Received: 30 October 2023

Revised: 11 January 2024

Accepted: 17 January 2024

DOI: 10.1002/biot.202300587

RESEARCH ARTICLE

The choice of ultra-low attachment plates impacts primary
human and primary canine hepatocyte spheroid formation,
phenotypes, and function
Chen Xing1
Aurino Kemas1
Evgeniya Mickols2
Per Artursson2
Volker M. Lauschke1,3,4

Kathrin Klein3,4

1

Department of Physiology and Pharmacology,
Karolinska Institute, Stockholm, Sweden

2

Department of Pharmacy, Uppsala
University, Uppsala, Sweden

3

Abstract
Organotypic three-dimensional liver spheroid cultures in which hepatic cells retain
their molecular phenotype and functionality have emerged as powerful tools for pre-Dr Margarete Fischer-Bosch Institute ofClinical Pharmacology, Stuttgart, Germany

clinical drug development. In recent years a multitude of culture systems have been4

developed; however, a thorough side-by-side benchmarking of the different meth-

University of Tübingen, Tübingen, Germany

ods is lacking. Here, we compared the performance of ten different 96- and 384-well
Correspondence
Volker M. Lauschke, Department of Physiology
and Pharmacology, Karolinska Institutet,
SE-171 77 Stockholm, Sweden.
Email: volker.lauschke@ki.se

microplate types to support spheroid formation and long-term culture. Specifically, weevaluated differences in spheroid formation kinetics, viability, functionality, expression patterns, and their utility for hepatotoxicity assessments using primary humanhepatocytes (PHH) and primary canine hepatocytes (PCH). All 96-well plates enabled

Funding information
Swedish Research Council, Grant/Award
Numbers: 2019-01837, 2021-02801; Ruth och
Richard Julins Stiftelse, Grant/Award Number:
2021-00158

formation of PHH liver spheroids, albeit with differences between plates in spheroid
size, geometry, and reproducibility. Performance of different 384-wells was less consistent. Only 6/10 microplates supported the formation of PCH aggregates. Interestingly,
even if PCH aggregates in these six microplates were more loosely packed than PHH
spheroids, they maintained their function and were compatible with long-term pharmacological and toxicological assays. Overall, Corning and Biofloat plates showed thebest performance in the formation of both human and canine liver spheroids withhighest viability, most physiologically relevant phenotypes, superior CYP activity andlowest coefficient of variation in toxicity assays. The presented data constitutes a valuable resource that demonstrates the impacts of current ultra-low attachment plates onliver spheroid metrics and can guide evidence-based plate selection. Combined, theseresults have important implications for the cross-comparison of different studies andcan facilitate the standardization and reproducibility of three-dimensional liver culture
experiments.

Abbreviations: ANOVA, analysis of variance; APAP, acetaminophen; BSEP, bile salt export pump; Cmax , peak serum concentration; CPZ, chlorpromazine; CV, coefficient of variation; CYP,
cytochrome P450; DMSO, dimethyl sulfoxide; ECM, extracellular matrix; FBS, fetal bovine serum; LC/MS-MS, liquid chromatography tandem mass spectrometry; NAM, new approach
methodology; NAPQI, n-acetyl-p-benzoquinonimine; PCA, principal component analysis; PCH, primary canine hepatocytes; PHH, primary human hepatocytes; SEM, standard error of the mean;
TRO, troglitazone; ULA, ultra-low attachment.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2024 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.
Biotechnol. J. 2024;19:2300587.
https://doi.org/10.1002/biot.202300587

www.biotechnology-journal.com

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XING ET AL.

KEYWORDS

cell culture, liver spheroids, medical biotechnology, metabolism, 3D cell culture

1

INTRODUCTION

absorption of small molecules [28] , thereby changing cellular exposure levels. To optimize spheroid cultures and improve cross-study and

The discovery of safe and effective medicines relies on the accurate

cross-center comparability, it is thus imperative to be aware of theclinical translation of findings based on preclinical models. The liver

differences caused by plate parameters.

is among the organ systems most commonly affected by adverse drug

To provide practical guidance for plate selection, we here comparedreactions, and hepatotoxicity is a main reason for the attrition of drug

the performance of ten different 96- and 384-well ULA microplates.

candidates and the withdrawal of drugs from the market.[1] Addition-

We generated spheroids from primary human hepatocytes (PHH) andally, the liver is a key determinant of the pharmacokinetics of newly

found considerable differences in spheroid formation kinetics, viability,

developed compounds and accurate prediction of formed metabolites,

functionality, gene expression profiles, and their utility in hepatotoxic

drug bioavailability, and clearance constitute integral steps in preclini-assessments. To facilitate utility for translational research, we fur-cal drug development. Animal tests have long been an essential part ofthermore generated primary canine hepatocyte (PCH) spheroids andthe preclinical testing pipeline with an estimated 190 million animals

analyzed inter-species differences. These investigations provide a basis

used in medical research globally per year [2] . Regulatory guidelines

for evidence-based microplate selection for liver spheroid studies andrequire safety and tolerability data from at least one rodent and one

aspire to promote standardization of ULA plate parameters.

non-rodent species, before first-in-man trials. Of these, Beagle dogs
are the most popular for preclinical safety testing and pharmacokinetic
studies.[3] However, it is becoming increasingly clear that animals are

2

MATERIAL AND METHODS

overall poor predictors of human drug efficacy and safety [4–6] . Thisawareness has resulted in the increased adoption of new approach

2.1

Spheroid culture

methodologies (NAMs) in preclinical pharmacology and toxicology[7] . These developments are further supported by recent changes in

Cryopreserved PHH were obtained from patients undergoing liver

legislation that end the blanket mandate for animal testing [8] .

resection at the Department of Surgery at Uppsala University Hospital,

To facilitate early prediction of hepatotoxicity and pharmacoki-

Sweden (Ethical Approval no. 2009/028, amended 2018/1108). PHH

netic properties, organotypic culture models of primary hepatocytes

were isolated as previously described [29] . In brief, the liver tissue washave emerged as versatile in vitro models [9–12] . Specifically, three-rinsed using Hypothermosol FRS (Biolife Solutions), cannulated, anddimensional liver spheroids are increasingly utilized as they allow for

perfused with collagenase and protease buffers to release cells fromthe maintenance of physiologically relevant phenotypes and functions

the tissue matrix. For cryopreservation, the hepatocytes were resus-

for multiple weeks [13–15] . Furthermore, liver spheroids have been

pended in CryoStor CS10 solution (BioLife Solutions) supplemented

well characterized as model systems for the prediction of toxicity

with 10% fetal bovine serum (FBS) and stored at -150◦ C until use. Cry-

[16,17] and clearance [18–20] where they outperform previous culture

opreserved PCHs from male Beagle dogs were commercially acquired

systems, such as sandwich or suspension cultures. Multiple differ-(BioIVT).

ent methodologies for liver spheroid culture have been described.

Spheroids were seeded and cultured as previously described [30] .

Spheroid formation techniques include hanging drops, agitation-basedSpecifically, cells were thawed in a 37◦ C water bath. Subsequently,

methods, as well as aggregation in ultra-low attachment (ULA) plates

the cell suspension was transferred to thawing medium (Williams E[21,22] . Particularly the latter enables the rapid, inexpensive and scal-

medium containing 27% isotonic Percoll) and centrifuged at 100 × g forable formation of thousands of spheroids of well-defined sizes in 96- or

10 min. The supernatant was aspirated, and hepatocytes were washed384-well plate formats. Once spheroids are formed, they can be main-

and resuspended in warm culture medium (Williams E medium withtained in different media with or without the addition of serum. For

2 mM L-glutamine, 100 units mL−1 penicillin, 100 µg mL−1 strepto-

media compositions, extensive careful benchmarking studies have con-mycin, 10 µg mL−1 insulin, 5.5 µg mL−1 transferrin, 6.7 ng mL−1 sodium

tributed to protocol standardization and have facilitated cross-study

selenite, 100 nM dexamethasone) with 10% fetal bovine serum (FBS).

reproducibility and comparability [23] . However, similar studies have

After cell counting, PHH were then seeded into ten different mod-not been conducted to compare different culture plates despite theels of 96- or 384-well ULA plates at a concentration of 1500 viablewide range of available products on the market.

PHH or PCH per well (Table S1). In 96-well plates, cells were cul-

Different plates differ in geometry, which in turn can impact

tured in 100 µL while spheroids were cultured in 80 µL of medium

spheroid formation kinetics and alter evaporation [24] . Furthermore,in 384-well plates. When the spheroids were sufficiently compact,

different coatings can alter surface properties and biomechani-medium was exchanged to serum-free culture medium. Notably, both

cal cues, which can influence viability, molecular phenotypes, andspheroid formation and maintenance occurs under scaffold-free condi-

stability.[25–27] Surface treatments and materials can also affect thetions. Spheroid exposures were started 7 days (PHH) or 10 days (PCH)18607314, 2024, 2, Downloaded from https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/biot.202300587 by Rwe Power Ag Frau Moers, Wiley Online Library on [19/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

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after seeding. Spheroids were maintained for up to 3 weeks with aacetonitrile and 0.1% formic acid (flowrate 0.5 mL min−1 ) on a Waters

medium change every 48 to 72 h.

BEH C18 column, 2.1 × 50 mm (1.7µm) at 60◦ C. Peaks were quantified
using MassLynx Software V4.2 with TargetLynx.

2.2

Gene expression analysis
2.6

Statistical analyses

RNA was isolated from ≥30 pooled spheroids per sample using theZymo Quick-RNA Microprep Kit (Zymo Research) and expression of

Statistical analyses were performed with GraphPad Prism (Version

selected genes was quantified using the Biomark HD system (Fluidigm)

9.5.1). No outlier elimination was performed, and all obtained datawith a FLEXsix integrated fluidic circuit (IFC, Fluidigm). The utilized

points were included into the analyses. p-values < 0.05 were consid-

gene expression assays are provided in Table S2. The gene expres-ered significant.

sion levels were calculated using the ΔΔCt method with two to threehousekeeper genes as reference.

3
2.3

Immunofluorescence

RESULTS

3.1
The choice of ULA microplates impacts PHH
spheroid formation kinetics and morphology

PHH spheroids were fixed overnight in 4% formaldehyde at 4◦ C, cryoprotected in 30% sucrose for 2 days at room temperature and then

To compare different microplates for liver spheroid formation, we first

embedded in Tissue-Tek OCT compound (Sakura). Spheroid cryosec-

monitored the aggregation of PHH into spheroids. Over the course

tions (8 µm) were stained for CYP3A4 (PAP011, 1:5000, Cypex) andof 7 days, PHH aggregated and compacted into spheroids with diam-albumin (sc51515, 1:200, Santa Cruz), and mounted in ProLong Gold

eters of 200 to 300 µm in almost all microplates (Figure 1A,B). The

antifade reagent with DAPI (ThermoFisher). Immunofluorescenceestablishment of three-dimensional liver spheroids as a standardized

images were acquired with an LSM 880 confocal microscope (Zeiss).

methodology in preclinical pharmacology and toxicology requires highreproducibility. To this end, it is crucial that cells assemble into a single spheroid in each well without the formation of satellites. Across

2.4

Assessment of cell viability

96-well plates, the cell suspensions aggregated predominantly (> 90%)
into one single spheroid (Figure 1C). In 384-well plates, aggregation

Cell viability was assessed using the CellTiter-Glo Luminescent

was overall less uniform and only Biofloat384 well plates showed uni-

Cell Viability Assay (Promega) on a SpectraMax iD3 Multi-Modeform formation of single spheroids. In contrast, in Greiner384 plates,

Microplate Reader (Molecular Devices). Chlorpromazine hydrochlo-cells formed multiple small spheroids with diameters ranging fromride (CPZ, Sigma) and troglitazone (TRO, Sigma) stock solutions were

70 to 100 µm, suggesting that these plates are of limited use forprepared in dimethyl sulfoxide (DMSO) while acetaminophen (APAP,

high-throughput pharmacological or toxicological experiments.

Sigma) was dissolved in culture medium. Exposure values (Cmax ) wereDuring aggregation, spheroid diameters decreased over time withobtained from the published literature [16] as follows: APAP = 136 µM,

similar kinetics across 96-well plates (Figure 1D). Among the 384-well

CPZ = 0.84 µM, and TRO = 6.08 µM.

plates, spheroids formed considerably faster in the Biofloat384 plateswith fully formed aggregates being visible after 3 days compared to
5 or 6 days in the other plate models (Figure 1E). Physical proximity

2.5

Metabolic profiling

may play an important role in the initial assembly of loose cell suspensions which depends on cell interaction through surface integrins [31] .

Single PHH spheroids were incubated for 4 h at 37◦ C with a cock-

The compact packing of PHHs after seeding facilitated by the specifictail of CYP substrates: 10 µM midazolam (CYP3A4), 5 µM dex-

geometry of the wells and the specific coating may thus account fortromethorphan (CYP2D6), 10 µM amodiaquine (CYP2C8), and 10 µM

the rapid organization of the cells into single spheroids in this plate

diclofenac (CYP2C9), while CPH were incubated with 10 µM midazo-

model.

lam (CYP3A4) and 5 µM dextromethorphan (CYP2D6). The reactions

To compare plate performance in imaging-based high-throughput

were stopped by mixing with ice-cold acetonitrile/water (60:40); 10

assays, we imaged entire plates with an automatic microscope on day

nM warfarin was used as internal standard. The respective metabo-

7 after seeding (Figure 1F). We did not observe systematic differences

lites 1-hydroxymidazolam, dextrorphan, N-desethylamodiaquine andwithin plates between columns or rows. Also, no clear edge effect was

4-hydroxy diclofenac were measured by liquid chromatography tan-observed. Overall, the morphology of spheroids agreed with the images

dem mass spectrometry (LC/MS-MS). Samples were centrifuged for 10

captured by manual acquisition. Spheroids could not be automatically

min at 4◦ C with 3500 × g, and supernatants were analyzed on a Waters

captured in PE384 plates since most spheroids were not located at

Acquity UPLC coupled to Waters Xevo TQ MS with electrospray ion-the center of the wells, likely due to the flat-bottom design. The otherization. Compounds were separated with a 2 min gradient elution ofplates were found to be compatible with automatic plate scans.

18607314, 2024, 2, Downloaded from https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/biot.202300587 by Rwe Power Ag Frau Moers, Wiley Online Library on [19/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

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XING ET AL.

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F I G U R E 1 Morphology and formation kinetics of human liver spheroids. (A) Time series of brightfield images showing PHH spheroid
formation. Scale bar = 200 µm. (B) Diameter of PHH spheroids on Day 7 after seeding in different types of plates. ** and **** indicate p < 0.01 andp < 0.0001 in an ANOVA test with Welch’s correction. (C) The fraction of wells in which cells aggregated into one single spheroid. * and ****
indicate p < 0.05 and p < 0.0001 in a Kruskal-Wallis test. Error bars indicate SEM. (D-E) Compaction kinetics of PHH over 7 days after seeding in
96-well plates (D) and 384-well plates (E). (F) High-throughput scan of spheroids in different types of plates. Only one representative row is shown
per plate.

3.2
Characterization of PHH spheroid phenotype,function, and stability in different ULA microplates

ATP levels were maintained throughout the whole investigated period.
In Biofloat384 and Biofloat96 plates, a significant drop in ATP levels could be observed at day 21 (p < 0.01). In all other plate models,

To assess the impact of the different microplates on long-term cul-

ATP content dropped significantly already after 14 days. In Greiner96,

ture of PHH spheroids, we first assessed cell viability for up to 3

PE96 and SBio384 plates, spheroid viability even dropped to lev-

weeks (Figure 2A,B). In Corning96 and Corning384 plates, the initialels < 25%, suggesting that these plates might be of limited use for18607314, 2024, 2, Downloaded from https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/biot.202300587 by Rwe Power Ag Frau Moers, Wiley Online Library on [19/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

4 of 12F I G U R E 2 Cell viability of human liver spheroids during 3 weeks of culture. (A-B) Viability is shown for PHH spheroids after 7 days, 14 days,and 21 days of culture in 96-well plates (A) and 384-well plates (B). n = 8 per plate and time point. *, **, and *** corresponds to p < 0.05, p < 0.01, andp < 0.001 in a one-way ANOVA, respectively. Error bars indicate SEM. The dotted lines indicate 80% cell viability. (C-D) The coefficient of variationis shown for the different replicates after 7 days, 14 days, and 21 days of culture in 96-well plates (C) and 384-well plates (D).

long-term liver spheroid experiments. The variability between individ-

In agreement with these observations, the expression signature ofual spheroids increased with culture time and was in general lower in

spheroids cultured in Nunc96 plates formed a unique cluster after PCA

96-well plates compared to 384-well plates (Figure 2C,D). Most plates

at 7 days (Figure 3B). After 14 days however, two distinct clusters

allowed reproducible measurements of cell viability (coefficient of vari-emerged, which separated transcriptional patterns in PE96, Nunc96,

ation [CV] < 20%) except for Greiner, PE and SBio plates in which CVs

PE384, and Sbio384 from the other microplate types.

exceeded 30%.

We complemented our expression analyses with measurements ofMaintenance of phenotype and functionality of liver spheroids ismetabolic activity. To this end, we incubated PHH spheroids after 7

critical for translational applications. We thus evaluated the expres-days of culture with a cocktail of probe substrates for the metabolic

sion of hepatic genes related to drug metabolism, ethanol catabolism,

enzymes (CYP1A2, CYP2C8, CYP2C9, and CYP3A4) and quantified

bile acid synthesis, and differentiation. Overall, drastic gene- and plate-the formed metabolites by LC-MS (Figure 3C). Striking differences

specific differences were observed (Figure 3A; Figure S1). Expres-between CYP activities were observed between microplates. PE96,

sion of the differentiation markers HNF1A, HNF4A, and ABCC2 did

Nunc96, PE384, and Sbio384 exhibited the lowest metabolic activ-

not markedly differ between microplates. ALB, CYP2E1, and ABCC2

ities for all substrates, with CYP2C9 and CYP3A4 activity being

showed only marginal inter-plate variability, whereas the key drug

below detection limit. In contrast, Biofloat384 and Corning384 showedmetabolizing enzymes CYP2D6 and CYP3A4, the bile acid genes

the highest activities, closely trailed by Greiner96, Corning96, andCYP7A1 and ABCB11, as well as the aldehyde dehydrogenase ADH1A

Biofloat96. Among these plates, Biofloat96 and Biofloat384 plates

exhibited pronounced differences. For the best performing plates

showed the lowest variability between the different measurements.

(Corning96, Corning384, Biofloat96, and Biofloat384), we moreover

These results indicate that the choice of microplate constitutes ansupplemented these results at RNA level by immunofluorescenceimportant parameter that can have pronounced impacts on results andstainings. Notably, we found that the hepatic markers CYP3A4

interpretation of PHH spheroid studies.

and albumin remained expressed at protein level throughout thespheroids without apparent differences between these plate types(Figure S2).
Particularly, spheroid culture in Nunc96 plates resulted in major

3.3
Microplates affect the outcomes ofhepatotoxicity assessments in PHH spheroids

transcriptional alterations after 7 days and, while these differences
equilibrated for CYP2D6 and CYP3A4 at later timepoints, expression

To test the impact of microplates on preclinical toxicity assays, PHH

of CYP7A1 and ABCB11 remained significantly different after 14 days.

spheroids were exposed to hepatotoxins with different modes of18607314, 2024, 2, Downloaded from https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/biot.202300587 by Rwe Power Ag Frau Moers, Wiley Online Library on [19/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

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XING ET AL.

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F I G U R E 3 The choice of culture plate affects molecular phenotypes and functions of human liver spheroids. (A) Matrix representation of
expression differences across ten important hepatic genes. The bottom left quadrant and top right quadrants indicate differences after 7 days and
14 days, respectively. n = 3 per plate and time point. Grey cells indicate p ≥ 0.05 while yellow, light orange, dark orange, and red indicate p < 0.05,
p < 0.01, p < 0.001, and p < 0.0001 in a one-way ANOVA, respectively. (B) Principal component analysis (PCA) can cluster microplate types basedon transcriptional differences. Note that the differences along principal component 1 (PC1) that distinguish plate types are much larger thanacross principal component 2 (PC2), which separate replicates. (C) Column plots showing the levels of acetaminophen, N-desethylamodiaquine,
4-hydroxy diclofenac, and hydroxymidazolam, which serve measurements of the metabolic activities of CYP1A2, CYP2C8, CYP2C9, and CYP3A4.

action. Specifically, we selected APAP, TRO, and CPZ as prototypic

evident for TRO and CPZ whose higher concentrations caused com-compounds that exert toxicity via reactive metabolites, mitochondrial

plete cell death. The largest variability between plates was observed

injury and cholestatic mechanisms, respectively. We evaluated toxicity

for intermediate TRO concentrations (5x cmax ) where no toxicity wasboth after a single dose (48 h exposure) and after 7 days of repeatedobserved in Nunc96 and Sbio384 (viability > 80%) whereas isogenic

exposures (Figure 4).

liver spheroids in Corning96 and Corning384 were completely dead.

As expected, spheroid sensitivity increased with prolonged expo-

Overall, the highest concentration of TRO and CPZ was required tosure for all compounds and plate types (Figure 4). Furthermore, we

detect in vitro toxicity in Nunc96 plates, whereas PHH spheroids in

observed clear concentration-dependency of toxicity. This was mostCorning384 and Biofloat384 were most sensitive with toxicity being

18607314, 2024, 2, Downloaded from https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/biot.202300587 by Rwe Power Ag Frau Moers, Wiley Online Library on [19/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

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F I G U R E 4 Toxicity of prototypic hepatotoxins in human liver spheroids. Cell viability was measured after a 48 h single exposure (A) and 7
days repeated exposures (B) for acetaminophen (APAP), troglitazone (TRO), and chlorpromazine (CPZ). n = 8 per plate, drug, and time point. Error
bars indicate SEM. *, **, and *** correspond to p < 0.05, p < 0.01, and p < 0.001 in one-sample t-tests, respectively.
clearly evident even at the lowest concentration tested after 7 days

parison, ATP levels dropped more rapidly in Biofloat96, Biofloat384,

exposure.

and Sbio384. To further characterize the molecular phenotype of PCH
aggregates, we evaluated the expression levels of the canine orthologues of eight hepatocyte-specific genes (Figure 5B). Specifically, we

3.4
Only few plate models support the formationof primary canine hepatocyte spheroids

selected the canine transporter genes ABCB11 and ABCC2, canine

Advanced organotypic cultures of primary hepatocytes of mouse, rat,

CYP2D15 (orthologue of human CYP2D6), and CYP3A12 (orthologue

minipig and cynomolgus monkey have been established to guide theof human CYP3A4). Upon aggregation (day 10), expression signatures

selection of preclinical model species [16,32–34] . However, despite thein Sbio384 plates were different from the other plates, particularly

popularity of Beagle dogs for preclinical drug safety testing, similar

due to altered expression of ALB and CYP1A2. However, unlike in PHH

cultures have not been established for primary canine hepatocytes

spheroids, differences between plates were comparable in magnitude(PCH). We thus tested the potential of the different ULA plates to sup-to differences between replicates (Figure 5C; 10 days). In contrast,

port PCH spheroid culture. In Corning96, PE96, Corning384, Sbio384,

after extended culture (day 17), Corning384 plates showed differential

Biofloat96, and Biofloat384 microplates, PCH formed aggregates that

expression of ABCB11, ABCC2, ALB, CYP1A2, CYP2D15, CYP3A12, andwere stable enough to be manipulated and transferred for endpoint

TF compared to the other microplates (Figure 5C; 17 days). At the phe-measurements. In contrast, in Greiner96, Nunc96, Greiner384, andnotypic level, Corning96 and Biofloat96 exhibited the highest levels

PE384 microplates, PCH did not form stable aggregates and theseof metabolic activity, whereas activities in Sbio384, Corning384, andplates were thus excluded from further analyses.

Biofloat384 were around two-fold lower (Figure 5D).

albumin (ALB) and transferrin (TF) as secretory markers, the hepatocyte master regulator HNF4A and the cytochrome P450s CYP1A2,

Spheroids generated in Corning96, PE96, and Corning384 main-

To evaluate hepatotoxicity in the different microplates, we exposed

tained relatively high ATP levels for 17 days with the latter showing

PCH aggregates to different concentrations of APAP, TRO, and CPZ

the highest ATP levels after 24 days of culture (Figure 5A). By com-

(Figure 5E,F). After exposure to APAP, PCH aggregates generally main-

18607314, 2024, 2, Downloaded from https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/biot.202300587 by Rwe Power Ag Frau Moers, Wiley Online Library on [19/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

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XING ET AL.

F I G U R E 5 Establishment and characterization of primary dog liver spheroids. (A) Viability for PCH aggregates is shown after 10 days, 17days, and 24 days of culture in 96-well plates and 384-well plates. Results are only shown for the six plate types that supported the formation of
PCH aggregates. n = 8 per plate and time point. *, **, and *** corresponds to p < 0.05, p < 0.01, and p < 0.001 in a one-way ANOVA, respectively.
Error bars indicate SEM. The dotted lines indicate 80% cell viability. (B) Matrix representation of expression differences across eight important
hepatic genes. The bottom left and top right quadrants indicate differences after 10 days and 17 days, respectively. n = 3 per plate and time point.
Grey cells indicate p ≥ 0.05 while yellow, light orange, dark orange, and red indicate p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 in a one-way
ANOVA, respectively. (C) Principal component analysis (PCA) of expression patterns of the eight analyzed hepatic genes. (D) Column plots showing
the levels of hydroxymidazolam and dextrorphan, which serve measurements of the metabolic activities of canine CYP3A12 (orthologue of human
CYP3A4) and CYP2D15 (orthologue of human CYP2D6). The sensitivity of human and dog spheroids is compared after a 48h single exposure (E)and 7 days repeated exposures (F) for acetaminophen (APAP), troglitazone (TRO), and chlorpromazine (CPZ). Three concentrations (1x, 5x, and20x cmax ) are tested per compound. Error bars indicate SEM. *, **, and *** correspond to p < 0.05, p < 0.01, and p < 0.001 in two-tailed
heteroscedastic t-tests, respectively.

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tained high viability except for spheroids generated in PE96 plates ininvestigated plates, resulting in spheroid formation already at 3 days

which 20x cmax concentration was clearly toxic. Across plates, spheroid

after cell seeding while 5 to 6 days were required in all other tested

sensitivity to TRO and CPZ was similar, with one exception. While TRO

plates. Interestingly, PCH aggregated more loosely compared to PHH

showed toxicity only after repeated exposure with the highest con-across all microplates. As a result, aggregation was slower (10 days vs.

centration in all plates, this drug was clearly hepatotoxic already at 5x

7 days) and only 6 out of 10 microplates supported the formation of

Cmax for spheroids generated in Biofloat96 plate. Compared to PHH

PCH aggregates.

spheroids, PCH aggregates were overall less sensitive to all compounds
tested, particularly after repeated exposure.

Cultivation of three-dimensional liver spheroids for multiple weeks
is essential for chronic disease modelling and repeated drug exposures.

To the best of our knowledge, these results constitute the first

Our data indicate that Corning and Biofloat microplates, both in 96-

demonstration that primary canine hepatocyte aggregates can be

and 384-well formats, are best suited for long-term culture. Which fac-stably cultured for multiple weeks. Culture in chemically definedtors are driving these differences however, remains unclear. Previous

serum-free media with identical compositions as for PHH spheroids

studies demonstrated that E-cadherin-mediated cell-cell interactions

facilitates the overall maintenance of molecular phenotypes and func-

inhibit apoptosis and promote cell survival in human liver spheroids

tions and is compatible with both acute and chronic toxicity tests,

compared to isolated cells or two-dimensional cultures [40] . Further

thus establishing these three-dimensional aggregates as a viable model

factors with potential impacts on cell viability include microstructure

for translational applications. However, the choice of microplate is

and leaching of compounds from the coating itself or the underlyingof tremendous importance to enable successful formation of PCH

bulk material [41–43] .

three-dimensional culture models.

In addition to changes in long-term viability, we also observedthat the choice of microplate affected gene expression patterns. We
have previously reported that primary hepatocytes undergo dynamic

4

DISCUSSION

transcriptomic changes during aggregation into spheroids which recapitulate molecular events observed during liver regeneration [44] .

Protocol standardization is imperative to facilitate the acceptanceBased on these findings, one can assume that differences in aggre-of three-dimensional tissue models as reliable preclinical assays. As

gation kinetics between plates could directly translate into alteredof September 2023, more than 4500 spheroid experiments can be

expression patterns. These findings have important implications as

retrieved from the MISpheroID database, indicating that the potential

they suggest that the use of different plates could have significantof the spheroid model in biomedical research is increasingly recognizedimpacts on the biological interpretations of the obtained results even[35] . Notably, comparison of the different selected spheroid methods

if otherwise identical protocols are used.

revealed diverse impacts on spheroid metrics, thus indicating the need

Different microplates furthermore affected the sensitivity of liverfor careful benchmarking. For liver spheroids, this should encompass

spheroids to hepatotoxins with different modes of action. APAP hepa-

the choice of cell model, seeding protocol, media composition as well

totoxicity is caused by its metabolic byproduct, the reactive metabolite

as microplates. Most of these parameters have been benchmarked

N-acetyl-p-benzoquinonimine (NAPQI). As NAPQI formation is depen-in recent studies. The phenotype and function of primary hepato-

dent on the hepatic enzymes CYP2E1 and CYP3A4, dedifferentiatedcytes have been shown to be superior to hepatoma cells and stem

and metabolically inactive hepatic cell models are largely protected

cell-derived hepatocytes [36–39] . Furthermore, the effects of differ-from APAP toxicity [45] . Here we find that APAP sensitivity of PHH

ent spheroid formation protocols and media compositions have been

spheroids was overall similar across plates, which is in agreement

extensively evaluated. Our study is the first to test the impacts of dif-with similar expression levels of CYP2E1 and CYP3A4. In contrast,

ferent microplates and demonstrates that they can have pronounced

CPZ and TRO exert toxicity at least in part by inhibiting the bile salt

impacts on formation kinetics, morphology, gene expression patterns,

export pump (BSEP; encoded by ABCB11) at the transcriptional and/or

compound toxicity and long-term stability. While effects on humanprotein level [46,47] . At high concentrations, both compounds wereliver spheroids were mostly quantitative, strong qualitative differences

clearly toxic across all microplates; however, toxicity of these com-were observed for canine hepatocyte spheroids, which could only bepounds at lower concentrations (CPZ 1x cmax and TRO 5x cmax ) differedsuccessfully formed in six out of the ten plates tested.

by > 10-fold, with spheroids cultivated in Nunc96 plates being the mostTo implement three-dimensional liver spheroids in preclinical phar-resistant. These results align with increased BSEP expression in thismacology and toxicology, reproducibility of spheroid formation plays

plate type, which might bestow hepatocytes with an increased capac-

a crucial role. This in turn requires that all seeded cells integrate into

ity to export bile salts and thus blunt CPZ and TRO toxicity. Differences

one structure and form a single spheroid in each well. The formationin the sensitivity to hydrophobic compounds between different platesof one spheroid per well could be achieved in all tested plates, except

may be also due to differences in compound absorption. Cell culture

for Greiner384 plates in which cell aggregation resulted in multiple

plastics are prone to absorption of hydrophobic small molecules [48] .

spheroids per well. In addition to reproducibility, rapid spheroid forma-

TRO and CPZ are both hydrophobic with logP values of 3.6 and 5.18,

tion is important, particularly in the context of using three-dimensional

respectively, whereas APAP is hydrophilic (logP = 0.51). Different sur-liver spheroids for drug screening and toxicological analyses. Com-

face treatments may reduce absorption, resulting in different freepaction of PHH seeded in Biofloat384 was the fastest among theconcentrations, which translates into altered compound sensitivity at

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XING ET AL.

XING ET AL.

the same nominal exposure concentration. Minimizing drug absorp-

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In summary, by comparing morphology, phenotype and functionof isogenic liver spheroids in side-by-side experiments, we demonstrated diverse impacts of the choice of ULA microplates. While mostplates supported the formation of human liver spheroids, large plate
differences were observed in hepatic gene expression and toxicity
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future liver spheroid experiments.
AUTHOR CONTRIBUTIONS
Conceptualization: Chen Xing, Aurino Kemas, Volker M. Lauschke;
Formal analysis: Chen Xing, Aurino Kemas, Kathrin Klein; Funding
acquisition: Per Artursson, Volker M. Lauschke; Investigation: Chen
Xing, Aurino Kemas, Evgeniya Mickols, Kathrin Klein; Project administration: Volker M. Lauschke; Resources: Per Artursson, Volker M.
Lauschke; Supervision: Aurino Kemas, Volker M. Lauschke; Visualization: Chen Xing, Volker M. Lauschke; Writing – original draft: Chen
Xing; Writing – review & editing: Aurino Kemas, Volker M. Lauschke.
ACKNOWLEDGMENTS
The project was financed by faCellitate GmbH. The study sponsor was involved in experimental planning but did not influence theinterpretation of the results. In addition, the authors received support from the Swedish Research Council [grant numbers:2019-01837
2021-02801 and 2023-03015], the Ruth och Richard Julins Foundation for Gastroenterology [grant number 2021-00158] and Robert
Bosch Foundation, Stuttgart, Germany. The authors are grateful to Igor
Liebermann (IKP Stuttgart) for conducting expression analyses.
CONFLICT OF INTEREST STATEMENT
VML is co-founder, CEO and shareholder of HepaPredict AB, as well as
co-founder and shareholder of PersoMedix AB.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from thecorresponding author upon reasonable request
ORCID
Volker M. Lauschke

https://orcid.org/0000-0002-1140-6204

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XING ET AL.

SUPPORTING INFORMATION
Additional supporting information can be found online in the Supporting Information section at the end of this article.

How to cite this article: Xing, C., Kemas, A., Mickols, E., Klein,
K., Artursson, P., & Lauschke, V. M. (2024). The choice ofultra-low attachment plates impacts primary human andprimary canine hepatocyte spheroid formation, phenotypes,
and function. Biotechnology Journal, 19, e2300587.
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