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Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

72059
10.1038/s41598-024-72059-x
Article
Improved functionality of hepatic spheroids cultured in acoustic levitation compared to existing 2D and 3D models
Rabiet Lucile lucile.rabiet@gmail.com

12
Jeger-Madiot Nathan 1
García Duván Rojas 1
Tosca Lucie 3
Tachdjian Gérard 3
Kellouche Sabrina 4
Agniel Rémy 4
Larghero Jérôme 2
Aider Jean-Luc jean-luc.aider@espci.psl.eu

1
Arakelian Lousineh lousineh.arakelian@aphp.fr

2
1 grid.464131.5 0000 0004 0370 1507 Laboratoire Physique et Mécanique des Milieux Hétérogènes (PMMH), CNRS, ESPCI, 7 Quai Saint-Bernard, 75005 Paris, France
2 grid.413328.f 0000 0001 2300 6614 Inserm U976, CIC-BT CBT501, AP-HP, Université Paris-Cité, Hôpital Saint-Louis, 1 avenue Claude Vellefaux, 75010 Paris, France
3 https://ror.org/04sb8a726 grid.413738.a 0000 0000 9454 4367 Service Histologie Embryologie Cytogénomique, Hôpital Antoine Béclère, 157 Rue de la Porte de Trivaux, 92140 Clamart, France
4 https://ror.org/043htjv09 grid.507676.5 Laboratoire ERRMECe, Maison Internationale de la Recherche, CY Cergy Paris Université, 1 rue Descartes, 95000 Neuville-sur-Oise, France
14 9 2024
14 9 2024
2024
14 215283 6 2024
3 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Hepatic spheroids are of high interest in basic research, drug discovery and cell therapy. Existing methods for spheroid culture present advantages and drawbacks. An alternative technology is explored: the hepatic spheroid formation and culture in an acoustofluidic chip, using HepaRG cell line. Spheroid formation and morphology, cell viability, genetic stability, and hepatic functions are analyzed after 6 days of culture in acoustic levitation. They are compared to 2D culture and non-levitated 3D cultures. Sizes of the 25 spheroids created in a single acoustofluidic microphysiological system are homogeneous. The acoustic parameters in our system do not induce cell mortality nor DNA damage. Spheroids are cohesive and dense. From a functional point of view, hepatic spheroids obtained by acoustic levitation exhibit polarity markers, secrete albumin and express hepatic genes at higher levels compared to 2D and low attachment 3D cultures. In conclusion, this microphysiological system proves not only to be suitable for long-term culture of hepatic spheroids, but also to favor differentiation and functionality within 6 days of culture.

Keywords

Acoustofluidics
Acoustic levitation
Spheroids
Microphysiological system
Hepatocytes
Subject terms

Tissue engineering
Biological physics
Drug development
Preclinical research
Cell polarity
Chromosomes
Multicellular systems
DNA damage and repair
Transcriptomics
Ecole Doctorale Frontières de l’Innovation en Recherche et Education (ED FIRE) – Programme BettencourtUniversité Paris‐Citéissue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

The liver is a vital organ with major properties, including protein synthesis and secretion, ion storage, and xenobiotics detoxification1,2. Despite its unique regenerative capacities in vivo,3 isolated primary hepatocyctes are rapidy depolarized, lose their functionality and long term viability in vitro, in 2D culture4.

Other hepatic cell models, including iPSC derived hepatoblasts and hepatic cell lines are also used for research purposes5. Among these cell lines, the bipotent HepaRG is of high interest6. In standard non-confluent 2D culture conditions, these cells proliferate and remain undifferentiated. At high confluence, cells differentiate along two sub-populations of hepatocyte-like and cholangiocyte-like cells. When further treated with dimethyl sulfoxide (DMSO), many genes including those of the detoxification enzymes are induced7–9. These properties have made HepaRG a useful tool for drug screening and basic research10.

It has been shown that 3D culture models including spheroids and organoids improve hepatic cell functionality11 and are promising tools for basic research, as well as drug screening. In vitro 3D cultures have been shown to be beneficial for both primary hepatocytes and hepatic cell lines12,13.

Several techniques have been developed to create spheroids, such as suspension cultures, hanging drops, low attachment surfaces, scaffolds, microgravity, microphysiological systems or magnetic levitation14,15. Otherwise promising, all these methods still face some of the following hurdles: heterogeneity in size, labor-intensive procedure, low throughput, poorly defined matrices and hydrogels, low cellular functionality, or a lack of technological maturity16–18. These drawbacks have slowed down the use of spheroids in the interested fields, such as cell therapy19–21 or drug development pipelines22,23.

Acoustic levitation represents an innovative technique for spheroid formation. It implies the use of ultrasound waves that, when reflected, create acoustic nodes that can trap small particles, as well as cells24,25. In our previous studies, we showed evidence that acoustic levitation allowed culturing different cell types, including HepaRG, up to 60 h26,27 keeping them viable and allowing them to self-organize into spheroids. In the current study, HepaRG were cultivated in acoustic levitation for a longer period of time and cell viability, genetic stability and functionality were evaluated. Spheroids obtained in acoustic levitation were compared to 2D cultures and spheroids obtained in ultra-low attachment (ULA) plates.

Results

Comparative morphological analysis and size reproducibility confirmation

Fig. 1 Comparative study during 6 days of cell culture. (a) Schematic of workflow in 2D, ultra-low attachment and acoustic levitation. Created with BioRender.com. (b) Schematic of the acoustofluidics setup for acoustic levitation. (c) Morphology of HepaRG cultures, viewed from the top: in 2D at low confluence at day 6, and at high confluence at day 14 (arrows represent differentiated hepatocyte-like cells); in ULA dishes; and in acoustic levitation. Scale bars: 400 μm. (d) Side-view of HepaRG spheroids in acoustic levitation. Scale bars: 200 μm. (e) Distribution of diameters, showing size homogeneity of the spheroids. Middle line is plotted at the median, the box extends from the 25th to 75th percentiles and whiskers show 10–90 percentiles, n = 12 for ULA and n = 50 for acoustic levitation. (f) Dynamics of spheroid formation in ultra-low attachment dishes and in acoustic levitation, calculated by percentage of initial spheroid area (from top-view). Error bars show mean ± SEM, n = 12 for ULA and n = 2 for acoustic levitation.

HepaRG cells were successfully cultured in 2D, in ULA and in acoustic levitation during 6 days (Fig. 1a). Long-term culture in levitation was performed inside an incubator, with precise control of flow and of acoustic radiation force (ARF) and constant visual monitoring (Fig. 1b). All cultures were imaged from the top (Fig. 1c) and cultures in acoustic levitation were also imaged from the side (Fig. 1d). As observed previously26,27 these cells organized themselves into spheroids in levitation. Spheroid formation was also obtained with HepaRG cells cultured in ultra-low attachment plates, whereas, in 2D cultures, cells grew in a monolayer as expected (Fig. 1c). In 2D, non-confluent HepaRG cells had an elongated aspect, whereas, at high confluence, two subpopulations of hepatocyte-like and cholangiocyte-like cells could be observed (Fig. 1c). Sizes and shapes of levitated spheroids were similar inside one acoustofluidic chip, and this homogeneity was observed throughout the self-organization (Fig. 1d). Spheroids’ diameters were homogeneous in levitation and comparable to ULA spheroids (Fig. 1e). The dynamics of the self-organization into spheroids in acoustic levitation and ultra-low attachment plates were comparable, showing similar compaction rates (Fig. 1f). Levitated spheroids remained cohesive after removal from the acoustofluidic chip, at day 6 (Fig. 1c).

Levitated spheroids maintain a high viability and retain their bipotent potential

Fig. 2 Validation of spheroid viability, adhesion and bipotent potential. (a) Cell viability evaluated by flow cytometry after dissociation and Live/Dead staining. Error bars show mean ± s.d., n = 3. (b) Scanning electron microscopy (SEM) images of spheroids obtained in ultra-low attachment dishes or in acoustic levitation. Arrows indicate microchannels at the surface of the spheroids. Scale bars: 20 μm (upper row) and 2 μm (bottom row). (c) Adhesion of HepaRG spheroids formed in acoustic levitation to a plastic culture flask. After initial proliferation, a bipotent phenotype (CL: cholangiocyte-like and HL: hepatocyte-like) is observed by optical microscopy, in a confluent cell monolayer, 14 days after the end of the levitation. Scale bars: 200 μm (upper row) and 50 μm (bottom row).

A direct viability assay performed on dissociated cells with the Live/Dead kit showed that adherent 2D cultures were almost 100% viable during the total period of 6 days of culture. The viability of cells in the spheroids formed in acoustic levitation and in ultra-low attachment plates were comparable (Fig. 2a).

Scanning electron microscopy (SEM) was performed on spheroids that attached to coverslips after the initial 6 days of culture. Spheroids formed in ultra-low attachment 24-well plates were more heterogeneous in shape and size. Only the smaller non-levitated spheroids were able to adhere to the cover glasses whereas almost all levitated samples attached. The results revealed that levitated spheroids were more compact than ULA spheroids (Fig. 2b). Tight interactions were observed between adjacent cells. Microchannels were spotted in between cells, at the surface of the levitated and non-levitated spheroids (Fig. 2b, arrows, upper row). Such pores might suggest the presence of apical biliary canaliculi. Micro-villi structures typical of hepatic cells were observed in both levitated and non-levitated spheroids (Fig. 2b, bottom row). Finally, when seeded on plastic, spheroids obtained by acoustic levitation and in ultra-low attachment plates were both able to adhere, spread and proliferate. Upon high confluence, after 2 weeks of culture, cells spread from spheroids differentiated into hepatocyte-like and cholangiocyte-like cells, indicating the preservation of their bipotency (Fig. 2c).

Acoustic levitation does not disrupt the genetic stability of HepaRG cells

The HepaRG karyotype is unaffected by 6 days of acoustic levitation

A preliminary karyotype analysis was performed on native HepaRG cells cultures in 2D, to study their basic characteristics. Analysis showed an abnormal karyotype with a supernumerary chromosome 7 of abnormal structure, and a derivative chromosome of a translocation between chromosome 12 and chromosome 22, resulting in the loss of chromosome 12 short arm (Fig. 3a). These results are in accordance with the previous description of the cell line: 46,XX,+del(7)(q11;q21)inv(7)(q21q36),-der22t(12;22)(p11;q11)6. This sample served as control for the comparison of other culture conditions. HepaRG cells after 6 days of culture in 2D monolayers, in ultra-low attachment plates or in acoustic levitation, followed by 7 days of 2D amplification, showed similar results, without additional chromosomal alterations.Fig. 3 Genetic stability of the three culture systems. (a) G-banded standard analysis of the HepaRG karyotype. The three culture models resulted in a female karyotype with an additional chromosome 7, a structural abnormality on one chromosome 12, and a missing chromosome 22 (arrows). (b–d) Array CGH results on native HepaRG genomic DNA. (b) Chromosome 7 profile with a complete trisomy and an interstitial deletion 7q11.23q21.11 of 6.45 Mb encompassing 49 RefSeq genes. View of whole chromosome 7 (left panel), short arm (middle panel), and long arm (right panel) ratio plots. (c) Chromosome 12 profile with a monosomy 12p of 34.16 Mb encompassing 329 RefSeq genes. View of whole chromosome 12 (left panel) and short arm (right panel) ratio plots. (d) Chromosome 5 profile with an interstitial duplication 5q31.2q32 of 11.14 Mb encompassing 148 RefSeq genes. View of whole chromosome 5 (left panel) and distal 5q region (right panel) ratio plots.

Array CGH

Array comparative genomic hybridization (CGH) on native HepaRG cells revealed a complete trisomy 7 (chr7:65,558-159,118,566, hg19) associated with an interstitial deletion 7q11.23q21.11 (chr7:73,265,298-79,725,034, hg19) of 6.45 Mb (Fig. 3b), and a monosomy 12p13.33p11.1 (chr12:194,249-34,360,030, hg19) of 34.16 Mb (Fig. 3c). An additional interstitial duplication 5q31.2q32 (chr5:138,139,653-149,283,415) of 11.14 Mb was observed (Fig. 3d). These results were in agreement with the karyotype and previous description6. The analysis of HepaRG cells after 6 days of culture in 2D monolayers, in ultra-low attachment plates or in acoustic levitation, followed by 7 days of 2D amplification, showed no further relevant copy number variation (CNV).

Both karyotype and array CGH analysis confirmed that ultrasounds used for acoustic levitation did not induce DNA damage or alteration.

Levitated spheroids are polarized and functional

Fig. 4 Hepatic cell functionality after 6 days in one of the three culture models. (a) Albumin gene expression and protein secretion evaluated by RT-qPCR and ELISA respectively. (b,c) Gene expression (RT-qPCR) of polarization markers, nuclear receptors, CYP450 drug-metabolizing and urea cycle enzymes. Error bars show mean ± s.d., n = 3. (d) Immunofluorescence of cryosectioned spheroids, after 6 days of acoustic levitation. Spheroids were stained for the F-actin label phalloidin (green, left), the polarization marker MRP2 (green, middle) or the drug-metabolizing enzyme CYP3A4 (red, right). All cryosections were stained with DAPI (blue). Arrows indicate intraspheroidal MRP2 clusters. Scale bars: 25 μm (middle), 50 μm (left and right).

Albumin (ALB) secretion is a hallmark of liver functionality. Therefore, ALB gene expression and protein secretion were quantified by RT-qPCR and by ELISA, respectively (Fig. 4a). ALB was not expressed in 2D non-confluent cultures whereas it was significantly overexpressed in spheroids formed in ultra-low attachment plates (mean RQ 6.2). This expression was even stronger in levitated spheroids (mean RQ 15.8). Albumin quantification by ELISA confirmed these results at protein secretion level. No albumin was detected in 2D non-confluent cultures’ supernatant whereas the total quantity detected in ULA spheroids culture medium was 2.7 μg and for levitated spheroids 10.53 μg.

The same overexpression tendency of polarization markers including MRP2, MRP3 and BSEP was observed in spheroids compared to non-confluent 2D HepaRG cells (Fig. 4b).

As for the expression of nuclear receptors, no difference was detected for AhR between the three models, whereas PXR and CAR were strongly overexpressed in the spheroids. CAR was overexpressed 45.5 folds in ULA spheroids and 72.4 folds in levitated ones (Fig. 4c).

Drug-metabolizing and urea cycle enzymes, including Cyp1A1, Cyp1A2, CYP2C9, Cyp2E1, Cyp3A4 and ARG1 were strongly overexpressed in spheroids compared to non-confluent 2D cultures. These gene expressions were much stronger in levitated spheroids than ULA ones (Fig. 4c).

These results indicate a more mature and functional profile of spheroids compared to 2D samples.

Immunostaining was also performed on levitated spheroids. Phalloidin staining showed a cohesive spheroid, with the actin cytoskeleton mainly concentrated at cell cortex. Moreover, we showed that spheroids expressed Cyp3A4, and that MRP2 was found at the surface of the spheroid, as well as more scarcely inside, demonstrating a stronger polarization at the surface compared to the core of the spheroids (Fig. 4d).

Discussion

Our acoustofluidic device was first used as a tool to form rapidly cohesive spheroids, from an initial cell suspension. The formation of spheroids in acoustic levitation was comparable to the formation of spheroids in standard low attachment plates. As previously reported, in acoustic levitation, self-organization relied strongly on the cell type characteristics27. Therefore, it is expected that levitated and non-levitated spheroids displayed similar dynamics of formation. Nevertheless, a higher circularity was obtained in acoustic levitation, which might be explained by the influence of the transverse component of the ARF. The impact of this force has been reported in previous works28,29. This could also be observed by SEM, which showed that levitated spheroids were more compact than ULA ones. Our study highlighted a satisfactory homogeneity in shape and size of levitated spheroids. It is comparable to the reproducibility of spheroids formed in low attachment 96-well plates, a technique that is well-known for its reproducibility30. Spheroids obtained in acoustic levitation were rapidly cohesive, and remained this way even after interruption of the ultrasonic trapping. Long-term culture in acoustic levitation, up to 6 days, did not affect the cohesiveness of the spheroids, thus assuring biomimetic cell-cell interactions.

We previously reported the proof of concept of cell culture in acoustic levitation for 24 h26. Nevertheless, prolonged exposition to ultrasounds during 6 days brought specific concerns such as increase in temperature, creation of pores in the plasma membrane, shear stress, cell lysis or DNA damage31,32.

These effects are generally witnessed with high intensity ultrasounds, which is clearly not the case for our system, where low intensity ultrasounds were used (voltage < 5 Vpp and power < 0.1 W) allowing a very gentle manipulation of cells. Viability of the levitated spheroids was excellent and comparable with 2D and ULA controls. Genetic stability was maintained, confirming that acoustic levitation did not induce further chromosomal damage or genetic alterations. This genetic stability is an important point, especially if this culture system is further developed for being used for therapeutic purposes33,34.

HepaRG cells are an excellent tool for drug testing. However, in order to differentiate in 2D culture, they have to attain a very high confluence, for at least 14 days35. Furthermore, culture in the presence of DMSO is required to induce detoxification enzymes, but this stimulation is not stable overtime and enzyme expression levels drop upon removal of DMSO. Therefore, the development of technologies allowing a DMSO-free and stable cell differentiation could be beneficial for drug screening purposes. Our results showed that 3D culture of HepaRG as spheroids induced a strong overexpression of detoxification enzymes as well as polarization markers, compared to nonconfluent 2D cultures. This effect was much emphasized in levitated spheroids. This could be partly due to a rapid cell aggregation in acoustic levitation, mimicking a high confluence. It has thus been reported that a stable overexpression of CAR receptor reduces the requirement for DMSO treatment of HepaRG, for induced drug metabolism36. These results indicate that there may be a chain of gene expression control between CAR and metabolic enzymes, which is stable overtime in 3D HepaRG cultures.

In conclusion, we show that acoustic levitation is an innovative tool well adapted to HepaRG long-term and gentle cell culture. It allows a rapid aggregation of cells, leading to their fast and increased maturation, without inducing any genetic damage. When compared to classic ULA spheroids, these levitated spheroids had globally a much higher expression of liver specific genes. In the future, it would be interesting to culture other hepatic models such as primary hepatocytes or iPSC-derived hepatoblasts, in order to study the effects of acoustic levitation on cell proliferation and functionality. This innovative cell culture tool could be up-scaled for the development of reproducible hepatic spheroids that could be used for drug screening, disease modeling or cell therapy.

Methods

HepaRG culture and amplification

Cells and spheroids were cultivated in incubators at 37∘C and 5% CO2. For amplification, HepaRG cells (Biopredic, St. Grégoire, France) were cultivated in basal hepatic cell medium (Biopredic) with 10% of HepaRG growth medium supplemented with antibiotics. The initial cell density was 2.8×104 cells cm-2. Culture medium was renewed every 3 days. Every 14 days, cells were detached and passaged using 0.25% trypsin + EDTA (Gibco, UK).Fig. 5 Schematic of the acoustofluidic well used in the present study to form and culture HepaRG spheroids over 6 days. The walls of the well are made of PDMS. An oil layer is used as a matching layer between the upper wall of the well and the transducer. The reflector facing the transducer is a glass slide. A constant flow rate is applied, allowing a total renewal of the medium after 3 days. The typical sizes of spheroids, as well as the internode distance obtained with a 2.2 MHz acoustic wave, are also displayed on the schematic.

Acoustic levitation

To create an acoustic force field inside a cavity, one has to create an acoustic standing wave by placing a reflector facing the transducer at a distance h matching the resonance condition (h=nλac/2, with λac the acoustic wavelength). The particles or cells inside the cavity are then submitted to the acoustic radiation force (ARF). The axial component of the ARF (Fac) forces them to move toward the acoustic pressure nodes (levitation planes).

The ARF can be written as37:1 Fac=π4E0kdp3FYsin(2kz)ez,

where E0 is the mean acoustic energy density, dp the diameter of the object, FY the acoustic contrast factor and k the acoustic wave number38. This expression is valid for spherical particles much smaller than the acoustic wavelength (Rayleigh approximation). Usually, one can consider that the acoustic levitation plane is located at the acoustic pressure node, but for dense and/or large objects, this condition is not necessarily verified and the axial position of the levitation planes may be lower than the acoustic pressure node. These non-standard conditions can lead to the triggering of self-acoustophoresis of metallic nanorods39 or displacement of large levitated objects40. In our case, once the spheroids have been formed in levitation, they remain at a stable equilibrium position.

Acoustofluidic Chips

The acoustofluidic chips were fabricated in polydimethylsiloxane (PDMS, 1:10 curing agent:base ratio, RTV 615, Neyco, Vanves), a transparent, gas permeable and biocompatible material that does not strongly reflect acoustic waves. PDMS was poured in a plexiglass mold, heated at 70∘C for 24h, then bonded with a plasma cleaner on a microscope glass slide (acoustic reflector). Each acoustofluidic chip is comprised of two chambers: an acoustofluidic well and its preceding passive bubble trap. There are connected by PDMS channels. The formation of bubble is non existent inside the well. However, due to the continuous medium renewal, bubbles frequently arrive with the flow and are stopped inside the passive bubble trap chamber (Fig. 1b) before arriving in contact with the cells or the acoustic field. The bubble trap is a cylindrical well (height h= 12 mm and diameter d= 5 mm), where gaz bubble are trapped at the top, while liquid medium exits at the bottom.

Formation and culture of spheroids in acoustic levitation

In our acoustofluidic chip (Fig. 5), a 2 MHz ultrasound broadband transducer was used to create a multi-modes acoustic standing wave in a h= 10 mm high well. Ultrasonic standing waves of a few megahertz can produce an Acoustic Radiation Force (ARF) that can be used to manipulate small particles as well as mammalian cells. The acoustic frequency Fac (and corresponding acoustic wavelength λac) was tuned to match the resonance condition (h=n λac/2). One million cells were injected in each chip. Ultrasonic transducers (SignalProcessing ™) were powered by a waveform generator (Handyscope HS5, TiePie Engineering™) with a sinusoidal waveform of constant amplitude of A = 3.5 Vpp, which is the smallest possible amplitude to maintain the spheroids in levitation.

The acoustic frequency used was Fac = 2.2 MHz for these resonant cavities, a value contained in the levitation range of 1.5–2.5 MHz, as shown in previous studies41,42. After emission by the transducer, the acoustic wave travels through oil (acoustic matching layer), a PDMS membrane and the bulk of the well filled with cell medium, before reaching a glass slide, which plays the role of the reflector on the other side of the well (Fig. 5). The height of the cavities (h= 10 mm) lead to the formation of 30 levitation planes, i.e. 25–30 spheroids per chip (Supplementary Fig. 1). Levitated spheroids were formed and cultured in eight chips in parallel.

A syringe pump perfused the chip with a constant flow rate of 10 μL h-1 (Pump 11 Elite, Harvard Apparatus™) in order to renew the medium every three days. Timelapses showing the evolution of the spheroids inside the chip were obtained using Dino-Lite Pro 630-1165 USB Microscopes (Dinolite™). Snapshots were taken every 10 minutes during the duration of the culture. After 6 days, the ultrasound were turned off and the spheroids were collected from the chip by aspiration. They were immediately transferred into a 96-well plate and were placed in an Incucyte S3 imaging system (Sartorius™) for immediate imaging of the top view of the spheroids.

Geometrical analysis

In order to quantify the dynamics of formation of the spheroids as well as the possible evolution of their size and shape over the time of the culture, diameter of the spheroids were computed from the snapshots using the ImageJ software. To characterize the time evolution of the shape of the spheroids during the first three days, we computed the area from ImageJ analysis of the top views of the spheroids.

Cell viability

Levitated spheroids were aspirated from the culture devices and were placed in a 15 mL centrifugation tube (Falcon). ULA spheroids were also collected in 15 mL centrifugation tubes. Spheroids were centrifuged at 284 g for 5 min at 20∘C and the supernatant was eliminated. Monolayers and spheroids were then dissociated into single cells with 0.25% trypsin during 15 min. After suspension in PBS (Eurobio, Courtaboeuf, France), all conditions were stained for 5 min with calcein and ethidium bromide (LIVE/DEAD mammalian viability kit, Montlucon, France). Viability of individual cells was evaluated by flow cytometry (AttuneÂ® NxT Acoustic Focusing Cytometer).

Scanning electron microscopy

After 6 days of culture, ULA spheroids or levitated spheroids were collected and placed on round cover glasses (12 mm diameter) inside a 24-well plates (Corning, Falcon, NY, USA) for 24 hours. Adherent spheroids were fixed with glutaraldehyde 2.5% and PFA 2%, diluted in cacodylate 0.1 m buffer. After dehydration in several ethanol baths of increasing concentration, supercritical drying with carbon dioxide was performed. Spheroids were coated with platinum 4 nm. The sample preparation preserved the 3D structure of the spheroids. Scanning electron microscopy was conducted using a field emission gun scanning electron microscope (GeminiSEM300, Carl Zeiss) with an acceleration voltage of 2 keV under high vacuum. Secondary electrons were collected. Scan speed and line averaging were adjusted during observation.

Spheroids readhesion and spreading on plastic

After 6 days in levitation, spheroids were transferred in 96-well plates (Corning, Falcon, NY, USA) with fresh medium, and placed in an Incucyte S3 imaging system (Sartorius) at 37∘C. Images were taken every 10 min during 14 days to assess the adhesive potential of levitated spheroids.

Genetic stability

Genetic stability was evaluated by karyotype and array comparative genomic hybridization (CGH). Cells originating from the same culture batch were cultivated in acoustic levitation, in 24-well ultra-low attachment plates or in 25 mm2 flasks during 6 days (three replicates for each), then cultivated in adhesive 6-well plates (Corning, Falcon, NY, USA) for amplification during 7 days.

Karyotype

While plates were still not confluent, 20 μL of colchicin (20 mg L-1 ; EUROBIO) were added in each 6-well plate well, followed by an incubation of 2 h at 37∘C. Cells were detached with 0.25% trypsin during 5 min (37∘C), transferred into a 15 mL tube and centrifuged at 284g for 5 min at 20∘C. Supernatant was replaced by 8 mL of preheated KCL 0.075 m and incubated during 20 min at 37∘C. 2 mL of 4∘C Carnoy (3:1 methanol:acetic acid) were added for prefixation of the samples. After centrifugation, the supernatant was eliminated and samples were fixed in 8 mL of Carnoy. Samples were kept at 4∘C before analysis. Karyotype was studied using standard procedures (G-banding by using trypsin and Giemsa staining (GTG).

Array comparative genomic hybridization (CGH)

Cells were dissociated with 0.25% trypsin during 5 min (37∘C). DNA was extracted from the samples using Mini-Kit PureLink® Genomic DNA Kits K1820-02, according to the manufacturer’s instructions, and eluted in 50 μL of PureLink Genomic Elution Buffer. DNA quantity and purity were evaluated using NanoDropLite (Thermo scientific™).

DNA integrity was assessed on a 1% agarose gel. Genomic imbalances were analyzed by array CGH using 180K oligonucleotide arrays (Agilent Technologies, Massy, France). Genomic DNAs from 2D cultures and spheroids were compared with native HepaRG genomic DNA. Hybridization was performed according to the manufacturer’s protocol. Images were processed with Feature Extraction software (10.7.3.1), and data analysis was performed with Genomic Workbench V5.0.14 (Agilent Technologies). The genomic positions were determined using version 19, Build37 of the human Genome Browser (University of California, Santa Cruz, CA; http://genome.ucsc.edu/). The Aberration Detection Method 2 algorithm was used for statistical analysis. Copy number alterations were considered important if they were defined by four or more oligonucleotides and spanned at least 39 kb and were not identified in the Database of Genomic Variants (http://projects.tcag.ca/cgi-bin/variation/gbrowse/hg19).

Secreted albumin quantification

Supernatants of levitated samples (1 million of cells) were collected at the outlet of the chip during the 6 days of medium renewal. Supernatants of non-levitated samples (2D culture and ULA spheroids ; 1 million of cells) were collected during medium renewal at day 3 and day 6, and pooled. Albumin quantification in the supernatants was performed with Human Serum Albumin DuoSet ELISA (RnD Systems™), according to the manufacturer’s instructions. The optical density at 450 nm was detected using a microplate reader (Varioskan™LUX multimode, Thermo Scientific™, Waltham, MA USA). Supplemented HepaRG cell culture medium served as negative control. The concentration (μg mL-1) was multiplied by the total volume of the supernatant in order to calculate the total albumin secreted by the cells (μg).

RNA extraction

RNA extraction was performed based on a trizol/chloroform method. RNA quantity and purity were evaluated using NanoDropLite (Thermo scientific™). Gene expression was then evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

Reverse transcription (RT)

RT was performed with 500 ng of RNA and high capacity cDNA reverse transcription kit (Thermofisher™) in 0.5 mL tubes (Easy strip Snap Tubes, Thermofisher™) in a SimpliAmp Thermal Cycler (Applied Biosystems), using the following program: 10 min at 25∘C, 120 min at 37∘C, 5 min at 85∘C, followed by a cooling step at 4∘C.

Quantitative polymerase chain reaction (qPCR)

The cDNAs were diluted to 1/50 in nuclease free water and were used for qPCR (5 ng of cDNA per well) with RNEasy Plus Mini Kit (Quiagen). TaqMan™probes were used for the amplification. The reactions were performed in 384 reaction plates (Applied Biosystem™) in a final volume of 20 μL per well. The reagents were dispatched in the wells using an Epmotion 5073 robot (Eppendorf).The expression of genes related to polarity (apical cell membrane transporters), detoxification (nuclear receptors and cytochromes), albumin and the urea cycle were analyzed. RPLP0 was used as housekeeping gene. The list of the TaqMan™probes used for these experiments are found in Table 1. Table 1 TaqMan™probes used for the qPCR analysis.

Gene symbol	Protein full name	Reference	
RPLP0	Ribosomal protein lateral stalk subunit P0	Hs99999902	
MRP2/ABCC2	Multi-drug resistance protein 2	Hs00166123	
MRP3/ABCC3	Multi-drug resistance protein 3	Hs00978452	
BSEP/ABCB11	Albumin	Hs00609411	
AhR	Aryl Hydrocarbon Receptor	Hs00169233	
PXR/NR1I2	Pregnane X receptor	Hs01114267	
CAR/NR1I3	Constitutive androstane receptor	Hs00901571	
CYP1A1	Cytochrome P450 family 1 subfamily A member 1	Hs01054796	
CYP1A2	Cytochrome P450 family 1 subfamily A member 2	Hs00167927	
CYP2C9	Cytochrome P450 family 2 subfamily C member 9	Hs02383631	
CYP2E1	Cytochrome P450 family 2 subfamily E member 1	Hs00559367	
CYP3A4	Cytochrome P450 family 3 subfamily A member 4	Hs00604506	
ALB	Bile salt export pump	Hs00994811	
ARG1	Arginase 1	Hs00163660	

All results were expressed as relative quantification (RQ) compared to one of the control samples which consists of HepaRG cells cultivated during 6 days in 2D (initial seeding density of 28,000 cells cm-2). Statistical test: t-test (n=3). An RQ difference of factor 2 was considered as significant.

Cryosection

Levitated spheroids at day 6 were fixed in PFA 4% during 30 min, then stored at 4∘C in PBS. The supernatant was replaced by a 15% sucrose (Sigma-Aldrich) solution in PBS for 1 h, then by a 30% sucrose solution for 48 h. Spheroids were withdrawn from the sucrose solution, and deposited inside a silicon mold filled with ≈ 1 mL of optimal cutting temperature (OCT) cryofix gel (Biognost) cryo-embedding matrix. The samples were immediately placed on dry ice and frozen. Samples were removed from the mold, placed in cold 2 mL tubes and stored at -80∘C. Spheroids were cut (25 μm sections) with a cryostat (Leica CM 1950) and deposited on polylysin-coated glass slides. Sections were stored at -80∘C until staining.

Immunofluorescence

Samples were saturated and permeabilized for 15 min in PBS containing 3% bovine serum albumin (BSA) and 0.05% Triton, followed by a 30 min incubation in O.3M glycin. They were then incubated for 1 h with the primary antibodies (dilution 1:200, 200 μL per slide). After three washes in PBS-BSA and 0.1% Tween-20, samples were incubated during 30 min with phalloidin-FITC (1:500) or secondary antibodies (1:500). Cell nuclei were stained with DAPI (1:2000). After three washes, cover slides were mounted with ProLongTM Gold antifade reagent (Life Technologies). The primary and secondary antibodies are listed in Table 2. Images were acquired using a confocal microscope LSM 780 system. Table 2 Primary and secondary antibodies used for immunofluorescence.

Antibody	Supplier	Reference	
Rabbit anti-CYP3A4	Invitrogen	PA1-343	
Goat anti-rabbit IgG-Alexa Fluo™555	Invitrogen	A21428	
Mouse anti-MRP2	Santa Cruz	SC-59609	
Goat anti-mouse IgG1- Alexa Fluor™488	Invitrogen	A21121	

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72059-x.

Acknowledgements

We thank Biopredic International for having kindly provided the HepaRG cell line, Dr. Niclas Setterblad and the Technological Core Facility of the Saint-Louis Research Institute, and the Fondation Rothschild for their material and technical support. We thank Dr. Sabine Gerbal-Chaloin, Dr. Martine Daujat and Laurence Françoise for their implications in this study. We thank Dr. Stéphane Brunet for his lecture and advice on the manuscript presentation. We are grateful to Ecole Doctorale Frontières de l’Innovation en Recherche et Education (ED FIRE) - Programme Bettencourt and Université Paris-Cité for the funding of the L. Rabiet thesis. We also thank the Région Ile-de-France (DIM ELICIT) and the French National Research Agency (RHU program ANR-16-RHUS-0005) for providing financial support for this research.

Author contributions

L.R., L.A., N.J., D.R.G., L.T., G.T., S.K., R.A., J.L. and J.-L.A. conceived and planned the experiments. L.R., L.A., N.J., D.R.G., L.T., G.T., S.K. and R.A. performed the experiments. J.-L.A., J.L. and L.A. supervised the project. All authors discussed the results and contributed to the final manuscript.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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