Combined Toxic Effects of BPA and Its Two Analogues BPAP and BPC in a 3D HepG2 Cell Model.

Bisphenol A (BPA) is one of the most commonly used substances in the manufacture of various everyday products. Growing concerns about its hazardous properties, including endocrine disruption and genotoxicity, have led to its gradual replacement by presumably safer analogues in manufacturing plastics. The widespread use of BPA and, more recently, its analogues has increased their residues in the environment. However, our knowledge of their toxicological profiles is limited and their combined effects are unknown. In the present study, we investigated the toxic effects caused by single bisphenols and by the combined exposure of BPA and its two analogues, BPAP and BPC, after short (24-h) and prolonged (96-h) exposure in HepG2 spheroids. The results showed that BPA did not reduce cell viability in HepG2 spheroids after 24-h exposure. In contrast, BPAP and BPC affected cell viability in HepG2 spheroids. Both binary mixtures (BPA/BPAP and BPA/BPC) decreased cell viability in a dose-dependent manner, but the significant difference was only observed for the combination of BPA/BPC (both at 40 µM). After 96-h exposure, none of the BPs studied affected cell viability in HepG2 spheroids. Only the combination of BPA/BPAP decreased cell viability in a dose-dependent manner that was significant for the combination of 4 µM BPA and 4 µM BPAP. None of the BPs and their binary mixtures studied affected the surface area and growth of spheroids as measured by planimetry. In addition, all BPs and their binary mixtures studied triggered oxidative stress, as measured by the production of reactive oxygen species and malondialdehyde, at both exposure times. Overall, the results suggest that it is important to study the effects of BPs as single compounds. It is even more important to study the effects of combined exposures, as the combined effects may differ from those induced by single compounds.

GDNF drives rapid tubule morphogenesis in a novel 3D in vitro model for ADPKD.

Cystogenesis is a morphological consequence of numerous genetic diseases of the epithelium. In the kidney, the pathogenic mechanisms underlying the program of altered cell and tubule morphology are obscured by secondary effects of cyst expansion. Here, we developed a new 3D tubuloid system to isolate the rapid changes in protein localization and gene expression that correlate with altered cell and tubule morphology during cyst initiation. Mouse renal tubule fragments were pulsed with a cell differentiation cocktail including glial-derived neurotrophic factor (GDNF) to yield collecting duct-like tubuloid structures with appropriate polarity, primary cilia, and gene expression. Using the 3D tubuloid model with an inducible Pkd2 knockout system allowed the tracking of morphological, protein, and genetic changes during cyst formation. Within hours of inactivation of Pkd2 and loss of polycystin-2, we observed significant progression in tubuloid to cyst morphology that correlated with 35 differentially expressed genes, many related to cell junctions, matrix interactions, and cell morphology previously implicated in cystogenesis.This article has an associated First Person interview with the first author of the paper.

Lethal Electric Field Thresholds for Cerebral Cells With Irreversible Electroporation and H-FIRE Protocols: An In Vitro Three-Dimensional Cell Model Study.

The lethal electric field (LEF) thresholds for three typical cerebral cells, including a malignant glioblastoma (GBM) cell line and two cell lines from the healthy blood-brain barrier (BBB), treated by irreversible electroporation (IRE) or high-frequency irreversible electroporation (H-FIRE) protocols were investigated in an in vitro three-dimensional (3D) cell model. A conventional IRE protocol (90 pulses, 1 Hz, and 100-µs pulse duration) and three novel H-FIRE protocols (1-3-1, 0.5-1-0.5, and 1-1-1) were used to treat the cerebral cells in both 3D single-cell and two-cell models. The electrical conductivity of the 3D cell model under different electric field strengths were characterized with the method of electrochemical impedance spectroscopy (EIS). Based on EIS, a numerical electrothermal model of electroporation was built for the determination of the LEF threshold with different protocols and temperature monitoring. Cell viability was assessed by fluorescence staining 6 h after the treatment. The results showed no thermal lethal effect on cells when these protocols were used. The LEF threshold for GBM cells was significantly lower than that of the healthy BBB cells. These results suggest the possibility of selective ablation of human cerebral GBM by IRE and H-FIRE treatments with no injury or reversible injury to healthy cells, and the potential use of IRE or H-FIRE for transient disruption of the BBB to allow chemotherapy to reach the tumor.

A Genetically Encoded Bioluminescence Intracellular Nanosensor for Androgen Receptor Activation Monitoring in 3D Cell Models.

In recent years, there has been an increasing demand for predictive and sensitive in vitro tools for drug discovery. Split complementation assays have the potential to enlarge the arsenal of in vitro tools for compound screening, with most of them relying on well-established reporter gene assays. In particular, ligand-induced complementation of split luciferases is emerging as a suitable approach for monitoring protein-protein interactions. We hereby report an intracellular nanosensor for the screening of compounds with androgenic activity based on a split NanoLuc reporter. We also confirm the suitability of using 3D spheroids of Human Embryonic Kidney (HEK-293) cells for upgrading the 2D cell-based assay. A limit of detection of 4 pM and a half maximal effective concentration (EC50) of 1.7 ± 0.3 nM were obtained for testosterone with HEK293 spheroids. This genetically encoded nanosensor also represents a new tool for real time imaging of the activation state of the androgen receptor, thus being suitable for analysing molecules with androgenic activity, including new drugs or endocrine disrupting molecules.

Development of in vitro 3D cell model from hepatocellular carcinoma (HepG2) cell line and its application for genotoxicity testing.

The evaluation of genotoxicity plays an important role within hazard identification and risk assessment of chemicals and consumer products. For genotoxicity assessment, in vitro hepatic cells are often used as they have retained certain level of xenobiotic metabolic activity. However, current protocols are designed for the use on 2D monolayer models that are associated with several limitations due to the lack of numerous biological functions, which results in the loss of many hepatic properties. In this respect, an attractive alternative are three-dimensional (3D) models. The aim of our study was to develop physiologically more relevant 3D cell model (spheroids) from the human hepatocellular carcinoma (HepG2) cell line for genotoxicity testing. The spheroids were prepared by the forced floating method, which had been optimized for the production of a large number of uniform spheroids. The sensitivity of the spheroids to detect genotoxicity was determined by the comet assay after the exposure of spheroids to non-cytotoxic concentrations of model indirect acting genotoxic compounds, namely polycyclic aromatic hydrocarbon (B(a)P), mycotoxin (AFB1), two heterocyclic aromatic amines (PhIP and IQ) and a direct acting etoposide (ET). All five tested compounds concentration dependently induced DNA damage. Higher sensitivity of 3D cell model compared to 2D monolayer culture was noticed particularly for detection of the genotoxicity of the heterocyclic aromatic amines and BaP. Deregulation of mRNA expression (qPCR) by genotoxic compounds revealed that HepG2 cells in 3D express important genes encoding phase I and II metabolic enzymes, as well as DNA damage responsive genes in an inducible form. The newly developed HepG2 3D model shows improved sensitivity for detecting genotoxic compounds compared to 2D cultures and can provide a suitable experimental model for genotoxicity assessment.

Advanced three-dimensional in vitro liver models to study the activity of anticancer drugs.

The liver is one of the most important organs in the human body. It performs many important functions, including being responsible for the metabolism of most drugs, which is often associated with its drug-induced damage. Currently, there are no ideal pharmacological models that would allow the evaluation of the effect of newly tested drugs on the liver in preclinical studies. Moreover, the influence of hepatic metabolism on the effectiveness of the tested drugs is rarely evaluated. Therefore, in this work we present an advanced model of the liver, which reflects most of the morphologically and metabolically important features of the liver in vivo, namely: three-dimensionality, cellular composition, presence of extracellular matrix, distribution of individual cell types in the structure of the liver model, high urea and albumin synthesis efficiency, high cytochrome p450 activity. In addition, the work, based on the example of commonly used anticancer drugs, shows how important it is to take into account hepatic metabolism in the effective assessment of their impact on the target organ, in this case cancer. In our research, we have shown that the most similar to liver in vivo are 3D cellular aggregates composed of three important liver cells, namely hepatocytes (HepG2), hepatic stellate cells (HSCs), and hepatic sinusoidal endothelial cells (HSECs). Moreover, we showed that the cells in 3D aggregate structure need time (cell-cell interactions) to improve proper liver characteristic. The triculture model additionally showed the greatest ability to metabolize selected anticancer drugs.

Construction and applications of the EOMA spheroid model of Kaposiform hemangioendothelioma.

BACKGROUND: Kaposiform hemangioendothelioma (KHE) is a rare intermediate vascular tumor with unclear pathogenesis. Recently, three dimensional (3D) cell spheroids and organoids have played an indispensable role in the study of many diseases, such as infantile hemangioma and non-involuting congenital hemangiomas. However, few research on KHE are based on the 3D model. This study aims to evaluate the 3D superiority, the similarity with KHE and the ability of drug evaluation of EOMA spheroids as an in vitro 3D KHE model. RESULTS: After two days, relatively uniform morphology and high viability of EOMA spheroids were generated by the rotating cell culture system (RCCS). Through transcriptome analysis, compared with 2D EOMA cells, focal adhesion-related genes such as Itgb4, Flt1, VEGFC, TNXB, LAMA3, VWF, and VEGFD were upregulated in EOMA spheroids. Meanwhile, the EOMA spheroids injected into the subcutaneous showed more obvious KMP than 2D EOMA cells. Furthermore, EOMA spheroids possessed the similar characteristics to the KHE tissues and subcutaneous tumors, such as diagnostic markers (CD31 and LYVE-1), cell proliferation (Ki67), hypoxia (HIF-1α) and cell adhesion (E-cadherin and N-cadherin). Based on the EOMA spheroid model, we discovered that sirolimus, the first-line drug for treating KHE, could inhibit EOMA cell proliferation and downregulate the VEGFC expression. Through the extra addition of VEGFC, the effect of sirolimus on EOMA spheroid could be weakened. CONCLUSION: With a high degree of similarity of the KHE, 3D EOMA spheroids generated by the RCCS can be used as a in vitro model for basic researches of KHE, generating subcutaneous tumors and drug screening.

Adverse (geno)toxic effects of bisphenol A and its analogues in hepatic 3D cell model.

Bisphenol A (BPA) is one of the most widely used and versatile chemical compounds in polymer additives and epoxy resins for manufacturing a range of products for human applications. It is known as endocrine disruptor, however, there is growing evidence that it is genotoxic. Because of its adverse effects, the European Union has restricted its use to protect human health and the environment. As a result, the industry has begun developing BPA analogues, but there are not yet sufficient toxicity data to claim that they are safe. We investigated the adverse toxic effects of BPA and its analogues (BPS, BPAP, BPAF, BPFL, and BPC) with emphasis on their cytotoxic and genotoxic activities after short (24-h) and prolonged (96-h) exposure in in vitro hepatic three-dimensional cell model developed from HepG2 cells. The results showed that BPFL and BPC (formed by an additional ring system) were the most cytotoxic analogues that affected cell viability, spheroid surface area and morphology, cell proliferation, and apoptotic cell death. BPA, BPAP, and BPAF induced DNA double-strand break formation (γH2AX assay), whereas BPAF and BPC increased the percentage of p-H3-positive cells, indicating their aneugenic activity. All BPs induced DNA single-strand break formation (comet assay), with BPAP (≥0.1 µM) being the most effective and BPA and BPC the least effective (≥1 µM) under conditions applied. The results indicate that not all of the analogues studied are safer alternatives to BPA and thus more in-depth research is urgently needed to adequately evaluate the risks of BPA analogues and assess their safety for humans.

Development of three-dimensional primary human myospheres as culture model of skeletal muscle cells for metabolic studies.

Introduction: Skeletal muscle is a major contributor to whole-body energy homeostasis and the utilization of fatty acids and glucose. At present, 2D cell models have been the most used cellular models to study skeletal muscle energy metabolism. However, the transferability of the results to in vivo might be limited. This project aimed to develop and characterize a skeletal muscle 3D cell model (myospheres) as an easy and low-cost tool to study molecular mechanisms of energy metabolism. Methods and results: We demonstrated that human primary myoblasts form myospheres without external matrix support and carry structural and molecular characteristics of mature skeletal muscle after 10 days of differentiation. We found significant metabolic differences between the 2D myotubes model and myospheres. In particular, myospheres showed increased lipid oxidative metabolism than the 2D myotubes model, which oxidized relatively more glucose and accumulated more oleic acid. Discussion and conclusion: These analyses demonstrate model differences that can have an impact and should be taken into consideration for studying energy metabolism and metabolic disorders in skeletal muscle.

Imaging and detecting intercellular tensile forces in spheroids and embryoid bodies using lipid-modified DNA probes.

Cells continuously experience and respond to different physical forces that are used to regulate their physiology and functions. Our ability to measure these mechanical cues is essential for understanding the bases of various mechanosensing and mechanotransduction processes. While multiple strategies have been developed to study mechanical forces within two-dimensional (2D) cell culture monolayers, the force measurement at cell-cell junctions in real three-dimensional (3D) cell models is still pretty rare. Considering that in real biological systems, cells are exposed to forces from 3D directions, measuring these molecular forces in their native environment is thus highly critical for the better understanding of different development and disease processes. We have recently developed a type of DNA-based molecular probe for measuring intercellular tensile forces in 2D cell models. Herein, we will report the further development and first-time usage of these molecular tension probes to visualize and detect mechanical forces within 3D spheroids and embryoid bodies (EBs). These probes can spontaneously anchor onto live cell membranes via the attached lipid moieties. By varying the concentrations of these DNA probes and their incubation time, we have first characterized the kinetics and efficiency of probe penetration and loading onto tumor spheroids and stem cell EBs of different sizes. After optimization, we have further imaged and measured E-cadherin-mediated forces in these 3D spheroids and EBs for the first time. Our results indicated that these DNA-based molecular tension probes can be used to study the spatiotemporal distributions of target mechanotransduction processes. These powerful imaging tools may be potentially applied to fill the gap between ongoing research of biomechanics in 2D systems and that in real 3D cell complexes.

Curcumin nanoparticles combined with 3D printed bionic tumor models for breast cancer treatment.

Compared with conventional therapeutic approaches, nanomedicines are attracting a growing interest due to their better targeting ability, higher delivery efficiency, and good water solubility. However, conventional drug efficacy assessment methods are based on a two-dimensional (2D) culture approach of single cells to obtainin vitrotherapeutic effects, which may not be representative of actual tumors. Based on the above considerations, the three-dimensional (3D) cell culture models became a better choice since they can increase the complexity ofin vitrosystems and provide a biomimetic microenvironment that is closer to thein vivonative than 2D cultures. In our study, curcumin nanoparticle (CurNPs) with good water solubility and good tumor therapeutic effects were prepared by combining polymeric non-ionic surfactant (Pluronic F127) with curcumin. The hybrid scaffolds based on nano-clay, sodium alginate, and gelatin were also prepared, which showed good printability and excellent biocompatibility. We then studied the therapeutic effects of CurNPs on metastatic breast cancer using a 3D tumor model fabricated with scaffold-bound metastatic breast cancer (MDA-MB-231) cells. It was showed that the 3D cell model presented better cell proliferation effect while compared with 2D version. Additionally, there was good enhanced permeability and retention effect when CurNPs entered with better accumulate in 3D cell 'tumor' sites which represented more realistic response of a more real tumor treatment effect for breast cancer cells. Our study indicated that the combinational of nanomaterials with 3D cell 'tumor' models provided an alternative and better platform for drug screening and has great potential be used as safe and effective treatment screening for breast cancer.

[Establishment of a culture system for human nasal mucosa organoids with controllable differentiation].

OBJECTIVE: To establish a culture system for human nasal mucosal organoids with controllable differentiation to reproduce the structure and function of the source tissue through staged expansion-differentiation culture. METHODS: Fresh samples of surgically resected middle turbinate and nasal polyp tissues were collected, from which the nasal mucosa epithelial cells were isolated by enzymatic digestion and filtration for continuous culture at the air-liquid interface for expansion (EO group) or staged culture for expansion and differentiation (DO group). Immunohistochemical staining was used to characterize the structure, cellular composition and ciliary function of nasal mucosal organoids in the two groups. The secretion function of the differentiated nasal mucosal organoids in DO group was evaluated using PAS staining. RESULTS: Both of the two organoid culture systems yielded vacuolar or solid spherical 3D organoids, and their diameters increased progressively with time. On day 16 of culture, more vacuolar organoids occurred in DO group, while more solid spherical organoids were seen in EO group, and the proportion of vacuoles was significantly greater in DO group than in EO group [(54.67±13.26)% vs (21.67±8.57)%, P < 0.05]. Short tandem repeat (STR) test of the nasal mucosal organoids and the source tissue showed a 100% match between them. On day 21 of culture, scanning and transmission electron microscopy of the nasal mucosal organoids identified ultrastructure of cilia in DO group and short villi structure in most of the organoids in EO group. Immunohistochemical staining showed positivity for P63 (basal cells), ß-tubulin (ciliated columnar cells), and MUC5AC (goblet cells) in the organoids. Compared with those in EO group, the organoids in DO group showed significantly greater percentages of ciliated cells [(7.95±1.81)% vs (27.04±5.91)%, P < 0.05] and goblet cells [(14.46±0.93)% vs (39.85±5.43)%, P < 0.05) with a similar percentage of basal cells [(56.91±14.12)% vs (53.42±15.77)%, P > 0.05]. The differentiated nasal mucosal organoids in DO group were positively stained for glycogen. CONCLUSION: The staged expansion-differentiation culture method allows more stable and prolonged growth of the cultured cells in vitro to produce organoids with controllable differentiation closely resembling the morphological structure and functions (ciliary function and secretory function) of the source tissue.

Synthesis and anticancer activity in vitro and in vivo evaluation of iridium(III) complexes on mouse melanoma B16 cells.

Combining the ligand NPIP (2-(2-nitrophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline) with piq (1-phenylisoquinoline) and bzq (benzo[h]quinolone) gave [Ir(piq)2(NPIP)](PF6) (Ir1), and [Ir(bzq)2(NPIP)](PF6) (Ir2). The newly synthesized complexes were characterized by high-resolution mass spectrometry (HRMS), 1H NMR and 13C NMR. The complexes showed high antiproliferative activity against B16 cells. Three-dimensional (3D) cell model in vitro was used to evaluate the inhibitory effect of iridium (III) complex on B16 cells. The cellular uptake, mitochondrial localization, and intracellular distribution of the drugs confirmed that the iridium (III) complexes targeted the mitochondria, and the complexes can lead to the loss of mitochondrial membrane potential (MMP), increases the intracellular ROS content, further induces apoptosis. We also found that Ir1 and Ir2 can trigger the release of damage-associated molecular patterns (DAMPs) (cell surface calreticulin (CRT), heat-shock protein 70 (HSP70) and high mobility group box 1 (HMGB1)). In addition, Ir1 and Ir2 inhibited glutathione (GSH) synthesis and thus induced oxidative stress, Ir1 and Ir2 promoted malondialdehyde (MDA) production which is the stable metabolite of lipid peroxidation products. Finally, mice xenograft assay was performed to demonstrate that the complex shows higher antitumor activity in vivo than cisplatin. The inhibitory rates for cisplatin and Ir1 are 38.95% and 69.67%, respectively.

HepG2 spheroids as a biosensor-like cell-based system for (geno)toxicity assessment.

3D spheroids developed from HepG2 cells were used as a biosensor-like system for the detection of (geno)toxic effects induced by chemicals. Benzo(a)pyrene (B(a)P) and amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) with well-known mechanisms of action were used for system validation. HepG2 spheroids grown for 3 days were exposed to BaP and PhIP for 24 and 72 h. The growth and viability of spheroids were monitored by planimetry and Live/Dead staining of cells. Multi-parametric flow cytometric analysis was applied for simultaneous detection of specific end-effects including cell cycle analysis (Hoechst staining), cell proliferation (KI67 marker), and DNA double-strand breaks (ℽH2AX) induced by genotoxic compounds. Depending on the exposure concentration/time, BaP reduced spheroid growth, affected cell proliferation by arresting cells in S and G2 phase and induced DNA double-strand breaks (DSB). Simultaneous staining of ℽH2AX formation and cell cycle analysis revealed that after BaP (10 µM; 24 h) exposure 60% of cells in G0/G1 phase had DNA DSB, while after 72 h only 20% of cells contained DSB indicating efficient repair of DNA lesions. PhIP did not influence the spheroid size whereas accumulation of cells in the G2 phase occurred after both treatment times. The evaluation of DNA damage revealed that at 200 µM PhIP 50% of cells in G0/G1 phase had DNA DSB, which after 72-h exposure dropped to 40%, showing lower repair capacity of PhIP-induced DSB compared to BaP-induced. The developed approach using simultaneous detection of several parameters provides mechanistic data and thus contributes to more reliable genotoxicity assessment of chemicals as a high-content screening tool.

Induction of CYP450 by illicit drugs: Studies using an in vitro 3D spheroidal model in comparison to animals.

Information regarding the metabolism of illicit drugs is under urgent need for toxicological assessment. Its development, however, is limited by the currently available animal models. To this end, we proposed three-dimensional (3D) HepaRG spheroids as an in vitro model to study the effects of illicit drugs on hepatic cytochrome P450 (CYP450) enzymes and potential drug-drug interactions (DDIs). By comparing the results from animal and cell experiments, we confirmed the significant impact of heroin, morphine, tetrahydrocannabinol, and fentanyl on CYP450 enzymes, and the 3D spheroids results were in good agreement with the animal results for 2B6, 2C19, 2D6. Using 3D HepaRG spheroids, we demonstrated DDIs between heroin as a 2B6 perpetrator and clinical medicine for cancer, depression, and illicit drug withdrawal. Specifically, the clearance rate of 5.4 µM bupropion was increased by 214 % under DDI with 5 µM heroin, highlighting the importance of DDI pre-screening and individualized medication guidance for illicit drug users. This research contributes to the growing body of evidence regarding the metabolic toxicity of illicit drugs and suggests 3D HepaRG spheroids as a high-throughput and cost-efficient platform for DDI analysis.

The Development of 3D Bovine Intestinal Organoid Derived Models to Investigate Mycobacterium Avium ssp Paratuberculosis Pathogenesis.

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of Johne's Disease, a chronic enteritis of ruminants prevalent across the world. It is estimated that approximately 50% of UK dairy herds are infected with MAP, but this is likely an underestimate of the true prevalence. Infection can result in reduced milk yield, infertility and premature culling of the animal, leading to significant losses to the farming economy and negatively affecting animal welfare. Understanding the initial interaction between MAP and the host is critical to develop improved diagnostic tools and novel vaccines. Here we describe the characterisation of three different multicellular in vitro models derived from bovine intestinal tissue, and their use for the study of cellular interactions with MAP. In addition to the previously described basal-out 3D bovine enteroids, we have established viable 2D monolayers and 3D apical-out organoids. The apical-out enteroids differ from previously described bovine enteroids as the apical surface is exposed on the exterior surface of the 3D structure, enabling study of host-pathogen interactions at the epithelial surface without the need for microinjection. We have characterised the cell types present in each model system using RT-qPCR to detect predicted cell type-specific gene expression, and confocal microscopy for cell type-specific protein expression. Each model contained the cells present in the original bovine intestinal tissue, confirming they were representative of the bovine gut. Exposure of the three model systems to the K10 reference strain of MAP K10, and a recent Scottish isolate referred to as C49, led to the observation of intracellular bacteria by confocal microscopy. Enumeration of the bacteria by quantification of genome copy number, indicated that K10 was less invasive than C49 at early time points in infection in all model systems. This study shows that bovine enteroid-based models are permissive to infection with MAP and that these models may be useful in investigating early stages of MAP pathogenesis in a physiologically relevant in vitro system, whilst reducing the use of animals in scientific research. Bos taurus: urn:lsid:zoobank.org:act:4C90C4FA-6296-4972-BE6A-5EF578677D64.

Development of a device useful to reproducibly produce large quantities of viable and uniform stem cell spheroids with controlled diameters.

Three-dimensional cellular aggregates can mimic the natural microenvironment of tissues and organs and obtaining them through controlled and reproducible processes is mandatory for scaling up and implementing drug cytotoxicity and efficacy tests, as well as tissue engineering protocols. The purpose of this work was to develop and evaluate the performance of a device with two different geometries fabricated by additive manufacturing. The methodology was based on casting a microwell array insert using a non-adhesive hydrogel to obtain highly regular microcavities to standardize spheroid formation and morphology. Spheroids of dental pulp stem cells, bone marrow stromal cells and embryonic stem cells showing high cell viability and average diameters of around 253, 220, and 500 µm, respectively, were produced using the device with the geometry considered most adequate. The cell aggregates showed sphericity indexes above 0.9 and regular surfaces (solidity index higher than 0.96). Around 1000 spheroids could be produced in a standard six-well plate. Overall, these results show that this method facilitates obtaining a large number of uniform, viable spheroids with pre-specified average diameters and through a low-cost and reproducible process for a myriad of applications.

A Simple Method for Generating, Clearing, and Imaging Pre-vascularized 3D Adipospheres Derived from Human iPS Cells.

Beige/brite/brown-like adipocytes (BAs), dispersed in white adipose tissue, represent promising cell targets to counteract obesity and associated diseases. However, there are major limitations for a BA-based treatment of obesity, among which the main ones are the rareness of BAs in adult humans and the lack of a relevant cell culture condition for modeling the development of BAs. We describe in this chapter the capacity of human induced pluripotent stem cells-derived BA progenitors (hiPSC-BAPs) to self-organize in spheroids and a method for their differentiation at a high efficiency in hiPSC-derived 3D adipospheres containing UCP1-expressing cells. Enrichment of adipospheres with human dermal microvascular endothelial cells (HDMECs) allows to better mimic native adipose tissue. To observe the accumulation of lipid droplets, organization of the extracellular matrix and expression of adipogenic markers on the surface of hiPSC-adipospheres, we detail how to combine Oil Red O staining with immunostaining both imaged by fluorescence microscopy. Furthermore, to have a global view of pre-vascularized network formed by HDMECs inside of hiPSC-adipospheres, we describe a method which consists of the whole adipospheres fixation, multicolor immunostaining, clearing, and imaging.

Rapid Evaluation of Novel Therapeutic Strategies Using a 3D Collagen-Based Tissue-Like Model.

2D cell cultures are commonly used to rapidly evaluate the therapeutic potential of various treatments on living cells. However, the effects of the extracellular matrix (ECM) including the 3D arrangement of cells and the complex physiology of native environment are missing, which makes these models far from in vivo conditions. 3D cell models have emerged in preclinical studies to simulate the impact of the ECM and partially bridge the gap between monolayer cultures and in vivo tissues. To date, the difficulty to handle the existing 3D models, the cost of their production and their poor reproducibility have hindered their use. Here, we present a reproducible and commercially available "3D cell collagen-based model" (3D-CCM) that allows to study the influence of the matrix on nanoagent uptake and radiation effects. The cell density in these samples is homogeneous. The oxygen concentration in the 3D-CCM is tunable, which opens the opportunity to investigate hypoxic effects. In addition, thanks to the intrinsic properties of the collagen, the second harmonic imaging microscopy may be used to probe the whole volume and visualize living cells in real-time. Thus, the architecture and composition of 3D-CCMs as well as the impact of various therapeutic strategies on cells embedded in the ECM is observed directly. Moreover, the disaggregation of the collagen matrix allows recovering of cells without damaging them. It is a major advantage that makes possible single cell analysis and quantification of treatment effects using clonogenic assay. In this work, 3D-CCMs were used to evaluate the correlative efficacies of nanodrug exposure and medical radiation on cells contained in a tumor like sample. A comparison with monolayer cell cultures was performed showing the advantageous outcome and the higher potential of 3D-CCMs. This cheap and easy to handle approach is more ethical than in vivo experiments, thus, giving a fast evaluation of cellular responses to various treatments.

Hepatocellular carcinoma (HepG2/C3A) cell-based 3D model for genotoxicity testing of chemicals.

The major weakness of the current in vitro genotoxicity test systems is the inability of the indicator cells to express metabolic enzymes needed for the activation and detoxification of genotoxic compounds, which consequently can lead to misleading results. Thus, there is a significant emphasis on developing hepatic cell models, including advanced in vitro three-dimensional (3D) cell-based systems, which better imitate in vivo cell behaviour and offer more accurate and predictive data for human exposures. In this study, we developed an approach for genotoxicity testing with 21-day old spheroids formed from human hepatocellular carcinoma cells (HepG2/C3A) using the dynamic clinostat bioreactor system (CelVivo BAM/bioreactor) under controlled conditions. The spheroids were exposed to indirect-acting genotoxic compounds, polycyclic aromatic hydrocarbon [PAH; benzo(a) pyrene B(a)P], and heterocyclic aromatic amine [PhIP]) at non-cytotoxic concentrations for 24 and 96 h. The results showed that both environmental pollutants B(a)P and PhIP significantly increased the level of DNA strand breaks assessed by the comet assay. Further, the mRNA level of selected genes encoding metabolic enzymes from phase I and II, and DNA damage responsive genes was determined (qPCR). The 21-day old spheroids showed higher basal expression of genes encoding metabolic enzymes compared to monolayer culture. In spheroids, B(a)P or PhIP induced compound-specific up-regulation of genes implicated in their metabolism, and deregulation of genes implicated in DNA damage and immediate-early response. The study demonstrated that this model utilizing HepG2/C3A spheroids grown under dynamic clinostat conditions represents a very sensitive and promising in vitro model for genotoxicity and environmental studies and can thus significantly contribute to a more reliable assessment of genotoxic activities of pure chemicals, and complex environmental samples even at very low for environmental exposure relevant concentrations.