# A perfused iPSC-derived proximal tubule model for predicting drug-induced kidney injury

Michelle Lechtenberg, Coraline Chéneau, Kevin Riquin, Leopold Koenig, Carlos Mota, Franck Halary, Eva-Maria Dehne

► To cite this version:

Michelle Lechtenberg, Coraline Chéneau, Kevin Riquin, Leopold Koenig, Carlos Mota, et al. A perfused iPSC-derived proximal tubule model for predicting drug-induced kidney injury. Toxicology in Vitro, 2025, 105, pp.106038. 10.1016/j.tiv.2025.106038. inserm-05039878

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# A perfused iPSC-derived proximal tubule model for predicting drug-induced kidney injury

Michelle Lechtenberg<sup>a,1</sup>, Coraline Chéneau<sup>b,1</sup>, Kevin Riquin<sup>b</sup>, Leopold Koenig<sup>a</sup>, Carlos Mota<sup>c</sup>, Franck Salary<sup>b,1</sup>, Eva-Maria Dehne<sup>a,*,1</sup>

## ARTICLE INFO

Editor: Dr. P. Jennings

### Keywords:

- Drug-induced kidney injury

- Microphysiological system

- Proximal tubule

- iPSC

- Metallothioneins

## ABSTRACT

The kidney is frequently exposed to high levels of drugs and their metabolites, which can injure the kidney and the proximal tubule (PT) in particular. In order to detect nephrotoxicity early during drug development, relevant *in vitro* models are essential. Here, we introduce a robust and versatile cell culture insert-based iPSC-derived PT model, which can be maintained in a microphysiological system for at least ten days. We demonstrate the model's ability to predict drug-induced PT injury using polymyxin B, cyclosporin A, and cisplatin, and observe that perfusion distinctly impacts our model's response to xenobiotics. We observe that the upregulation of metallothioneins that is described *in vivo* after treatment with these drugs is reliably detected in dynamic, but not static *in vitro* PT models. Finally, we use our model to alleviate polymyxin-induced nephrotoxicity by supplementing the antioxidant curcumin. Together, these findings illustrate that our perfused iPSC-derived PT model is versatile and well-suited for *in vitro* studies investigating nephrotoxicity and its prevention. Reliable and user-friendly *in vitro* models like this enable the early detection of nephrotoxic potential, thereby minimizing adverse effects and reducing drug attrition.

## 1. Introduction

The kidney plays a critical role in removing waste and toxins from the bloodstream and is regularly exposed to high levels of drugs and their metabolites. The proximal tubule (PT) is particularly prone to injury due to its central role in the reabsorption and excretion of drugs. This constant exposure can result in serious damage and may cause acute kidney injury (AKI), chronic kidney disease, or end-stage renal disease (Perazella and Rosner, 2022). Drug-induced AKI is a common adverse effect, accounting for at least 14% of all hospitalized AKI cases (Hoste et al., 2015; Kwiatkowska et al., 2021). Although nephrotoxicity is considered an important safety aspect during drug development, it is difficult to monitor in preclinical studies (Bonventre et al., 2010; Clark and Steger-Hartmann, 2018; Radi, 2019). In order to predict the nephrotoxic potential of new therapeutic candidates more accurately and to gain insights into toxicity mechanisms, relevant *in vitro* kidney models are essential (Pognan et al., 2023). Early detection of nephrotoxicity

during drug development minimizes adverse effects and reduces drug attrition, ultimately leading to safer treatment options (Soo et al., 2018).

Traditional two-dimensional cultures of primary or immortalized PT cells constitute a basic and reproducible method for nephrotoxicity testing. These cultures, however, often show low expression levels of key transporters, significantly limiting their ability to predict *in vivo* nephrotoxicity (Jenkinson et al., 2012; Telang et al., 2024; Caetano-Pinto et al., 2022). Additional challenges when using primary cells include their restricted availability and donor-to-donor variability. Advanced three-dimensional models derived from induced pluripotent stem cells (iPSCs) or adult stem cells, on the other hand, incorporate multiple cell types and relevant physiological stimuli into a complex environment well-suited for personalized medicine, but struggle with reproducibility and long-term stability (Schutgens et al., 2019; Phipson et al., 2019; Przepiorski et al., 2018; Jensen and Little, 2023). As the PT is continuously perfused *in vivo* and experiences shear stress, the incorporation of flow into *in vitro* kidney models is highly beneficial and improves not

<footnote><sup>a</sup> TissUse GmbH, Berlin, Germany</footnote>

<footnote><sup>b</sup> INSERM, Nantes Université, Center for Research in Transplantation and Translational Immunology, UMR 1064, F-44000 Nantes, France</footnote>

<footnote><sup>c</sup> Department of Complex Tissue Regeneration, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, 6229 ER Maastricht, the Netherlands</footnote>

<footnote>* Corresponding author at: TissUse GmbH, Oudenarder Straße 16, 13347 Berlin, Germany.
E-mail address: eva.dehne@tissuse.com (E.-M. Dehne).</footnote>

<footnote n="1">¹ Authors contributed equally.</footnote>
only transporter expression, but also polarization and ciliation (Ross et al., 2021; Nieskens et al., 2020; Vriend et al., 2020). Several micro-
physiological systems (MPS) comprise a hollow, perfusable tube
embedded in extracellular matrix (ECM) that enables the culture of a
monolayer of PT cells in an in vivo-like architecture. While these intri-
cate models incorporate advanced physical cues, such as ECM stiffness
and shear stress, in a highly advanced manner, they are rarely easy to
use or versatile (Sakolish et al., 2023; Nguyen et al., 2023).

Here, we aimed to address these challenges by developing a robust and versatile cell culture (CC) insert-based iPSC-derived PT model suitable for long-term culture within an MPS. We thoroughly characterized the model and examined the impact of perfusion by comparing PT models cultured in the HUMIMIC Chip2 24-well with models cultured in multi-well plates. We subsequently investigated whether our perfused PT model could predict drug-induced PT injury by applying three drugs with distinct mechanisms of entry into PT cells: the anti-biotic polymyxin B, the immunosuppressant cyclosporin A, and the chemotherapeutic agent cisplatin (Perazella and Rosner, 2022; Perazella, 2019; Wu et al., 2018; Yu et al., 2023). Finally, we examined whether co-administration of the antioxidant curcumin could alleviate polymyxin-induced nephrotoxicity in our model, as previously described in studies using rodents (Vazin et al., 2020).

## 2. Results

2.1. Establishment of a perfused iPSC-derived PT model

We differentiated proximal tubule-like cells (PTL) from iPSCs ac-
cording to Chandrasekaran et al (Chandrasekaran et al., 2021). with
minor adaptations and characterized the obtained cells at different
timepoints throughout the differentiation. On day 14, we cryopreserved
PTL and examined the effect of cryopreservation on the subsequent
progress of the differentiation. We then seeded PTL onto CC inserts to
generate a barrier model and either cultured the inserts statically in
multi-well plates or transferred them into the HUMIMIC Chip2 24-well
on day 14 + 2 for dynamic culture (Fig. 1A, B).

We monitored the progress of the differentiation by flow cytometry throughout the course of three independent differentiations and used megalin, an endocytic receptor expressed in the brush border of PT cells, as a marker to evaluate the cells' identity (Nielsen et al., 2016). The percentage of megalin-positive cells increased steadily from 0.6 ± 0.2 % on day 0 to 87.1 ± 7 % on day 14, and then continuously decreased to 31.1 ± 6 % on day 28 (Fig. 1C, Supplementary Fig. 1). Aiming for a population rich in megalin-expressing cells, we thus cryopreserved PTL on day 14 with a megalin-positive population of 80.2 ± 7 %. To ensure that cryopreservation did not disturb the differentiation process, we compared freshly differentiated and cryopreserved cells over time. Directly after thawing, PTL exhibited 89.6 % megalin-positive cells. On day 21 of the differentiation or 7 days after thawing, 64.8 % or 63.0 % of freshly differentiated or cryopreserved cells were megalin-positive. By day 28, the megalin-positive population decreased to 37.0 % and 24.7 %, respectively. When we seeded cryopreserved PTL onto CC inserts on day 14, however, we detected an enrichment of megalin-positive cells. 98.4 ± 0.4 % of cells expressed megalin 12 days after seeding (Fig. 1D).

PTL formed tight junctions, as shown by ZO-1 expression (Fig. 1E), and when cultured on CC inserts, created a confluent monolayer of viable cells that generated an intact barrier with transepithelial electrical resistance (TEER) values of 2580 ± 240 Ω x cm² on day 14 + 4. Despite the gradual decrease in TEER, the model maintained a tight barrier until day 14 + 12 (1380 ± 140 Ω x cm²) and remained highly viable (Fig. 1F, H). Furthermore, PTL cultured on CC inserts were proliferative on day 14 + 2, as shown by Ki67 expression, and exhibited approximately one primary cilium per cell, visualized by ARL13b staining (Fig. 1G, Supplementary Fig. 2).

We then examined whether the developed PT model could be
cultured in an MPS for an extended period of time. With medium

exchanges performed every 48 h, the dynamic PT model exhibited a
steady glucose consumption and lactate production over the course of
10 days, indicating a metabolic equilibrium. Lactate dehydrogenase
(LDH), which is released into the supernatant upon cell death, was
detected at stable concentrations of approximately 10 U/L in the me-
dium throughout the culture period, indicating a consistent turnover of
cells. Static culture led to an increased lactate production and release of
the cytotoxicity marker LDH compared to dynamic culture (Fig. 1I-K).

Next, we analyzed expression levels of selected PT markers and off-
target markers in dynamic and static day 14 + 12 PT models and
normalized them to day 14 + 2 PT models to examine the impact of
culture duration and perfusion (Fig. 1L). In line with previous reports
(Chandrasekaran et al., 2021), we did not detect the expression of
several characteristic PT markers, including AQP1 (aquaporin 1),
SLC5A2 (Na+/glucose cotransporter 2), SLC22A2 (organic cation
transporter 2), SLC22A6 (organic anion transporter 1), SLC22A8
(organic anion transporter 3), and SLC47A1 (multidrug and toxin
extrusion protein 1). The multidrug resistance (–associated) protein-
encoding genes ABCB1, ABCB8, ABCC1, ABCC2, ABCC4, ABCC5,
ABCC6, and ABCC10, as well as ABCG2 (breast cancer resistance pro-
tein) were expressed in all samples and were mostly downregulated over
time with no impact of perfusion. We detected an upregulation of the
dehydroppeptidase-encoding gene DPEP1 over time with higher expres-
sion levels in static than in dynamic PT models. The SLC-transporters
SLC2A1 (GLUT-1), SLC2A2 (GLUT-2), SLC4A4 (electrogenic sodium
bicarbonate cotransporter 1), SLC15A2 (H+/peptide transporter),
SLC22A3 (organic cation transporter 3), SLC22A4 (novel organic cation
transporter 1), SLC22A5 (novel organic cation transporter 2), and
SLCO4C1 (organic anion transporting polypeptide 4c1) were expressed
and not affected by perfusion, while SLC22A1 (organic cation trans-
porter 1) expression was upregulated in dynamic and downregulated in
static PT models. We further detected an upregulation of multiple
junctional proteins over time and the absence of markers that are typi-
cally associated with podocytes, the loop of Henle and distal tubule.
Only the collecting duct marker AQP3 (aquaporin 3) was expressed and
upregulated over time.

2.2. Polymyxin B induces linear concentration-dependent nephrotoxicity

After confirming that the developed PT MPS model could be cultured for at least 10 days, we investigated whether it could reliably detect the effect of known nephrotoxins on the PT. We subjected our PT model to a repeated exposure regimen (Fig. 1A) with varying concentrations (25 µM, 50 µM, and 100 µM) of the antibiotic polymyxin B (PMB). Dynamic models were transferred into the MPS on day 14 + 2, while static controls remained in multi-well plates. Medium exchanges were performed every 48 h, and from day 14 + 4 on, each medium exchange entailed the application of PMB. All applied PMB concentrations led to an increased LDH release, with 100 µM PMB causing a distinct LDH peak after the first PMB exposure. Overall, we observed an immediate concentration-dependent increase in LDH levels regardless of static or dynamic culture (Fig. 2A).

We further detected a concentration-dependent decrease in barrier integrity. By the end of the culture on day 14 + 12, TEER values decreased to 71.5 ± 9 % or 54.0 ± 11 % in dynamic and static culture, respectively, upon exposure to 25 µM PMB. 50 µM PMB caused a decrease to 27.1 ± 15 % or 21.0 ± 7 % and PT models treated with 100 µM PMB exhibited TEER values that equaled 7.7 ± 3 % or 3.2 ± 1 % of TEER values measured in vehicle-treated controls (Fig. 2B).

Next, we used the CellTiter-Glo 3D assay to quantify the number of viable cells, measured by detecting ATP content, on day 14 + 12. For dynamic PT models, we observed a decrease in viable cells with all applied PMB concentrations, while static PT models exhibited a decrease in viable cells in response to 50 µM PMB and 100 µM PMB (Fig. 2C).

A live/dead staining further confirmed the observed linear
concentration-dependent effect of PMB on the PT model. With
<figure><img src="image_4.png" /><figcaption>(B)</figcaption></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>(C)</figcaption></figure>

Megalin⁺ cells

<figure><img src="image_7.png" /><figcaption>(D)</figcaption></figure>

Megalin⁺ cells

<figure><img src="image_8.png" /><figcaption>(E)</figcaption></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>(F)</figcaption></figure>

TEER

<figure><img src="image_11.png" /><figcaption>(G)</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>(H)</figcaption></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>(I)</figcaption></figure>

Glucose consumption

<figure><img src="image_15.png" /><figcaption>(J)</figcaption></figure>

Lactate secretion

<figure><img src="image_16.png" /><figcaption>(K)</figcaption></figure>

LDH release

<figure><img src="image_17.png" /><figcaption>(L)</figcaption></figure>

<figure><img src="image_18.png" /></figure>

(caption on next page)
**Fig. 1.** Establishment of a perfused iPSC-derived PT model. (A) Timeline of PTL differentiation and subsequent culture in the Chip2 24-well for repeated exposure nephrotoxicity testing. (B) Schematic view of the cell culture insert within the Chip2 24-well. PTL are located on the lower side of the standing insert. (C) Percentage of megalin-positive cells over time. Megalin expression was measured by flow cytometry. Data are presented as mean ± s.d. (N = 3 independent differentiations). (D) Percentage of megalin-positive cells in freshly differentiated PTL (n = 1–3 replicates), PTL from the same differentiation that were cryopreserved on day 14 (n = 1), and cryopreserved PTL that were seeded onto cell culture (CC) inserts on day 14 (n = 3 replicates). (E) ZO-1 staining with DAPI counterstaining of day 14 PTL cultured in a multi-well plate. Scale bar: 100 µm. (F) TEER in Ω x cm² of PTL cultured on CC inserts over time. Data are presented as mean ± s.d. (n = 7 replicates). (G) ARL13b (yellow, primary cilia) and Ki67 (magenta, proliferation) staining with DAPI counterstaining of PTL cultured on CC inserts on day 14 + 2. Scale bar: 100 µm. (H) Live/dead staining of entire CC insert showing a viable layer of cells using Calcein-AM red orange (red, viable cells) and the absence of dead cells using CellToxGreen (green, dead cells). Scale bar: 1 mm. (I-K) Glucose consumption, lactate secretion and LDH release of dynamic (circles) and static (triangles) PT models over time. Data are presented as mean ± s.d. (N = 3 independent experiments with 4–8 replicates each; \*p < 0.05, \*\*p < 0.01). (L) Heat map showing expression levels of selected proximal tubule markers and off-target markers in dynamic and static day 14 + 12 PT models detected by bulk RNA sequencing. Data were normalized to day 14 + 2 PT samples and are presented as mean log₂ fold change (n = 4–5 replicates). Abbreviations: CHIR: CHIR99021, RI: ROCK-inhibitor Y-27632, EGF: epidermal growth factor, HC: hydrocortisone, FGF9: fibroblast growth factor 9, ME: medium exchange, FCS: fetal calf serum, CC insert: cell culture insert, TEER: transepithelial electrical resistance, LDH: lactate dehydrogenase. ARL13b: ADP-ribosylation factor-like protein 13B. ZO-1: zonula occludens-1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

increasing concentrations of PMB, the membrane area fraction of CalceinAM-positive living cells decreased, while the membrane area fraction of CellToxGreen-positive dead cells increased (Fig. 2E).

Using bulk RNA sequencing, we identified genes encoding metallothioneins (MTs) as being among the most significantly upregulated genes following PMB exposure compared to vehicle controls. MTs are small cysteine-rich proteins that bind heavy metals and protect cells from oxidative stress. We detected an upregulation of *MT1E*, *MT1F*, *MT1X*, *MT2A*, *MT1G*, *MT1H*, and *MT1M* upon exposure to 25 µM, 50 µM, and 100 µM PMB in dynamic PT models. In static PT models, however, we only detected an upregulation of *MT1E*, *MT1F*, *MT1X* and *MT2A* after PMB treatment. The expression of *MT1M*, which exhibited the strongest upregulation in dynamic PT models, *MT1G*, and *MT1H* was not affected in static PT models (Fig. 2D).

## 2.3. Curcumin co-administration ameliorates PMB-induced nephrotoxicity

To investigate whether our PT model could replicate the finding that curcumin (CCM) alleviates drug-induced AKI in rodents (Vazin et al., 2020), we performed an acute exposure experiment in which we subjected our PT model to 50 µM PMB with or without 5 µM CCM for 48 h. In three independent experiments, CCM co-administration ameliorated PMB-induced nephrotoxicity. LDH levels increased significantly upon PMB exposure and decreased from 55.6 ± 14 U/L to 41.3 ± 9 U/L and 63.5 ± 15 U/L to 52.8 ± 12 U/L upon addition of CCM in dynamic and static culture, respectively (Fig. 3B). CCM co-administration further improved the barrier integrity of PMB-treated PT models. TEER values increased from 39.3 ± 7% to 53.0 ± 5% and 35.5 ± 6% to 53.5 ± 12% in the dynamic and static condition, respectively (Fig. 3C). The supplementation of CCM also reduced lactate secretion in dynamic PT models (Fig. 3A) Other researchers have reported that CCM reduces oxidative stress by inhibiting the formation of reactive oxygen species (ROS) or scavenging ROS (Vazin et al., 2020). We did not, however, detect an effect of CCM on cellular ROS levels in our PT model, despite a repeated brief PMB ± CCM exposure prior to ROS measurement to account for the short half-life of ROS and CCM (Nelson et al., 2017). While PMB exposure increased ROS levels to 147.9 ± 26% or 166.3 ± 21% in dynamic and static cultures relative to controls, the addition of CCM did not lead to a reduction of ROS levels (Fig. 3D). Next, we investigated the effect of CCM supplementation on the expression of metallothioneins. While *MT2A* and *MT1F* expression levels were significantly increased in PMB-treated PT models, they were not affected by CCM co-administration at the examined timepoint (Fig. 3E, F).

## 2.4. Exposure to subtoxic cisplatin and cyclosporin A concentration affects MT expression

We further used our PT model to assess the effect of two additional drugs known to cause PT injury. We tested three concentrations each of

cisplatin (CP; 2.5 µM, 5 µM, 10 µM) and cyclosporin A (CsA; 5 µM, 10 µM, 20 µM) and utilized the same repeated exposure regimen as for PMB (Fig. 1A). Dynamic models were transferred into the MPS on day 14 + 2, while static controls remained in multi-well plates. Medium exchanges were performed every 48 h, and from day 14 + 4 on, each medium exchange entailed the application of CP or CsA. LDH levels of CP-treated models were low overall (Fig. 4A, C), but increasing CP concentrations led to a steady decrease in TEER values in dynamic PT models. In static PT models, however, TEER increased upon treatment with 2.5 µM and 5 µM CP, and decreased after exposure to 10 µM CP (Fig. 4E). Static PT models treated with 20 µM CsA exhibited elevated LDH levels and no TEER, whereas dynamic PT models treated with 20 µM CsA showed decreased barrier integrity but no increase in LDH release. Treatment with 5 µM and 10 µM CsA did not lead to increased LDH levels or decreased TEER values in dynamic or static PT models (Fig. 4B, D, F).

While the three examined drugs caused heterogenous responses regarding LDH release and TEER, we identified a common pattern of MT expression. Exposure to the medium and high concentrations of CP and CsA led to an upregulation of at least one of the metallothionein-encoding genes *MT1E*, *MT1F*, *MT1X*, and *MT2A* in dynamic PT models, but not in static models (Fig. 4G, H). An upregulation of these genes was also detected upon PMB exposure in dynamic and static PT models (Fig. 2D). We further detected a second gene cluster comprising *MT1G*, *MT1H*, and *MT1M*. Their increased upregulation in dynamic culture compared to static culture after PMB exposure appeared to be conserved among the examined substances with minor differences. CP treatment did not affect *MT1G*, *MT1H*, and *MT1M* expression in dynamic PT models but caused their downregulation in static models (5 µM and 10 µM). CsA, on the other hand, induced their upregulation in dynamic models (5 µM) and their downregulation in static PT models (10 µM and 20 µM).

## 3. Discussion

Drug-induced nephrotoxicity is a common adverse effect that can result in AKI and drug attrition. In order to reduce the prevalence of adverse effects and to pave the way to safer medication, nephrotoxicity needs to be detected early during drug development. This requires *in vitro* models that faithfully recapitulate essential features of the human kidney, while omitting others for the sake of simplicity and reproducibility.

In this study, we report the development of a robust and versatile perfused iPSC-derived PT model well-suited to investigate nephrotoxicity and its prevention if the presented limitations regarding SLC-transporter expression are considered. We provide evidence that perfusion impacts drug-induced PT injury and show that the *in vivo*-like upregulation of metallothioneins upon drug exposure is detected reliably in perfused, but not static PT models, for two out of three tested substances. We further confirm the versatility of our model by demonstrating an alleviation of polymyxin-induced nephrotoxicity upon
<figure><img src="image_19.png" /><figcaption>**Fig. 2.** Polymyxin B induces linear concentration-dependent nephrotoxicity. Three concentrations of polymyxin B (PMB) were applied to dynamic (circles, solid lines) or static (triangles, dotted lines) PT models every 48 h for eight days: 25 µM PMB (rose), 50 µM PMB (pink), or 100 µM PMB (magenta). Vehicle controls are shown in black. (A) LDH release over time. Data are presented as mean ± s.d. (n = 7 replicates). (B) TEER measured on day 14 + 12 normalized to the respective vehicle control (n = 7 replicates). (C) Viability measured on day 14 + 12 using the CellTiter-Glo 3D viability assay. Data are normalized to the respective vehicle control (n = 3 replicates). (D) Heat map showing expression levels of metallothioneins detected by bulk RNA sequencing on day 14 + 12. Data are normalized to vehicle controls and presented as mean log₂ fold change (n = 3 replicates) with pₐₘ₅ < 0.05 unless specified otherwise (ns). (E) Live/dead staining of entire CC inserts using Calcein-AM red orange (red, viable cells) and CellToxGreen (green, dead cells) on day 14 + 12. Scale bar: 1 mm. Percentages indicate % of total area of the seeded membrane. Abbreviations: LDH: lactate dehydrogenase, TEER: transepithelial electrical resistance, PMB: polymyxin B. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

curcumin co-administration, as described in rodents (Vazin et al., 2020).

First, we focused on the characterization of iPSC-derived PTL over time and investigated the impact of cryopreservation and CC insert culture on megalin expression. While megalin expression is highest in PT within the human body, it is also expressed at low levels in several extrarenal absorptive epithelia (Lundgren et al., 1997). Together with cubilin, megalin forms a complex responsible for the uptake of proteins and certain drugs via endocytosis (Nielsen et al., 2016). We simultaneously assessed using lotus tetragonolobus lectin (LTL) as a PT-specific

marker to track the progress of the differentiation, since it is used for magnetic-activated cell sorting in various protocols (Aceves et al., 2022; Banan Sadeghian et al., 2023). LTL binds glycoproteins located on the PT brush border, but the Fucα1-3(Galβ1-4)GlcNAc structure it binds is also known as SSEA-1, a common pluripotency marker (Zhang et al., 2016; Noro et al., 2015). Flow cytometry analysis suggested that almost 100 % of undifferentiated cells of the employed iPSC line bound LTL, so we selected megalin as a more suitable flow cytometry marker to assess the identity of our iPSC-derived PTL. Like Chandrasekaran et al. already
<figure><img src="image_20.png" /><figcaption>**Fig. 3.** Curcumin co-administration ameliorates polymyxin B-induced nephrotoxicity. Dynamic or static PT models were treated with 50 µM polymyxin B (PMB) and/or 5 µM curcumin (CCM) for 48 h starting on day 14 + 4. (A-C, E, F) Each colour (black, pink, green) represents one independent experiment. Large squares depict the mean of each experiment, while small circles represent the individual replicates (N = 3 independent experiments with seven replicates each for A-C, 1-3 replicates for D, and 3-4 replicates for E and F; \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001). (A, B) Lactate secretion and LDH release measured in medium supernatant on day 14 + 6. (C) TEER measured on day 14 + 6 normalized to the respective vehicle control. (D) Reactive oxygen species (ROS) MFI presented as % of vehicle measured on day 14 + 6. Data are presented as mean ± s.d. (E, F) Gene expression levels of MT2A and MT1F detected by RT-qPCR. Data are presented as mean delta threshold cycle (dCT). Abbreviations: LDH: lactate dehydrogenase, TEER: transepithelial electrical resistance, PMB: polymyxin B, CCM: curcumin, ROS: reactive oxygen species, MFI: mean fluorescence intensity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

described, we detected megalin expression from day 3 onwards (Chandrasekaran et al., 2021). The three independent differentiations progressed at individual rates, with megalin expression reproducibly peaking on day 14, while expression levels exhibited a high deviation on day 7 and day 21. The varying speed at which the cells matured into PTL and dedifferentiated afterwards was a result of the inherent heterogeneity of iPSCs (Hayashi et al., 2019). Although this is generally tolerated, we aimed to cryopreserve a large quantity of PTL in order to reduce the number of factors that would impact the outcome of our downstream nephrotoxicity assay. We therefore cryopreserved day 14 PTL and showed that freshly differentiated PTL of the same differentiation exhibited a similar megalin-positive population as frozen and thawed PTL. The reduced megalin-positive population observed on day 28, however, indicated a loss of phenotype, greatly limiting the duration of nephrotoxicity assays. Interestingly, the culture of PTL on CC inserts not only prevented this loss of phenotype, but enriched the megalin-positive population, thus allowing for a culture duration of at least 14 + 12 days. We hypothesize that polarization along the apicobasal axis resulting from culturing PTL on CC inserts rather than in multi-well plates (Piroli et al., 2019; Vaidziulytė et al., 2019; Chung et al., 2018; Meijer et al., 2024) prevented dedifferentiation by providing essential physiological cues.

When cultivated on a CC insert, PTL formed a monolayer of cells that continued to proliferate over time. PTL did not form a tight barrier two days after seeding, but by day 14 + 4, TEER values averaged 2580 ± 240 Ω x cm². These TEER values greatly exceeded the TEER of other in

vitro PT models, indicating a much tighter barrier. Within the kidney, the tightest barrier is encountered in the collecting duct (Denker and Sabath, 2011). Paracellular permeability decreases from the PT towards the collecting duct as the expression profiles of claudins change. The low expression of pore-forming claudin 2, high expression of barrier-forming claudin 3 and unusually high TEER values suggest that the developed PT model exhibited barrier properties that are usually associated with the collecting duct (Kiuchi-Saishin et al., 2002; Hou et al., 2013). This is further in line with the lack of *AQP1* expression and the high expression levels of *AQP3*, which is usually expressed in the collecting duct (Knepper et al., 1996).

We further demonstrated that our PT model exhibited primary cilia, which is crucial to detect shear stress and thus essential when examining the effect of perfusion on our model (Raghavan and Weisz, 2016). *In vivo*, the apical side of PT cells is continuously perfused and experiences shear stress estimated to range from 0.5 dyne/cm² to 1.2 dyne/cm² in healthy individuals (Ross et al., 2021). PT models cultured within the HUMIMIC Chip2 24-well, however, are exposed to 0.2 × 10⁻³ dyne/cm² to 1.8 × 10⁻³ dyne/cm² (Hübner et al., 2018). Despite the fact that the shear stress the cells experience in the employed MPS is significantly lower than *in vivo*, it affected the cells nevertheless, as also known from other studies applying subphysiological shear stress (Specioso et al., 2022). We observed lower lactate production in dynamic PT models than in static models, which, together with a similar glucose consumption, indicated a switch to aerobic glycolysis in dynamic models. Transitioning from aerobic to anaerobic metabolism is a common sign of
<figure><img src="image_21.png" /><figcaption>**Fig. 4.** Exposure to subtoxic cisplatin and cyclosporin A concentrations. Three concentrations of cisplatin (CP; 2.5 µM, 5 µM, 10 µM) and cyclosporin A (CsA; 5 µM, 10 µM, 20 µM) were applied to dynamic (circles, solid lines) or static (triangles, dotted lines) PT models every 48 h for eight days. Vehicle controls are shown in black, while the low concentration of each compound is shown in rose, the medium concentration in pink and the high concentration in magenta. (A-D) LDH release over time for dynamic (A, B) and static (C, D) PT models treated with cisplatin (A, C) or cyclosporin A (B, D). Data are presented as mean ± s.d. (n = 4 replicates). (E, F) TEER measured on day 14 + 12 normalized to the respective vehicle control (n = 4 replicates). (G, H) Heat maps showing expression levels of metallothioneins detected by bulk RNA sequencing on day 14 + 12. Data are normalized to vehicle controls and presented as mean log₂ fold change with padj < 0.05 (n = 4 replicates). Abbreviations: LDH: lactate dehydrogenase, TEER: transepithelial electrical resistance, CP: cisplatin, CsA: cyclosporin A. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

cellular stress that is primarily caused by hypoxia and nutrient limitation (Vander Heiden et al., 2009). The constant perfusion of cell culture medium within MPS enhances the availability of oxygen and nutrients to cells that can thereby transition into an aerobic state. The elevated levels of LDH secreted by static models further underlined the increased stress

cells experienced and responded to with cellular damage (Kumar et al., 2018). Finally, we noticed that dynamic models exhibited a lower deviation of glucose consumption, lactate production, and LDH release values among three independent experiments than static models. The standardized design of the MPS guarantees that all models experience
the same environment, whereas multi-well plates are affected by the edge effect. The outer wells of multi-well plates are more prone to evaporation, and metabolic activity of cells cultured in outer wells of a 96-well plate is reported to be reduced by 35 % compared to cells cultured in central wells (Mansoury et al., 2021).

Lastly, we examined expression levels of characteristic PT markers in
dynamic and static day 14 + 12 PT models and normalized them to day
14 + 2 PT models to examine the impact of culture duration and
perfusion. Bulk RNA sequencing revealed that the extended culture
duration had a stronger effect on the expression of the examined
markers than their dynamic cultivation. For most of the selected
markers, expression levels of dynamic and static samples were similar.
Although some critical SLC-transporters were not expressed, other SLC-
transporters and various ABC-transporters were expressed in dynamic
and static PT models. Since many in vitro PT models struggle with the
expression of certain SLC-transporters, particularly organic anion
transporters, unless they are transduced to overexpress these proteins,
the lack of expression of SLC-transporters was not unexpected
(Chandrasekaran et al., 2021; Nieskens et al., 2016; Aschauer et al.,
2015; Tobin et al., 2023). Chandrasekaran et al. did not detect the
expression of multiple essential transporters, including organic cation
transporter 2 (OCT2), by RNA sequencing, while a consecutive study by
Meijer et al. nevertheless demonstrated the presence and functionality of
OCT2 by immunofluorescence, western blotting, and a directional
transport assay (Chandrasekaran et al., 2021; Meijer et al., 2024). This
suggests that, in addition to the known challenges PT models face with
organic cation and anion transporter expression, mRNA levels may not
accurately reflect the expression and activity of certain transporters, as
has also been reported for ABCB1 (Kosztyu et al., 2015).

After characterizing our healthy PT model, we assessed its use in
predicting drug-induced proximal tubule injury. We applied three drugs
known to cause drug-induced AKI with distinct entry routes into the PT
to evaluate our model's response to different nephrotoxins. Commonly
prescribed drugs that cause AKI in over 30 % of treated patients include
PMB, CP, and CsA (Oliota et al., 2019; Zhu et al., 2023; da Silva et al.,
2014). PMB, a last-line antibiotic against multidrug-resistant gram-
negative pathogens, undergoes extensive glomerular filtration and is
subsequently reabsorbed into PT cells through the apical endocytic re-
ceptor megalin. The chemotherapeutic agent CP is administered in the
treatment of various types of cancer and is imported into PT cells
through the basolateral transporter OCT2. CsA, an immunosuppressant
widely used to prevent transplant rejection and treat autoimmune dis-
eases, accumulates within PT cells and inhibits its own efflux, with its
entry mechanism remaining unclear. Despite their differing entry routes,
these drugs share similar mechanisms inducing toxicity, including
oxidative stress, mitochondrial dysfunction, autophagy, and apoptosis
(Wu et al., 2018; Zavascki and Nation, 2017; Wilmes et al., 2015). We
used three concentrations of each drug and selected the concentration
ranges based on literature. Although the EC₅₀ greatly depends on the
employed cell source and its transporter expression, we considered
studies using perfused in vitro systems with PT cells of various origin and
positioned ourselves in the lower range of applied concentrations to
include subtoxic concentrations.

The selected concentration range of PMB caused a linear
concentration-dependent toxic effect in dynamic and static PT models
that we observed in multiple readouts. With an increasing concentration
of PMB, the viability and barrier integrity decreased, while the LDH
release increased. Bulk RNA sequencing revealed that genes encoding
MTs were among the most differentially expressed genes in all condi-
tions. MTs are small, cysteine-rich proteins that protect cells from
oxidative stress and bind physiological (Zn, Cu) and toxic (Cd, Hg) heavy
metals. When oxidized, the thiolate clusters release metal ions, which
are thereby available to metal-dependent enzymes and transcription
factors. In addition to their role in heavy metal detoxification, MTs
therefore also modulate the activity of numerous Zn-dependent proteins
(Zulijani et al., 2021). With their expression being regulated by

oxidative stress and heavy metals, an upregulation is usually associated
with improved cell survival whilst also indicating that the cells are
stressed. Acharya et al. employed plasma MT levels to predict whether
patients suffering from acute-on-chronic liver failure would develop AKI
within the next 7 days (Acharya et al., 2023). Furthermore, Weber et al.
detected an increased expression of MT1E, MT1F, MT1G, MT1H, MT1M,
MT1X, and MT2A in primary human PT cells cultured in an MPS when
exposed to 50 µM PMB (Weber et al., 2018). We observed an upregu-
lation of MT1E, MT1F, MT1G, MT1H, MT1M, MT1X, and MT2A in dy-
namic PT models in response to all tested PMB concentrations. In static
PT models, however, we did not detect any upregulation of MT1G,
MT1H, and MT1M. These genes were specifically affected by MPS cul-
ture, with an MT1M fold change of 48.5, 68.6, and 55.7 in dynamic and
no effect in static PMB-treated PT models. This clustering of MT1G,
MT1H and MT1M was also detected upon CP and CsA exposure.

For CP and CsA, we did not observe linear concentration-dependent
toxicity with the applied concentrations. LDH values were low overall,
except in static PT models exposed to 20 µM CsA. TEER values, on the
other hand, showed a linear decrease with an increasing CP concen-
tration in dynamic PT models. In static models, however, the barrier
appeared to tighten first and then loosen when treated with 10 µM CP. A
similar phenomenon was observed in dynamic and static PT models
treated with CsA. 5 µM and 10 µM CsA led to a slight increase in TEER
values, whereas 20 µM CsA disrupted the barrier. Homan et al. also
described a tightening of their cell layer upon treatment with CsA, fol-
lowed by a massive loss of barrier integrity in cells treated with
increasing CsA concentrations, while Vormann et al. described a reor-
ganization of actin filaments in PT cells treated with low CP concen-
trations (Homan et al., 2016; Vormann et al., 2018). Wilmes et al.
further reported increased TEER values and a decreased expression of
pore-forming claudin 2 in a static PT model exposed to CsA (Wilmes
et al., 2014). We did not, however, detect changes in claudin expression
profiles in response to CsA or CP treatment. We hypothesize that all
employed concentrations of CP and CsA were subtoxic and therefore did
not elicit a strong LDH release, but nevertheless injured our PTL. Shear
stress, CsA, and CP can all induce the reorganization of the cytoskeleton
through different mechanisms, which can enhance or diminish each
other. This resulted in varying effects of CP and CsA on the barrier
integrity of dynamic and static PT models. We also attributed the toxic
effect of 20 µM CsA on static, but not dynamic PT models to these in-
teractions (Duan et al., 2008; Faul et al., 2008; Descazeaud et al., 2012;
Arany et al., 2012).

Again, bulk RNA sequencing showed that MTs were among the most
differentially expressed genes upon xenobiotic exposure. Interestingly,
the medium and high CP and CsA concentrations led to an upregulation
of at least one of the metallothionein-encoding genes MT1E, MT1F,
MT1X, and MT2A in dynamic PT models, but not in static models. For
both CP and CsA, an upregulation of MTs that accompanies nephro-
toxicity or hepatotoxicity is reported in rodents (Rezzani et al., 2005;
Sogawa et al., 2013; Tikoo et al., 2009). We speculate that the genes
MT1E, MT1F, MT1X, and MT2A are a sensitive group of biomarkers
indicating kidney injury, and that their expression is impacted by
perfusion.

Lastly, we investigated the impact CCM co-administration had on
PMB-induced nephrotoxicity in our PT model. Supplementation of CCM
can alleviate nephrotoxic effects of colistin and other nephrotoxic
compounds in rodents by inhibiting oxidative stress, mitochondrial
dysfunction, inflammation, and apoptosis (Vazin et al., 2020; Dai et al.,
2020; Motaharinia et al., 2019), so we examined whether the developed
perfused system could replicate this finding. Co-administration of CCM
with PMB did in fact reduce LDH secretion and improve barrier integ-
rity, indicating an alleviation of PT injury. While the beneficial effect of
CCM is usually attributed to its antioxidative properties, a reduction of
ROS was not confirmed. A mechanism involving the temporary upre-
gulation of MTs and subsequently increased cytoprotective defenses
cannot be ruled out (Wahyudi et al., 2022). After 48 h, however, none of
the examined MTs were upregulated by CCM supplementation.

Together, these findings demonstrate that our perfused iPSC-derived PT model is well-suited for *in vitro* studies investigating nephrotoxicity and its prevention if the presented limitations regarding SLC-transporter expression are considered. It is robust, easy to use, compatible with personalized medicine, and versatile, as it can also be used in multi-organ cultures. We further provide evidence that metallothioneins are conserved biomarkers indicating PT injury and that their expression is affected by MPS culture. Looking ahead, our model can be extended in two important directions. First, by using multiple iPSC lines to accommodate a broad genetic background or patient-derived iPSCs to enable personalized medicine, and second, by adding other organ models, such as liver spheroids, into the MPS to include xenobiotic metabolization in the assay.

# 4. Material and methods

## 4.1. iPSC culture

The hiPSC line TISSUi007-A (Ramme et al., 2021) (TissUse GmbH, Berlin, Germany) was cultured in StemMACS iPS-Brew XF (Miltenyi Biotec, Bergisch Gladbach, Germany) on Matrigel GFR-coated (8 µg/cm²; Corning, Corning, NY, USA) multi-well plates. Cells were passaged once or twice a week using 0.5 mM EDTA (Corning, Corning, NY, USA) with daily medium exchanges in between.

## 4.2. Differentiation into proximal tubule-like cells (PTL)

To generate PTL from iPSCs, the protocol by Chandrasekaran et al (Chandrasekaran et al., 2021; Meijer et al., 2023), was employed. iPSCs of 70–80 % confluence were detached as single cells using Accutase (Corning, Corning, NY, USA) and were seeded onto Matrigel GFR-coated 6-well plates with a density of $4.0 \times 10^4$ cells/cm² in medium 1 (1:1 mixture of DMEM without glucose, l-glutamine and sodium pyruvate (Corning, Corning, NY, USA) and Ham's F-12 (Corning, Corning, NY, USA), 2 mM Glutagro (Corning, Corning, NY, USA), 5 µg/mL insulin (PAN-Biotech, Aidenbach, Germany), 5 µg/mL transferrin (Sigma-Aldrich, St. Louis, MO, USA) and 5 ng/mL sodium selenite (MP Bio-medicals, Irvine, CA, USA)) supplemented with 1 µM TTPNB (Tocris Bioscience, Bristol, UK), 3 µM CHIR99021 (Sigma-Aldrich, St. Louis, MO, USA) and 10 µM ROCK-inhibitor (Cayman Chemicals, Ann Arbor, MI, USA). After 42 h, the medium was exchanged with medium 1 supplemented with 1 µM TTPNB. Further 30 h later, the medium was switched to medium 2 (medium 1 with 36 ng/mL hydrocortisone 21-hemisuccinate (Sigma-Aldrich, St. Louis, MO, USA), 10 ng/mL EGF (Miltenyi Biotec, Bergisch Gladbach, Germany) and 1 % penicillin/streptomycin (Corning, Corning, NY, USA)) supplemented with 10 ng/mL FGF9 (Miltenyi Biotec, Bergisch Gladbach, Germany). After 48 h, and every 2–3 days henceforward, the medium was exchanged with medium 2. The cells were passaged once on day 14 and were either cryopreserved, seeded into multi-well plates, or onto cell culture inserts.

For passaging, the cells were washed with PBS- once and were then incubated with 0.25 % Trypsin with 2.21 mM EDTA (Corning, Corning, NY, USA) at 37 °C for 5 min. After transferring the cell suspension into a centrifugation tube containing PBS+ with 2 % FCS (Corning, Corning, NY, USA), the suspension was centrifuged. The pellet was resuspended in medium 2 supplemented with 1 µM GW788388 (LKT Labs, St. Paul, MN, USA), 10 µM ROCK-inhibitor and 2 % FCS, and the single cell suspension was seeded into Matrigel GFR-coated multi-well plates ($1.2 \times 10^5$ cells/cm²) or onto the lower side of Matrigel GFR-coated cell culture inserts ($4.8 \times 10^5$ cells/cm²). After 4 h and every 2–3 days henceforward, the medium was exchanged with medium 2 supplemented with 1 µM GW788388.

In order to cryopreserve PTL on day 14, cells were harvested as described during passaging. The cell pellet, however, was resuspended in freezing medium consisting of 80 % medium 2, 10 % DMSO (VWR,

Radnor, PA, USA) and 10 % FCS to yield a concentration of $2 \times 10^6$ cells/mL. Aliquots of 1 mL were prepared, frozen and stored in liquid nitrogen. When thawing cryopreserved PTL, cryovials were carefully defrosted and the cell suspension was quickly transferred into medium 2. After centrifugation, the pellet was resuspended in medium 2 supplemented with 1 µM GW788388, 10 µM ROCK-inhibitor and 2 % FCS. PTL were either seeded into multi-well plates or onto cell culture inserts. After 4 h, the medium was exchanged with medium 2 supplemented with 1 µM GW788388. Every 2–3 days henceforward, the medium was exchanged using medium 2 containing 1 µM GW788388.

Minor adaptations to the protocol by Chandrasekaran et al. included the seeding densities and cultivating the cells on Matrigel GFR.

## 4.3. Cell culture (CC) insert culture

Millicell standing inserts (polycarbonate membrane, pore size: 0.4 µm, surface area: 0.6 cm²; PIHP01250; Merck Millipore, Burlington, MA, USA) were coated prior to cell seeding. Since the cells were cultured on the lower side of the CC insert, the inserts were turned upside down and a 120 µL drop of coating solution (76 µg/mL Matrigel GFR) was positioned on the membrane. After 4 h of incubation at RT, CC inserts were transferred into a 24-well plate containing medium 2 and were stored at 37 °C for up to 2 days. Prior to cell seeding, CC inserts were turned upside down again. A 120 µL drop of cell suspension containing $2.4 \times 10^6$ cells/mL in medium 2 supplemented with 1 µM GW788388, 10 µM ROCK-inhibitor and 2 % FCS was positioned on the membrane. The cells were allowed to adhere for 2–4 h at 37 °C before CC inserts were transferred into a 24-well plate containing 500 µL/well of medium 2 supplemented with 1 µM GW788388. An additional 250 µL of medium was added to the apical compartment of the insert. Every 2–3 days henceforward, the medium was exchanged.

## 4.4. HUMIMIC Chip2 24-well culture

After an equilibration phase of 2 days after seeding (day 14 + 2), CC inserts were transferred into the 24-well compartment of the HUMIMIC Chip2 24-well (TissUse GmbH, Berlin, Germany). The total medium volume in each circuit amounted to 750 µL, with 250 µL below the insert, 250 µL in the apical compartment of the insert and 250 µL in the 96-well compartment of the chip. The chips were connected to a HUMIMIC Starter (TissUse GmbH, Berlin, Germany) and were pumped with a frequency of 120 bpm and pressure and vacuum set to ±600 mbar. The entire medium volume was exchanged every 48 h, and samples for supernatant analysis were collected during each medium exchange. For dynamic and static PT models, the apical and basolateral supernatant samples were pooled, amounting to a total volume of 750 µL. The concentration of glucose, lactate and LDH in those medium supernatant samples was measured using an Indiko clinical chemistry analyzer (Thermo Fisher Scientific, Waltham, MA, USA). Substance exposure commenced on day 14 + 4 and was performed with every medium exchange henceforward.

At the end of the culture, the cell culture inserts were removed from the chips and were used for TEER measurements using an EVOM2 instrument with chopstick electrodes (both World Precision Instruments, Saratosa, FL, USA). Afterwards, the PT models were subjected to flow cytometry for megalin and ROS measurement, RT-qPCR, bulk RNA sequencing, immunofluorescence staining, live/dead staining or the CellTiter-Glo 3D viability assay. Static controls were treated similarly to dynamic samples. They were cultured in 24-well plates with 500 µL of medium below the CC insert and 250 µL in the apical compartment of the insert.

## 4.5. Repeated exposure nephrotoxicity assay

After two days of equilibration within the MPS (day 14 + 4), the PT models were treated with polymyxin B (25 µM, 50 µM, 100 µM; CAS:
1405-20-5; Sigma-Aldrich, St. Louis, MO, USA), cisplatin (2.5 µM, 5 µM, 10 µM; CAS: 15663-27-1; Sigma-Aldrich, St. Louis, MO, USA), or cyclosporin A (5 µM, 10 µM, 20 µM; Sandimmun, Novartis, Basel, Switzerland). To emulate a repeated dosing regimen, the cells were treated with the compounds every 48 h for 8 days until the end of the culture on day 14 + 12. The static controls were treated similarly. Compounds were prepared as listed in Supplementary Table 2.

## 4.6. Polymyxin B-curcumin co-administration assay

To assess whether curcumin co-administration could ameliorate polymyxin B-induced nephrotoxicity, an acute exposure was emulated. PT models were transferred into the MPS on day 14 + 2 and were pre-incubated with 5 µM curcumin (CAS: 458-37-7; Thermo Fisher Scientific, Waltham, MA, USA). On day 14 + 4, 50 µM polymyxin B and/or 5 µM curcumin were applied once. The culture was terminated on day 14 + 6. The static controls were treated similarly. Compounds were prepared as listed in Supplementary Table 2.

## 4.7. Flow cytometry

Cells were harvested by washing PTL with PBS⁻ and incubating them with 0.25 % Trypsin with 2.21 mM EDTA at 37 °C for 5 min. The cell suspensions were diluted with PBS⁺ containing 2 % FCS and then centrifuged. The detached cells were either used for ROS measurement, or they were fixed prior to analyzing megalin expression. For fixation, cells were incubated with 4 % w/v formaldehyde (Thermo Fisher Scientific, Waltham, MA, USA) at RT for 20 min, followed by two washing steps with PBS⁺.

Megalin staining was performed on detached and formaldehyde-fixed PTL. They were resuspended in blocking buffer containing 5 % Human TruStain FcX (BioLegend, San Diego, CA, USA). After incubating the cells for 10 min, they were centrifuged and the cell pellets were resuspended in staining solution (PBS⁺ with 3 % FCS, 0.1 % Triton X-100, 1 µg/1 million cells Human Megalin/LRP2 Alexa Fluor 647-conjugated antibody, or 1 µg/1 million cells Mouse IgG1 Alexa Fluor 647-conjugated antibody as an isotype control (both Bio-Techne, Minneapolis, MN, USA)). The cells were centrifuged after 20 min of incubation at 4 °C and the pellets were resuspended in PBS⁺ with 3 % FCS for flow cytometry measurement using a MACSQuant 16 (Miltenyi Biotec, Bergeisch Gladbach, Germany).

The DCFDA/H2DCFDA - cellular ROS assay kit (Abcam, Cambridge, UK) was employed to quantify reactive oxygen species in living cells. The detached cells were resuspended in staining solution (50 µM DCFDA in 1× dilution buffer) and were incubated at 37 °C for 30 min. After centrifugation, the pellets were resuspended in medium 2 supplemented with 1 µM GW788388 and 50 µM polymyxin B with or without 5 µM curcumin. After 45 min of incubation at 37 °C, the cells were centrifuged, resuspended in PBS⁺ with 3 % FCS, and measured.

## 4.8. RNA isolation and RT-qPCR

PTL cultured on CC inserts were washed with PBS⁻ and were then lysed. RNA isolation was performed using the Nucleospin RNA XS Kit according to the manufacturer's instructions (Machery-Nagel, Düren, Germany). The reverse transcription of the extracted RNA into cDNA was performed using the TaqMan Reverse Transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) and a QuantStudio 5 RT-qPCR LightCycler (Thermo Fisher Scientific, Waltham, MA, USA). RT-qPCR was conducted using the SensiFAST SYBR Lo-Rox kit (Bioline, Memphis, TN, USA) and a QuantStudio 5 RT-qPCR LightCycler. TBP or EEF1A1 were used as endogenous control genes. The ΔΔCt method was used, with TBP or EEF1A1 serving as endogenous control genes and dynamic or static vehicle controls being used for normalization. Primer sequences are listed in Supplementary Table 1.

## 4.9. RNA isolation and bulk RNA sequencing

RNA was extracted according to the manufacturer's instructions using the QIAGEN RNEasy Plus Mini Kit (QIAGEN, Venlo, The Netherlands). For 3' digital gene expression profiling, the RNA sequencing protocol was performed according to our implementation of the protocol from Soumillon et al (Kilens et al., 2018; Soumillon et al., 2014). Briefly, the libraries were prepared from 10 ng of total RNA. The mRNA poly(A) tails were tagged with universal adapters, well-specific barcodes, and unique molecular identifiers during template-switching reverse transcription. Barcoded complementary DNAs from multiple samples were then pooled, amplified, and tagmented using a transposon fragmentation approach, which enriches for 3' ends of complementary DNA. A library of 350 to 800 bp was sequenced on Nova seq6000 at the GenoA platform facility (Nantes University, Nantes, France). An average of 5 million 75 bp single-end reads were obtained for each sample. Samples were demultiplexed and aligned to the hg38 genome using the DGE bioinformatics pipeline. Post-sequencing, data demultiplexing was performed using Illumina bcl2fastq. The resulting FASTQ files were analyzed using the 3' Sequencing RNA Profiling pipeline (Charpentier et al., 2021; Chaumette et al., 2022) and DESeq2 R package (Love et al., 2014).

## 4.10. Immunofluorescence (IF) staining

First, PTL were incubated with 4 % PFA at RT for 20 min, followed by two washing steps with PBS⁺. For the staining, the cells were incubated over night at 4 °C with a primary antibody solution (PBS⁺ with 1 % goat serum, 0.1 % Triton X-100, and a 1:100 dilution of primary antibodies binding ZO-1, ARL13b (both Proteintech, Rosemont, IL, USA), or Ki67 (eBioscience, San Diego, CA, USA)). The cells were washed twice with PBS⁺ and were then incubated with a secondary antibody solution (PBS⁺ with 500 µg/mL DAPI and a 1:200 dilution of Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, DyLight 594 and Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, DyLight 488 (both Thermo Fisher Scientific, Waltham, MA, USA)) for 1 h at 37 °C. The cells were washed twice with PBS⁺ before imaging using a Keyence BZ-X700E fluorescence microscope or a Nikon A1RS confocal scanning laser microscope.

## 4.11. Live/dead staining

A staining solution (medium 2 with 5 µg/mL CellTrace Calcein Red-Orange, AM (Thermo Fisher Scientific, Waltham, MA, USA) and a 1:1000 dilution of CellTox Green (CellTox Green Cytotoxicity Assay; Promega, Madison, WI, USA)) was added to PT models. After 20 min of incubation at 37 °C, the staining solution was aspirated and the cells were washed twice with medium 2. Images were acquired using the Keyence BZ-X700E fluorescence microscope. The mean fluorescence intensity (MFI) was quantified using ImageJ and was normalized to the total area.

## 4.12. CellTiter-Glo 3D cell viability assay

The CellTiter-Glo solution was prepared by mixing equal parts of CellTiter-Glo 3D cell viability assay (Promega, Madison, WI, USA) reagent and medium 2. CC inserts were transferred into a 24-well plate containing 400 µL CellTiter-Glo solution. The plate was incubated on an orbital shaker (30 rpm) in the dark at RT for 5 min and was further incubated in the dark at RT without shaking for 15 min. The samples were diluted in medium 2 and their luminescence was measured using a CLARIOStar plate reader.

## 4.13. Statistical analyses

The GraphPad Prism software was employed to plot data and
perform comparative analyses between the means of different condi-
tions. To compare two paired means, a paired two-tailed Student's t-test
was used. A repeated measure two-way ANOVA was performed when
comparing means of data from three independent experiments with two
independent variables (e.g. dynamic/static culture and treated/un-
treated condition). Each group was compared with every other group
using uncorrected Fisher's LSD.

Funding

This project was funded by the European Union under Grant
Agreement No. 964452 (BIRDIE) and 964537 (RISK-HUNT3R). RISK-
HUNT3R is part of the ASPIS cluster. Views and opinions expressed
are those of the authors and do not necessarily reflect those of the Eu-
ropean Union. Neither the European Union nor the granting authority
can be held responsible for them.

CRediT authorship contribution statement

**Michelle Lechtenberg:** Conceptualization, Methodology, Investi-
gation, Formal analysis, Data curation, Visualization, Writing – original
draft. **Coraline Chêneau:** Conceptualization, Methodology, Investiga-
tion, Formal analysis, Writing – review & editing. **Kevin Riquin:** Formal
analysis, Writing – review & editing. **Leopold Koenig:** Methodology,
Writing – review & editing. **Carlos Mota:** Conceptualization, Writing –
review & editing. **Franck Salary:** Conceptualization, Writing – review
& editing. **Eva-Maria Dehne:** Supervision, Project administration,
Conceptualization, Writing – review & editing.

Declaration of competing interest

M.L., L.K., and E.D. are employees of TissUse GmbH. All authors
declare they have no competing interests.

Acknowledgements

We thank F. Coulon, K. Hunt, J. Wiest, and R. Stolberg for their
technical assistance, as well as J. Dietzfelbinger for creating the graph-
ical illustration of Fig. 1B.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.tiv.2025.106038.

Data availability

Data will be made available on request.

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