow of cells between compartments during the initial cell loading process, allowing independent localized biochemical treatments to each compartment, enabling independent elution of supernatant from each cell compartment, and allowing biochemicals to diffuse between the chambers to enable cell-cell communication. The number of microchannels, along with the height and width, remained same throughout all experiments as to not affect the diffusion rate of molecules. The device contains an on-chip reservoir block placed on top of the cell culture chamber layer and is comprised of multiple 4 mm diameter and 2 mm deep reservoirs, where each reservoir is aligned in such a way that they are placed on top of the inlets and outlets of each cell culture chamber in the main cell culture layer. The center culture chamber has one reservoir on top of it and the outermost culture chamber has four reservoirs on top of it, while the two middle culture chambers have two reservoirs each on top of them, to provide sufficient cell culture medium to all parts of the cell culture chambers evenly. The feto-maternal interface -OOC was fabricated in polydimethyl siloxane (PDMS, 1:10 mixture, Sylgard 184; DowDuPont, Midland, MI, United States) using a two-step photolithography master mold fabrication process, followed by a soft lithography process of replica-molding the final PDMS device from the master mold (Park et al., 2010). The bonding of the PDMS layer onto the glass substrate was improved by treating the PDMS layers with oxygen plasma (Harrick Plasma, Ithaca, NY, United States), followed by bonding the layer onto a glass substrate. This process was repeated to bond the PDMS reservoir layer on top of the device. The feto-maternal interface -OOC device was stored dry (for up to 1 month) and before using the feto-maternal interface -OOC, the devices were sterilized with 70% ethanol for 15 min.

Before using the feto-maternal interface-OOC, the devices were washed three times with PBS, filled with type IV basement membrane collagen Matrigel (Corning Matrigel basement membrane matrix, DEV-free; 1:25 in media), and incubated at 37°C with 5% CO₂ overnight. Diluted type IV collagen basement membrane Matrigel was used to fill the microfluidic channels between the AMC and the CTC compartments, mimicking the amnion and chorion basement membranes in utero. These Matrigel-filled microchannels will allow localized drug treatment of each cell layer and independently take supernatant from each layer. These channels also allow biochemical to diffuse between the layers and permit cell migration and transition, as seen in the fetal membrane-decidua feto-maternal interface.

After Matrigel loading, the cell chambers were rinsed two times with PBS to remove extra Matrigel, and the devices were loaded with immortalized cells in the different culture chambers. Immortalized cells were then trypsinized and loaded into the device, starting from the center chamber to the outside. Cell loading concentration in each chamber mimicked those of in utero cell ratios of the FM tissue (60,000 DECs for chamber one, 200,000 CTCs + 5% primary collagen + 25% Matrigel for chamber two, 62,500 AMCs + 20% primary collagen + 25% Matrigel for chamber three, and 120,000 AECs for chamber four). Following cell seeding, both the feto-maternal interface-OOC devices and the 24-well plates were incubated at 37°C with 5% CO₂ overnight before Rosuvastatin treatment.

Rosuvastatin was obtained from Sigma-Aldrich (St Louis, MO). Rosuvastatin calcium salt was prepared as a stock of 10 mg/ml in serum-free media, and was added in a concentration of 200 nmol/L. The dose of rosuvastatin was chosen based on the maximum concentrations achievable in tissues after therapeutic doses, as previously reported by our laboratory (Ayad et al., 2018). Next day, rosuvastatin (200 ng/ml) was added to the maternal DEC chamber, and the supernatant from each chamber (AEC, AMC, CTC, and maternal DEC) was collected after 4 h. We chose this time point because previous studies from our laboratory have observed propagation of rosuvastatin from the maternal decidual chamber to the AEC chamber of the feto-maternal interface-OOC within 4 h after introducing the drug (unpublished data). To confirm the kinetics of Rosuvastatin in OATP2B1 knockdown CTCs, the concentration of rosuvastatin in the media collected from each of the chamber was analyzed by mass spectrometry (see below for details).

Targeted liquid chromatography tandem mass spectrometry (LC-QQQ) analysis was performed on a TSQ Altis mass spectrometer (Thermo Scientific, Waltham, MA) coupled to a binary pump UHPLC (Vanquish, Thermo Scientific). Scan parameters for target ions as per the Selective Reaction Monitoring (SRM Table) for Rosuvastatin were used. The injection volume was 10 µL. Chromatographic separation was achieved on a Hypersil GOLD™ C18 HPLC column (Dimensions: Particle size: 5 μm; Diameter: 50 mm; and Length: 2.1 mm) (Thermo Scientific) maintained at 30°C using a solvent gradient method. Solvent A was 0.1% formic acid in water. Solvent B was 0.1% formic acid in acetonitrile. The gradient method used was 0–1 min (20% B to 60% B), 1-2 min (60% B to 95% B), 2-4 min (95% B), 4–4.1 min (95% B to 20% B), and 4.1–5 min (20% B). The flow rate was 0.5 ml min⁻¹. Sample acquisition and data analysis was performed using the Trace Finder 4.1 software (Thermo Scientific).

Statistical analysis and significance were determined by using the Prism 9 software (Graph Pad, San Diego, CA). Statistical analyses for normally distributed data were performed using analysis of variance (ANOVA) with the Tukey Multiple Comparisons Test. Significance is shown as *p < 0.05 and **p < 0.01. All data are shown as mean ± standard error of the mean (SEM).

FMs are divided into morphologically distinct amnion and chorion membranes, each with its own well-defined microarchitecture (Menon et al., 2019). First, OATP2B1 localization was confirmed in different layers of FMs by IHC (n = 3). OATP2B1 expression was localized on the basolateral surface of AECs and on both the apical and basolateral surfaces of CTCs (Figure 1A). In line with previous studies, OATP2B1 was also localized on the basolateral surface of placental syncyiotrophoblasts (Figure 1A). Next, we determined the protein expression of OATP2B1 in FMs by western blotting (n = 6). The expression level of OATP2B1 was similar in FMs as in both maternal and fetal sides of the placenta (used as a positive control in our experiment) (Figure 1B). These results show that OATP2B1 location and expression are relatively similar in the FM and placenta.

To test whether OATP2B1 is expressed on the surface of fetal membrane cells, flow cytometry analysis was performed on AECs, CTCs, AMCs, and maternal DECs. As shown in Figure 2A, cells expressing OATP2B1 receptor bind to a conjugated antibody and show a distinct shift in flow peak towards right (blue) from the Isotype control stained cells (red). In line with our IHC data, flow cytometry analyses showed differential expression of OATP2B1 on the surface of FM cells (Figure 2A). The surface expression of OATP2B1 on the CTCs was significantly higher compared to the maternal DECs (p < 0.5) (Figure 2A). Furthermore, western blot analysis showed OATP2B1 expression in only AECs and CTCs of the FM, but not in AMCs and DECs (Figure 2B).

Next, the zombie violet uptake assay was used to determine the functional role of OATP2B1 in FM cells (Patik et al., 2018; Patik et al., 2015). Zombie violet is a fluorescent viability dye that is taken up by dead cells; however, the presence of OATP can mediate uptake of zombie violet dye in living cells at an extracellular pH of 5.5 (Patik et al., 2018). As an internal control, we have used the propidium iodide (PI) which will stain also dead cells. As shown in Figure 3A, uptake of the zombie violet dye by OATP2B1 receptor showed a distinct shift in flow peak towards right (blue) from the live cells (red). The uptake of zombie violet dye by fetal membrane AECs and CTCs was comparable to the dye uptake observed in the placental BeWo cells (Figures 3A,B). Uptake of the dye by the FM and placental cells suggest that OATP2B1 expressed on cell surfaces of AECs, CTCs and BeWo are functional.

Previous reports show that CSE-induced oxidative stress promotes cellular senescence, cellular transitions, and a pro-inflammatory environment (increased concentrations of pro-inflammatory cytokines) (Polettini et al., 2018; Richardson et al., 2020a). Moreover, a pro-inflammatory environment at the feto-maternal interface has been reported in preterm birth, preeclampsia, and fetal growth restriction (Aouache et al., 2018). Therefore, to determine the effect of CSE-induced oxidative stress on OATP2B1 expression at the feto-maternal interface, FM and placenta explants were exposed to CSE for up to 24 h (0, 4, 8, 24, 48 h) and analyzed for OATP2B1 expression by western blotting. Oxidative stress did not have a significant impact on OATP2B1 expression in the FM (Figure 4A); however, CSE led to a significant decrease in OATP2B1 expression in placental explants (p < 0.05) (Figure 4B). These results suggest that oxidative stress or a pro-inflammatory in utero environment during pregnancy can lead to differential OATP2B1 expression at the FM-decidua parietalis interface and the placenta-decidua basalis interface.

To validate the contribution of OATP2B1 in drug transport, the zombie violet uptake assay was conducted after silencing OATP2B1. A reduction in OATP2B1 expression was determined by western blot analysis in CTCs and BEWO cells (Figure 5A). Knocking down OATP2B1 in CTCs reduced the uptake of zombie violet dye (as indicated by the zombie violet-stained blue peak overlapping with the unstained red peak), suggesting that OATP2B1 in CTCs could play an essential role in the uptake of substrates within the FM (Figure 5B). Interestingly, silencing OATP2B1 had little to no effect on the zombie violet dye uptake in BeWo cells (Figure 5B). We speculate that the differences in uptake results between the FM and placental cells after OXTP2B1 knockdown indicate that possibly in placental BeWo cells other uptake receptors (such as OATP1B1) may also contribute to substrate uptake, whereas in FM CTCs OATP2B1 may play a more predominant role in the uptake of substrates from the maternal DEC.

To confirm OATP2B1’s role in drug transport, especially in CTCs, the feto-maternal interface-OOC device was used to study the contribution of OATP2B1 in the transport of rosuvastatin through the FM layers. Here, OATP2B1-silenced CTCs were used and compared to normal CTCs. We used stain as the model drug, since they are competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMG-CoA reductase) and have been tested in multiple models of pregnancy complications, such as preterm birth and preeclampsia (Istvan, 2003; Basraon et al., 2012). Specifically, we selected rosuvastatin because it is more hydrophilic than other statins (such as pravastatin) and requires protein transporters such as OATP2B1 to enter the cell to inhibit the HMG-CoA reductase enzyme (Climent et al., 2021). This drug was loaded into the DEC chamber (innermost chamber) and its propagation throughout the FM layers measured using mass spectrometry (Figure 6A). Mass spectrometry data suggests that 18.45% of rosuvastatin propagated from the maternal decidual chamber to the fetal CTC chamber under control conditions. siRNA-mediated silencing of OATP2B1 in CTCs however resulted in only 2.57% of rosuvastatin propagating from the DEC chamber to the CTC chamber (Figure 6B). Furthermore, OATP2B1 silencing also reduced rosuvastatin propagation from the DEC chamber to the AEC chamber, where only 0.32% of rosuvastatin was detected in the AEC chamber from OATP2B1 knockdown CTCs compared to 1.73% in non-targeted siRNA control CTCs (Figure 6B). These data further confirm our findings that OATP2B1 could potentially act as one of the most significant drug transport receptors in FM CTCs.