Development of three-dimensional (3D) spheroid cultures of the continuous rainbow trout liver cell line RTL-W1

Ecotoxicology and Environmental Safety 167 (2019) 250–258

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Development of three-dimensional (3D) spheroid cultures of the continuous rainbow trout liver cell line RTL-W1

T

Tobias Lammel , Georgia Tsoukatou, Johanna Jellinek, Joachim Sturve ⁎

Department of Biological and Environmental Sciences, University of Gothenburg, Medicinaregatan 18 A, Box 463, 413 90 Göteborg, Sweden

ARTICLE INFO

ABSTRACT

Keywords: Fish cell line In vitro model Aquatic toxicity Bioaccumulation Hepatocyte Cytochrome P450 1A

In vitro experimental systems based on continuous piscine cell lines can be used as an alternative to animal tests for obtaining qualitative and quantitative information on the possible fate and effect of chemicals in fish. However, their capability to reproduce complex metabolic processes and toxic responses as they occur in vivo is limited due to the lack of organ-specific tissue architecture and functions. Here we introduce a three-dimensional (3D) in vitro experimental system based on spheroidal aggregate cultures (spheroids) of the continuous rainbow trout liver cell line RTL-W1 and provide a first description of their structural and functional properties including growth, viability/longevity, metabolic activity, ultrastructure and cytochrome P450 1A (CYP1A) expression determined by bright-field, multi-photon fluorescence and transmission electron microscopy as well as RT-qPCR analysis. Our results show that RTL-W1 cells in 3D spheroids (ø ~ 150 µm) (including those in the interior) were viable, metabolically active and had higher basal and β-naphthoflavone-induced CYP1A expression levels than conventional 2D cell cultures. Furthermore, they displayed ultrastructural characteristics similar to differentiated hepatocytes. The available evidence suggests that 3D RTL-W1 spheroids may have enhanced hepatotypic functions and be a superior in vitro model to assess hepatic biotransformation, bioaccumulation and chronic toxicity compared to conventional cell monolayer cultures.

1. Introduction

information on hepatic biotransformation and clearance of chemicals, which can then be used to parameterize computational models (Fitzsimmons et al., 2007; Halder et al., 2014; Johanning et al., 2012; Lee et al., 2017; Lo et al., 2016; Nichols et al., 2013a, 2013b; Segner and Cravedi, 2001; Treu et al., 2015). However, their use for long-term toxicological studies such as bioaccumulation assessment of slowly metabolized compounds and chronic toxicity testing is limited, because they rapidly loose many of their xenobiotic metabolic competencies and become senescent with increasing culture time (Braunbeck and Segner, 2000; Segner and Cravedi, 2001). Culturing primary hepatocytes in a three-dimensional (3D) configuration, for example as spheroidal aggregate cultures (also referred to as “spheroids”), which restores “natural” cell shape and intercellular connectivity, can help overcoming these shortcomings (Kyffin et al., 2018; LeCluyse et al., 2012). So far, only few studies have applied this strategy with piscine cells. They demonstrate that fish (rainbow trout) primary hepatocytes cultured in 3D show improved longevity and enhanced hepatotypic functions including higher glucose production, albumin synthesis, vitellogen in excretion, cytochrome P450 (CYP) activity and ATP-binding cassette (ABC) transporter expression compared to 2D primary hepatocyte cultures (Baron et al., 2012; Cravedi et al.,

To ensure a high level of protection of aquatic ecosystems, chemicals and products placed on the European market need to be assessed with respect to their hazard to aquatic biota. For instance, industrial chemicals, which are regulated under REACH (the EU Regulation concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals), need to be tested for their acute toxicity, chronic toxicity and bioaccumulative potential in fish when produced in tonnages ≥ 1, ≥ 10 and ≥ 100 t/year, respectively. Due to ethical concerns related with fish in vivo tests, there are ongoing efforts to develop alternative test methods that can aid in reducing their use in environmental hazard assessment. However, while fish acute toxicity can be relatively well estimated using fish cell linebased cytotoxicity assays (Natsch et al., 2018; Tanneberger et al., 2013), chronic toxicity and bioaccumulation are still difficult to predict in a reliable manner with the existing in vitro models (Halder et al., 2014). Fish primary hepatocyte cultures are ascribed a high potential for use in regulatory bioaccumulation assessment (Fay et al., 2014a, 2014b; Mingoia et al., 2010), as they can provide quantitative ⁎

Corresponding author. E-mail address: [email protected] (T. Lammel).

https://doi.org/10.1016/j.ecoenv.2018.10.009 Received 12 June 2018; Received in revised form 29 September 2018; Accepted 2 October 2018 Available online 17 October 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.

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1996; Flouriot et al., 1995, 1993; Uchea et al., 2015, 2013). Furthermore, they were shown to metabolise pharmaceuticals at similar or higher rates compared to liver microsomal fractions (Baron et al., 2017). However, several practicability- and animal welfare-related factors argue against the use of primary hepatocyte-based in vitro models for long-term toxicity and bioaccumulation testing on a large scale in a regulatory context. First, their isolation from the animal and subsequent functional characterization for quality control purposes is time-intensive and costly. Second, the amount of cells that can be obtained from an individual fish is limited, i.e. studies need to be carried out using cells originating from several fish with different genetic and biochemical background introducing additional variability. Third, using primary hepatocytes as cellular source material for organotypic in vitro models still involves the killing of fish, i.e. fails the ultimate objective of a truly animal-free risk assessment. An alternative in vitro model to primary hepatocytes are continuous liver cell lines, which have the advantage that they can be infinitely sub-cultured and maintained in culture without undergoing senescence allowing the production of large amounts of cellular source material with identical genetic/biochemical background. This decreases the probability that responses to xenobiotic exposure vary across time and laboratories. It also allows repeating studies as many times as needed (e.g. to generate data for different experimental parameter settings and/or obtain data that are statistically robust) without coming in conflict with ethical principles, which demand to conduct the least number of experimental repetitions (Bols et al., 2005; Segner, 1998). However, a major shortcoming limiting the use of continuous liver cell lines in toxicological, and specifically metabolic studies is, that they often exhibit a de-differentiated phenotype associated with lower expression and/or activity levels of xenobiotic metabolizing enzymes and transporters compared to freshly isolated primary hepatocytes. One of the best characterized and most widely used piscine liver cell lines in environmental toxicology is the RTL-W1 cell line. It developed via spontaneous immortalization of cells obtained from non-tumorous liver tissue of an adult rainbow trout (Lee et al., 1993) and exhibits a number of important liver-typic functions including CYP and ABC transporters activities (Behrens et al., 2001; Creusot et al., 2015; Fischer et al., 2011; Stadnicka-Michalak et al., 2018; Thibaut et al., 2009). Recently, it has been characterized in detail regarding cytological, immunocytochemical, ultrastructural and growth properties (Malhao et al., 2013). Based on their findings the authors concluded that RTL-W1 cells closely resemble bile preductular epithelial cells, which are considered to function as stem cells in teleost liver (Malhao et al., 2013; Okihiro and Hinton, 2000). Furthermore, they demonstrated that RTL-W1 cells grown in form of 3D aggregate cultures on agar substrate regain structural and functional features of hepatocytes in their physiological environment (Malhao et al., 2013). This potential for RTL-W1 cells to differentiate into metabolically competent hepatocytes may allow developing advanced in vitro systems that benefit from all advantages of continuous liver cell lines while overcoming their main shortcoming (limited metabolic capabilities). The objective of this study was twofold: First, to investigate if it was possible to develop spheroidal aggregate cultures (spheroids) from RTLW1 cells and establish a methodological approach allowing their use in functional and toxicological studies. Second, to determine if culturing RTL-W1 cells as spheroids can restore a phenotype that resembles differentiated hepatocytes in terms of structure, function and response to model compounds.

(Stockholm, Sweden). AlamarBlue solution was purchased from ThermoFisher (Sweden). 2.2. Routine cell culture Cells of the rainbow trout liver cell line (RTL-W1) were cultured in 75 cm2 flasks (TC Flask T75, Sarstedt) in phenol red-free Leibovitz's L15 medium containing 5% fetal bovine serum (FBS) and 1% penicillin streptomycin (Gibco) at 19 °C. Cell cultures were subcultured (split ratio 1:2 or 1:3) when reaching confluence applying 0.2 g/L ethylenediaminetetraacetic acid (EDTA)/phosphate buffered saline (PBS) followed by 0.25% trypsin-EDTA solution (Gibco). 2.3. Spheroid culture 2.3.1. Spheroid production: Seeding, incubation and size measurements Two millilitre of a single cell suspensions (1 × 106 RTL-W1 cells/ml in L-15 supplemented with 10% FBS and 1% penicillin streptomycin (Gibco)) was added to polystyrene cell culture dishes (35 × 10 mm) with hydrophobic growth surface (Sarstedt). The culture dishes were placed and incubated on a GFL 3011 orbital rocking-shaker set to 45 rpm. Every 72 h 1.5 ml of the medium was replaced. Spheroid development was monitored daily and documented by taking images at regular intervals using a Zeiss Axiovert 135 or Nikon TMS inverted microscope. The spheroids’ size was determined measuring their projected area in the obtained images using ImageJ software (ImageJ 1.49, National Institutes of Health, USA). The number of cells per spheroid was estimated on the basis of the average diameter of single suspended RTL-W1 cells in suspensions (measured parameter), their volume (calculated parameter assuming ideal spherical shape) and the knowledge on the spheroid's diameter/volume. 2.3.2. Prevention of spheroid attachment to the culture dish by p-HEMA coating To ensure that spheroids did not attach to the bottom of the culture dishes during long-term incubation and/or static incubation conditions, the bottom of the wells was pre-coated with p-HEMA. For this purpose, p-HEMA granules were dissolved in ethanol to a final concentration of 2.5% (w/v). Once the p-HEMA was completely dissolved, the solution was used immediately. 500 µl were used for a 35 × 10 mm culture dish and 100 µl for 48-well plates in order to cover the bottom adequately. In order to ensure an even coating with the hydrogel as well as complete evaporation of the used ethanol, the culture dishes were placed on a hot plate set to 65 °C (set up inside the laminar flow hood) and swayed periodically until they were completely dry. Thereafter the culture dishes were washed with PBS and stored dry until used for spheroid culture. 2.3.3. Prevention of multi-spheroid cluster formation Different strategies were tested to prevent spheroid-spheroid collision and formation of heterogeneous multi-spheroid structures with increasing incubation time. 2.3.3.1. Incubation under static conditions. In order to reduce the frequency of spheroid-spheroid collision resulting in formation of multi-spheroid structures, spheroid stock cultures were removed from the orbital shaker at day 4 or 5 (see 2.3.1 Spheroid production) and subsequently incubated under static culture conditions (off-shaker incubation).

2. Material and methods

2.3.3.2. Isolation of single spheroids. In order to prevent spheroidspheroid collision and multi-spheroid cluster formation, individual spheroids were isolated from the stock and transferred into p-HEMA coated 48-well plates at day 4 or 5 using a micropipette equipped with a trimmed 200 µl tip, which were then incubated under static conditions at 19 °C.

2.1. Chemicals Ethanol (99.5%), dimethyl sulfoxide (DMSO), 7-ethoxyresorufin (ER), β-naphthoflavone (β-NF) and poly(2-hydroxyethyl methacrylate) (BioReagent, powder, suitable for cell culture) were from Sigma-Aldrich 251

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2.4. Analysis of cell viability and metabolic activity by multiphoton microscopy

resulting ΔΔCq-vales were used to calculate fold change (FC) in the CYP1A mRNA levels using the equation: FC = 2-ΔΔCq. FC expression levels were presented as mean ± standard deviation (SD) of three independent experimental repetitions (n = 3). The SD was calculated using the FC values calculated for each of the three experimental repetitions. Statistical comparisons between all samples/treatments (in vitro models and β-NF exposures) were performed on ΔCq values by One Way Analysis of Variance (One-way ANOVA) using SigmaPlot for Windows Version 12.0 (Systat Software, Inc.). Normal distribution (Shapiro-Wilk test) and equal variance (Brown-Forsythe test) of data was confirmed beforehand.

Spheroids (8–10 day-old; ø ~ 150 µm) were analysed with respect to cell viability and overall cellular metabolic activity by using alamarBlue and presence/inducibility of cytochrome P450 1A (CYP1A)-dependent ethoxyresorufin-O-deethylase (EROD) activity by using 7-ethoxyresorufin as biochemical reagents, respectively. For the alamarBlue assay spheroids were transferred to a culture dish containing 1.25% alamarBlue solution (prepared in L-15/ex) and incubated therein for 30 min on the shaker at room temperature in the dark. For the assessment of presence/inducibility of EROD activity spheroids were first exposed to 0.63 µM beta-naphthoflavone (β-NF) for 24 h and thereafter transferred to a culture dish containing 2 µM ethoxyresorufin solution (prepared in L-15/ex), in which they were incubated for 30 min on the shaker at room temperature in the dark. The solvent (=DMSO) concentration during β-NF exposure was 0.1%. Spheroids were fixed in 4% paraformaldehyde (30 min at room temperature) and stored in L-15/ex in the fridge (4 °C) until microscopy analysis. Production/fluorescence intensity of resorufin (the conversion product of both alamarBlue and ER) was imaged using a ZEISS LSM 710 NLO system coupled to a MaiTai multi-photon laser tuned to 750 nm and a GaAsP NND detector. The obtained images were analysed using ZEN lite microscope software from Zeiss.

2.6. Transmission electron microscopy (TEM) Spheroids from 15 day-old cultures were collected in a 1.5 ml Eppendorf tube and spun down at 300 g for 5 min. The supernatant was removed and replaced with 150 µl of modified Karnovsky's fixative. After 2 h the fixative was removed, replaced with 0.15 M sodium cacodylate buffer, and then post-fixed with 1% osmium tetroxide (OsO4) containing 1% potassium ferrocyanide for 1 h, stained with 0.5% uranylacetate for 5 min and thereafter step-wise dehydrated in a concentration series of ethanol. Subsequently the ethanol was gradually replaced with Agar 100 resin (Agar Scientific Ltd., UK). After polymerization ultrathin sections were produced from the resin blocks (including the spheroids) using a Leica UC60 ultramicrotome, collected on Cu slot grids, and examined with a Zeiss Leo 912 AB transmission electron microscope (Zeiss, Germany) operated at 120 kV. Images were acquired with a Veleta CCD camera and processed with ESI vision software.

2.5. Analysis of CYP1A expression levels 2.5.1. Culture and exposure Spheroidal aggregate cultures (15 day-old) and confluent monolayer cultures were exposed to 0.16, 0.63 and 2.5 µM β-NF as well as to medium only (L-15 incl. 10% FBS) for 24 h. Maximum care was taken to conduct the monolayer exposure in the same way as the spheroid exposure. Thus, the same spiking procedure was used to add β-NF. Furthermore, both the spheroid cultures and the monolayer cultures were exposed while being incubated on an orbital shaker preventing DMSO-dissolved β-NF from “sinking” to the bottom of the well (Tanneberger et al., 2010b) and thus ensuring homogenous distribution/availability.

3. Results 3.1. Spheroid formation and development Representative light microscopy images of the spheroids’ development are shown in Fig. 1. Spheroid formation was initiated via collision of single cells resulting in loose cell clusters within 24 h (Fig. 1A). The cell clusters increased in size by recruiting and binding more cells to their surface. After four days, compact spheroidal aggregates of uniform shape and size (ø ~ 80 µm) had developed (Fig. 1D). Spheroid size continued to increase the subsequent days reaching average diameters of ~ 140 µm after seven and ~ 150 µm after ten days (culture on orbital-rocking shaker) (Fig. 2). In parallel, the frequency of collisions between individual spheroids increased resulting in their merging to larger multi-spheroid structures of heterogeneous size and shape. Representative images of multi-spheroid structures present in the culture dish at day 7 and 12 (post-seeding) are shown together with individual spheroids, which were still abundantly present as well, in Fig. 1E–G and H. This uncontrolled formation of multi-spheroid structures considerably increased the size variability and heterogeneity in the spheroid culture (Fig. 2). Different approaches were explored to prevent the formation of multi-spheroid structures. The first approach consisted in switching from dynamic culture conditions to static culture conditions (i.e. offshaker incubation) from the moment compact spheroids of homogenous size had formed (day 4–5 post-seeding). The second approach consisted in transferring individual spheroids to a separate culture dish once they had reached a suitable size and exhibited a high enough structural integrity. Off-shaker incubation resulted in settling and attachment of the spheroids to the well bottom, followed by spheroid flattening and cells migrating out of the spheroid starting to re-form a monolayer (images not shown). Pre-coating the culture dishes with p-HEMA could successfully prevent spheroid attachment. Representative light microscopy images of spheroids cultured in p-HEMA coated dishes under static conditions from day 4 are shown in Fig. 1I (images taken at day 7) and

2.5.2. RNA extraction Cells and spheroids were collected and processed in a similar way. Adherent cells were carefully detached using a cell scraper and then transferred to a collection tube using a micropipette. Spheroids were directly transferred to a collection tube. Both cell and spheroid suspensions were centrifuged at 300 g for 5 min and washed with PBS. Thereafter, they were pelleted again by centrifugation and lysed in RLT buffer using QIAshredder spin-columns (Quiagen). Total RNA was isolated using the RNeasy Plus Mini Kit from Quiagen following manufacturer instructions. RNA quality and quantity was measured on a NanoDrop 2000c photospectrometer (ThermoFisher) 2.5.3. Gene expression analysis Relative CYP1A mRNA expression levels (cDNA copy numbers) were measured by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). Isolated total RNA was transcribed into cDNA using the iScript™ cDNA synthesis kit from BioRad. SsoAdvanced™ Universal SYBR Green Supermix and a CFX ConnectTM Real-Time System (Bio-Rad) was used for cDNA amplifcation. Primer sequences for CYP1A and β-actin (bACT) and conditions of optimized qPCR assays including primer concentration, primer efficiency and detailed program used to run qPCR reactions were described previously (Gräns et al., 2010; Wassmur et al., 2010). The obtained data were analysed as followed: The Cq values obtained for the target gene (CYP1A) were normalized against the Cq values obtained for the reference gene (β-ACT). All resulting ΔCq values were then normalized against the ΔCq values obtained for the no treatment control of the monolayer culture. The 252

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Fig. 1. Light microscopy images of spheroidal aggregates cultures of RTL-W1cells at different culture times and culture conditions. Images A-D show different “developmental stages” during the formation process of RTL-W1 spheroids (A: 1 day post-seeding; B: 2 days post-seeding; C: 3 days post-seeding, D: 4 days post-seeding). E-G are representative images of 7 day-old spheroid cultures including spheroids (E) and multi-spheroid structures (F-G) forming under dynamic culture conditions (on-shaker incubation). Image H shows an example of a large, fully merged multi-spheroid commonly observed in long-term spheroid cultures (here: day 12 post-seeding). Exemplary images of spheroid cultures incubated under static conditions (off-shaker; p-HEMA coated culture dishes) since day 4 are shown in I (day 7) and J-K (day 10). Image L shows a 20 day-old spheroid isolated and singly cultured from day 4. Scale bars in A-D and E-L correspond to 100 and 200 µm, respectively.

Fig. 1J and K (images taken at day 10). Size data obtained for these time points/conditions are displayed in Fig. 2. The results show that incubation under static conditions delayed the formation of multispheroid structures, but could not prevent it. The size and variability in spheroid size after 10 days was comparable to on-shaker conditions (Fig. 2). The isolation and transfer procedure did not affect spheroid integrity. The average size (ø) of singly cultured, 10 day-old spheroids (isolated at day 5) was ~ 150 µm showing that they increased in size even after isolation (Fig. 2). The spheroids could be cultured for at least three weeks without any observed cell death or loss of structural integrity. An image of a singly cultured spheroid maintained in culture for 20 days is exemplary shown in Fig. 1L.

alamarBlue-“stained” spheroids displayed similar fluorescence intensity compared to cells in the outer layers demonstrating that they were viable and metabolically active. 3.3. Cytochrome P450 1A expression RTL-W1 spheroids (8 day-old) exhibited CYP1A-dependent resorufin production/fluorescence upon exposure to the model arylhydrocarbon receptor (AhR) agonist β-naphthoflavone (β –NF) (Fig. 4A). Furthermore, the fluorescence intensity detected in the interior of the spheroid was comparable to the fluorescence intensity in the spheroid's periphery (Fig. 4B). These results demonstrate that CYP1A – a key xenobiotic metabolizing enzyme – was expressed, functional and inducible throughout RTL-W1 spheroids. Moreover, quantitative analysis of CYP1A mRNA expression in 15 day-old spheroids demonstrated that both the basal and induced expression level was significantly higher than in conventional RTL-W1 cell monolayer cultures (Fig. 5).

3.2. Cell viability inside mature RTL-W1 spheroids Fig. 3 shows that cells in the core of ~ 150 μm-in diameter large, 253

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Fig. 2. Box-plot showing the size and size variability of RTL-W1 spheroids as a function of culture time and condition. Vertical boxes with error bars show the median, 10th, 25th, 75th and 90th percentiles. Outliers are displayed as “+”. The number of spheroids (n) measured in “on-shaker” cultures was 200, 200, 100, 200, 100 and 129 for day 1, 2, 3, 4, 7 and 10 (post-seeding). The data set obtained for “off-shaker” cultures at day 7 and 10 (postseeding) had a size of n = 74 and n = 60, respectively. The number of individualized, singly cultured spheroids measured at day 10 (post-seeding) was n = 39. The scales to the right of the box-plot show the measured area (in µm2) and the corresponding calculated radius (in µm) and estimated cell number (#) of the spheroids.

cytoplasm of most cells was rich in vesicles, the larger of which probably being endosomal compartments, and mitochondria. Furthermore, free ribosomes and rough endoplasmatic reticulum were abundantly present (the latter often being dilated or irregular in shape). In some cells the rough endoplasmatic reticulum was strongly developed with its cisterns being arranged in a well-organized complex of several parallel or concentric layers. In addition, many cells –both in the interior and in the spheroid's periphery– featured a high number of electron-dense bodies probably representing autophagic compartments. 4. Discussion 4.1. Development of RTL-W1 spheroids and methodological considerations for future studies In this study we demonstrate –to the best of our knowledge for the first time- that it is possible to create spheroids from the continuous trout liver cell line RTL-W1. The methodological approach previously employed by other authors including Baron et al. (2012) and Uchea et al. (2013), that is, incubation of a cell suspension on an orbitalrocking shaker, was successfully used to initiate spheroid formation, but was unsuitable for long-term spheroid culture. The reason for this was that after spheroids have formed they started colliding with each other merging into multi-spheroids of heterogeneous size and shape difficult to control. Spheroids of different size and shape may have different structural and functional properties. For instance, there may be differences in the surface area available for chemical uptake and elimination, the supply with oxygen and nutrients, or the export of metabolic waste products –all affecting cell physiology and viability (Alvarez-Perez et al., 2005; Curcio et al., 2007; Glicklis et al., 2004; Groebe and Mueller-Klieser, 1991; Mueller-Klieser and Sutherland, 1984). Thus, a strong variability in spheroid size may translate into a decrease in reproducibility of functional and toxicological test results and is therefore undesirable, in particular for applications outside “exploratory” academic research such as regulatory toxicity testing. Although Baron et al. (2012) did not explicitly mention the formation of multi-spheroid structures in their paper, the reported increase in spheroid size and the shown light microscopy images suggest that this phenomenon might also become an issue for spheroids developed from trout primary hepatocytes when the culture period exceeds one week. In this study, we pursued different strategies aiming at preventing the formation of multi-spheroid structures. The first approach consisted in switching from dynamic incubation to static incubation at the time compact spheroids of homogenous size have formed (day 4–5). Although initial issues such as spheroid settling and attachment to the

Fig. 3. Viability of cells inside RTL-W1 spheroids. Multiphoton fluorescence microscopy image of a spheroid (ø ~150 µm, 10 day-old) incubated with the cell viability reagent alamarBlue. A. Focal plane (xy) showing the middle section of the spheroid. B and C show xz- and yz-orthogonal sections along the red and green lines displayed in A, respectively.

3.4. Ultrastructure of RTL-W1 spheroids Fig. 6 shows representative TEM images of 15 day-old spheroids. RTL-W1 cells in spheroids (15 day-old) were tightly packed and connected via cell-cell junctions, of which different types including tight junctions, adherens junctions and desmosomes could be identified. On several occasions adjacent cells were interdigitated. Furthermore, in the space between the plasma membranes of two or more adjacent cells small intercellular bodies with cytoplasma-like electron density, possibly cell protrusions from under- or overlaying cells, could be observed. The ultrastructural appearance of RTL-W1 cells was relatively homogenous throughout the spheroid and no differences could be discerned comparing cells in the centre with cells in the periphery of the spheroids, except that a few microvilli-like cell protrusions could be identified in cells forming the most outer layer of the spheroid. The 254

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Fig. 4. Presence and inducibility of EROD activity in RTL-W1 spheroids. Multiphoton fluorescence microscopy images of a spheroid (ø ~150 µm, 8 day-old) exposed to 7-ethoxyresorufin. A. Three-dimensional reconstruction of a z-stack of multiphoton fluorescence microscopy images showing Cyp1A-dependent resorufin production/fluorescence in the spheroid. B. Central focal plane (xy) and orthogonal sections along the xz and xy-axis through the middle of the spheroid. The coloured lines (green, red, blue) indicate the focal and section planes.

culturing and monitoring individual spheroids for up to at least three weeks. Size measurements conducted on isolated spheroids showed that their diameter further increased with time suggesting that RTL-W1 cells have maintained their proliferative activity. Individualization of spheroids opens up new possibilities for smaller functional studies, but has the disadvantage that it is quite laborious and thus not suitable for large scale toxicity testing. Therefore, we recommend exploring strategies that allow creating single RTL-W1 spheroids i.e. one spheroid per culture vessel, for example by seeding a defined number of cells into ultralow attachment plates as described by Bell et al. (2016) or using the hanging-drop technique by Kelm et al. (2003). 4.2. Functional characterization of RTL-W1 spheroids and comparison with conventional cell monolayer cultures The second objective of this study was to explore the spheroids’ potential for toxicological studies. The spheroids were assessed with respect to basic criteria that need to be met such as maintenance of viability over an appropriately long culture period as well as more specific properties such as hepatotypic phenotype and functionalities. Multiphoton microscopy analysis demonstrated that the viability and metabolic activity of cells in the interior of spheroids with diameters of ~ 150 µm was comparable to that of cells in the spheroid's periphery suggesting that gas, nutrient and waste exchange was not impaired. Also, the ultrastructural analysis of the spheroids by means of TEM did not indicate any necrotic core e.g. due to hypoxia or accumulation of metabolic waste products. This is consistent with the widely accepted assumption that cell necrosis because of oxygen exhaustion only occurs when the distance from the surface of the hepatocyte spheroid becomes larger than 150 µm (Funatsu et al., 2001; Glicklis et al., 2004; Kyffin et al., 2018). However, cells contained an increased number of electron dense bodies resembling autophagic compartments, which usually occur in response to stress (Kroemer et al., 2010). Since the electron dense bodies were equally abundant in cells in the interior and in the periphery of the spheroid hypoxia-induced apoptosis/phagocytosis can be excluded as reason. A possible explanation could be autophagy induction by 2-hydroxyethyl

Fig. 5. Basal and induced CYP1A mRNA expression levels in RTL-W1 spheroids and cell monolayer cultures. Bars and error bars show the mean and corresponding standard deviation (SD) of CYP1A mRNA levels measured in three independent experimental repetitions (n = 3). Statistically significant differences between CYP1A mRNA levels in spheroid cultures (grey bars) and conventional monolayer cultures (black bars) are indicated by asterisks (“*” = p < 0.05 and “***” = p < 0.001; ANOVA).

bottom of the culture dish under static incubation conditions could be resolved by using culture dishes pre-coated with p-HEMA as in Baron et al. (2012), this approach was of only limited success. Incubation under static conditions reduced the frequency of spheroid-spheroid interactions delaying the formation of large multi-spheroid structures, but could not fully prevent it. Yet, since the frequency of collisions between spheroids depends on their density in the suspension, our results suggest that appropriate dilution of the latter could help to delay multi-spheroid cluster formation maintaining homogeneity within the spheroid culture (i.e. spheroid size and shape) for a longer culture period. The second approach consisted in isolating individual spheroids once they have reached a suitable size and exhibited sufficient structural integrity for manipulation (day 4–5). This approach allowed 255

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Fig. 6. Transmission electron microscopy image showing the ultrastructure of RTL-W1 cells at the surface (A) and inside (B) of mature spheroids (15 dayold). NC = nucleus, MI = mitochondrion, RER = rough endoplasmatic reticulum, V = vesicle, G = golgi apparatus, TJ = tight junction, DS = desmosome, AJ = adherens junction, CJ = cellular junction, F = filaments, LY = lysosome, MB = multilamellar body, DB = dense body. Asterisk (*) = intercellular body of poor electron density (probably remainder of apoptotic cell). Black arrows = intercellular inclusions (probably cell protrusions from neighbour cells situated below or above the cutting plane). Scale bars in A and B correspond to 2 µm.

material type and surface area in the culture dish available for chemical absorption/partitioning, as well as the cellular uptake and elimination rates, amongst others (Gülden et al., 2015; Gülden and Seibert, 1997; Kramer et al., 2012; Tanneberger et al., 2010a). However, theoretical reflections on how these factors may differ and influence the cellular response in the two in vitro systems (cell monolayer and spheroid cultures) suggest that, if at all different, the local exposure concentration/availability of β-NF availability is rather likely to be higher in the monolayer culture (e.g. DMSO-dissolved β-NF sinks to the bottom of the well, less available surface area for chemical adsorption, all cells are directly exposed). Furthermore, differences in the local exposure concentration/availability of β-NF availability can neither explain the higher basal CYP1A levels in the control (which did not contain any βNF). A possibility that needs to be taken into consideration is that elevated CYP1A expression in spheroid cultures is related with the presence of p-HEMA. There is some indications in the literature that biodegradation of p-HEMA can occur upon direct contact with cells (macrophages) (Mabilleau et al., 2004), and that HEMA monomers can induce CYP1A expression (Samuelson et al., 2011). However, the contact of RTL-W1 spheroids with the p-HEMA-coated well bottom was limited, and there is no reports in literature that liver cells can degrade p-HEMA. In addition, our observations are in agreement with previous literature showing that basal expression levels of phase I and II xenobiotic metabolizing enzymes including CYP1A are elevated in 3D cultures of continuous liver cell lines compared to their 2D counterpart (Chang and Hughes-Fulford, 2009; Ramaiahgari et al., 2014; Rodd et al., 2017). Overall, our result support the hypothesis that culturing RTL-W1 cells in a configuration that more closely mimics the microenvironment of liver cells in vivo can aid in restoring a more hepatocyte-like phenotype, which may improve their potential for use in toxicological and metabolic studies in academic research and/or an environmental risk assessment context (e.g. for evaluating chronic toxicity or bioaccumulative potential of chemicals). Yet, future research needs to demonstrate, whether the observed increase in CYP1A expression at the transcriptional level also translates into elevated CYP1A enzyme activity, as well as how basal and chemical-induced enzyme activity levels in RTL-W1 spheroids compare to those measured in rainbow trout primary hepatocyte cultures (Behrens

methacrylate (HEMA) monomers released from the polymer coating. HEMA-induced autophagy has been reported in studies with mammalian cell models assessing the toxicity of pHEMA-containing dental resins (Teti et al., 2015; Yu et al., 2017). However, p-HEMA-based hydrogels have been widely used in cell biology research (including for spheroid culture) and there are no reports that they may induce an autophagic response. The appearance of electron dense bodies has also been reported by Malhao et al. (2013) for three-dimensional RTL-W1 aggregate cultures grown in agar. In fact, the RTL-W1 spheroids share many characteristics with the aggregate cultures grown by Malhao et al. (2013). Thus, our spheroids displayed a tissue-like architecture with adjacent cells being tightly associated via membrane digitations and junctional complexes. Furthermore, cells at the spheroids’ featured specialized epithelial junctions and some microvilli-like protrusions, which may reflect restoration of a polarized phenotype (Kelm et al., 2003; Malhao et al., 2013; Ramaiahgari et al., 2014). Furthermore, the cytoplasm of the cells was rich in organelles. The abundant presence of intracellular vesicles and endosomal compartments of different size are indicative of a well-developed, active intracellular transport system. Numerous cisternae of smooth and rough endoplasmic reticulum (RER) were observed. The RER was sometimes organized as large structures of multiple parallel or concentric layers. In addition, cells contained a high number of mitochondria. All these characteristics are typical for metabolically active cells including differentiated trout hepatocytes (Hampton et al., 1985; Rocha et al., 1994) and were not that distinct in TEM images of RTL-W1 cell monolayer cultures, –based on a qualitative judgment. These results corroborate the hypothesis of Malhao et al. (2013), that RTL-W1 cells adopt a more hepatocyte-like phenotype when cultured in a three-dimensional context. Further support for this hypothesis is given by our functional data, which demonstrated that both basal and chemical-induced mRNA expression levels of the key xenobiotic metabolizing enzyme CYP1A were significantly higher in spheroids than in conventional RTL-W1 cell monolayer cultures. When interpreting results obtained by two different in vitro experimental systems such as monolayer and spheroid cultures a number of factors that may influence the measured responses need to be taken into account. One critical factor is the local exposure concentration/ availability of the test compound (here: β-NF) for the cells. The local exposure concentration/availability depends on the solvent density, 256

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et al., 2001; Berbner et al., 1999; Nabb et al., 2006; Risso-de Faverney et al., 2000; Scholz and Segner, 1999; Uchea et al., 2013), liver subcellular fractions (Han et al., 2009), and intact liver tissue (Billiard et al., 2004; Hawkins et al., 2002; Katchamart et al., 2002; Lemaire et al., 1996; Malmstrom et al., 2004; Rabergh et al., 2000; Sandvik et al., 1997; Valdehita et al., 2012; Weber et al., 2002). In addition, it would be of outmost interest to perform direct comparisons of RTL-W1 cell-based and primary hepatocyte-based spheroids as used by Baron et al. (2012, 2017) and Uchea et al. (2013, 2015) in order to obtain information about their relative performance (longevity, hepatotypic functionalities etc.) and response to chemical insult.

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5. Conclusion This is the first study describing the development of spheroidal aggregate cultures from the continuous fish liver cell line RTL-W1. The spheroids fulfil a number of criteria essential to their use in functional studies and chemical toxicity testing. They possess a good structural integrity allowing manipulation in the laboratory (centrifugation, pipetting) and spheroids can be maintained in culture for at least three weeks. Cells in the core are viable and metabolically active suggesting that oxygen and nutrient supply as well as removal of metabolic waste products is sustained. Spheroids display a tissue-like architecture with many cells displaying hepatotypic ultrastructural characteristics. In addition, the key xenobiotic metabolizing enzyme CYP1A is expressed at significantly higher levels than in RTL-W1 cells cultured as monolayer. Altogether, the available evidence supports our hypothesis that 3D spheroid cultures of the RTL-W1 cell line may represent a more physiologically relevant in vitro experimental system to assess environmental toxicity, hepatic biotransformation and bioaccumulation of chemicals compared to conventional RTL-W1 cell monolayer cultures. Acknowledgments The authors acknowledge the Centre for Cellular Imaging at the University of Gothenburg and the National Microscopy Infrastructure, NMI (VR-RFI 2016-00968) for providing assistance in microscopy, as well as Prof. Dr. Kristin Schirmer (Department of Environmental Toxicology, EAWAG) for the kind provision of the RTL-W1 cell line. Competing interest statement The authors have no competing interests to declare. References Alvarez-Perez, J., et al., 2005. Microscopic images of intraspheroidal pH by 1H magnetic resonance chemical shift imaging of pH sensitive indicators. Magma 18, 293–301. Baron, M.G., et al., 2017. Pharmaceutical metabolism in fish: using a 3-D hepatic in vitro model to assess clearance. PLoS One 12. Baron, M.G., et al., 2012. Towards a more representative in vitro method for fish ecotoxicology: morphological and biochemical characterisation of three-dimensional spheroidal hepatocytes. Ecotoxicology 21, 2419–2429. Behrens, A., et al., 2001. Polycyclic aromatic hydrocarbons as inducers of cytochrome P4501A enzyme activity in the rainbow trout liver cell line, RTL-W1, and in primary cultures of rainbow trout hepatocytes. Environ. Toxicol. Chem. 20, 632–643. Bell, C.C., et al., 2016. Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease. Sci. Rep. 6, 25187. Berbner, T., et al., 1999. Induction of cytochrome P450 1A and DNA damage in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes by 2,3,7,8-tetrachlorodibenzo-pdioxin. Biomarkers 4, 214–228. Billiard, S.M., et al., 2004. In vitro and in vivo comparisons of fish-specific CYP1A induction relative potency factors for selected polycyclic aromatic hydrocarbons. Ecotoxicol. Environ. Saf. 59, 292–299. Bols, N.C., et al., 2005. Chapter 2 Use of fish cell lines in the toxicology and ecotoxicology of fish. Piscine cell lines in environmental toxicology. In: Mommsen, T.P., Moon, T.W. (Eds.), Biochemistry and Molecular Biology of Fishes. Elsevier, pp. 43–84. Braunbeck, T., Segner, H., 2000. Isolation and cultivation of teleost hepatocytes. In: Berry, M.N., Edwards, A.M. (Eds.), The Hepatocyte Review. Springer, Netherlands,

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