High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array

Culture of cells as three-dimensional (3D) aggregates can enhance in vitro tests for basic biological research as well as for therapeutics development. Such 3D culture models, however, are often more complicated, cumbersome, and expensive than two-dimensional (2D) cultures. This paper describes a 384-well format hanging drop culture plate that makes spheroid formation, culture, and subsequent drug testing on the obtained 3D cellular constructs as straightforward to perform and adapt to existing high-throughput screening (HTS) instruments as conventional 2D cultures. Using this platform, we show that drugs with different modes of action produce distinct responses in the physiological 3D cell spheroids compared to conventional 2D cell monolayers. Specifically, the anticancer drug 5-fluorouracil (5-FU) has higher anti-proliferative effects on 2D cultures whereas the hypoxia activated drug commonly referred to as tirapazamine (TPZ) are more effective against 3D cultures. The multiplexed 3D hanging drop culture and testing plate provides an efficient way to obtain biological insights that are often lost in 2D platforms.     

On-chip anticancer drug test of regular tumor spheroids formed in microwells by a distributive microchannel network

This paper proposes a new cytotoxicity assay in a microfluidic device with microwells and a distributive microfluidic channel network for the formation of cancer cell spheroids. The assay can generate rapid and uniform cell clusters in microwells and test in situ cytotoxicity of anticancer drugs including sequential drug treatments, long term culture of spheroids and cell viability assays. Inlet ports are connected to the microwells by a hydraulic resistance network. This uniform distribution of cell suspensions results in regular spheroid dimensions. Injected cancer cells were trapped in microwells, and aggregated into tumor spheroids within 3 days. A cytotoxicity test of the spheroids in microwells was subsequently processed in the same device without the extraction of cells. The in situ cytotoxicity assay of tumor spheroids in microwells was comparable with the MTT assay on hanging drop spheroids using a conventional 96-well plate. It was observed that the inhibition rate of the spheroids was less than that in the 2D culture dish and the effect on tumor spheroids was different depending on the anticancer drug. This device could provide a convenient in situ assay tool to assess the cytotoxicity of anticancer drugs on tumor spheroids, offering more information than the conventional 2D culture plate.     

Magnetic levitating polymeric nano/microparticular substrates for three-dimensional tumor cell culture

Herein, we describe magnetic cell levitation models using conventional polymeric microparticles or nanoparticles as a substrate for the three-dimensional tumor cell culture. When the magnetic force originating from the ring-shaped magnets overcame the gravitational force, the magnetic field-levitated KB tumor cells adhered to the surface area of magnetic iron oxide (Fe(3)O(4))-encapsulated nano/microparticles and concentrated clusters of levitated cells, ultimately developing tumor cells to tumor spheroids. These simple cell culture models may prove useful for the screening of anticancer drugs and their formulations.     

Three-Dimensional Aggregated Spheroid Model of Hepatocellular Carcinoma Using a 96-Pillar/Well Plate

A common method of three-dimensional (3D) cell cultures is embedding single cells in Matrigel. Separated cells in Matrigel migrate or grow to form spheroids but lack cell-to-cell interaction, which causes difficulty or delay in forming mature spheroids. To address this issue, we proposed a 3D aggregated spheroid model (ASM) to create large single spheroids by aggregating cells in Matrigel attached to the surface of 96-pillar plates. Before gelling the Matrigel, we placed the pillar inserts into blank wells where gravity allowed the cells to gather at the curved end. In a drug screening assay, the ASM with Hepatocellular carcinoma (HCC) cell lines showed higher drug resistance compared to both a conventional spheroid model (CSM) and a two-dimensional (2D) cell culture model. With protein expression, cytokine activation, and penetration analysis, the ASM showed higher expression of cancer markers associated with proliferation (p-AKT, p-Erk), tight junction formation (Fibronectin, ZO-1, Occludin), and epithelial cell identity (E-cadherin) in HCC cells. Furthermore, cytokine factors were increased, which were associated with immune cell recruitment/activation (MIF-3α), extracellular matrix regulation (TIMP-2), cancer interaction (IL-8, TGF-β2), and angiogenesis regulation (VEGF-A). Compared to CSM, the ASM also showed limited drug penetration in doxorubicin, which appears in tissues in vivo. Thus, the proposed ASM better recapitulated the tumor microenvironment and can provide for more instructive data during in vitro drug screening assays of tumor cells and improved prediction of efficacious drugs in HCC patients.     

Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits

We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 μm. The diffusive limit of competition for media occurred with a pitch of ≥1250 μm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research.     

A multicellular spheroid formation and extraction chip using removable cell trapping barriers

This paper presents a multicellular spheroid chip capable of forming and extracting three-dimensional (3D) spheroids using removable cell trapping barriers. Compared to the conventional macro-scale spheroid formation methods, including spinning, hanging-drop, and liquid-overlay methods, the recent micro-scale spheroid chips have the advantage of forming smaller spheroids with better uniformity. The recent micro spheroid chips, however, have difficulties in extracting the spheroids due to fixed cell trapping barriers. The present spheroid chip, having two PDMS layers, uses removable cell trapping barriers, thereby making it easy to form and extract uniform and small-sized spheroids. We have designed, fabricated and characterized a 4 × 1 spheroid chip, where membrane cell trapping barriers are inflated at a pressure of 50 kPa for spheroid formation and are deflated at zero gauge pressure for simple and safe extraction of the spheroids formed. In this experimental study, the cell suspension of non-small lung cancer cells, H1650, is supplied to the fabricated spheroid chip in the pressure range 145-155 Pa. The fabricated spheroid chips collect the cancer cells in the cell trapping regions from the cell suspension at a concentration of 2 × 10(6) ml(-1), thus forming uniform 3D spheroids with a diameter of 197.2 ± 11.7 μm, after 24 h incubation at 5% CO(2) and 37°C environment. After the removal of the cell trapping barriers, the spheroids formed were extracted through the outlet ports at a cell inlet pressure of 5 kPa. The cells in the extracted spheroids showed a viability of 80.3 ± 7.7%. The present spheroid chip offers a simple and effective method of obtaining uniform and small-sized 3D spheroids for the next stage of cell-based biomedical research, such as gene expression analysis and spheroid inoculation in animal models.     

Studies of anticancer drug cytotoxicity based on long‐term HepG2 spheroid culture in a microfluidic system

Cell‐on‐a‐chip systems have become promising devices to study the effectiveness of new anticancer drugs recently. Several microdevices for liver cancer culture and evaluation of the drug cytotoxicity have been reported. However, there are still no proven reports about high‐throughput and simple methods for the evaluation of drug cytotoxicity on liver cancer cells. The paper presents the results of the effects of the anticancer drug (5‐fluorouracil, 5‐FU) on the HepG2 spheroids as a model of liver cancer. The experiments were based on the long‐term 3D spheroid culture in the microfluidic system and monitoring of the effect of 5‐FU at two selected concentrations (0.5 mM and 1.0 mM). Our investigations have shown that the initial size of the spheroids has influence on the drug effect. With the increase of the spheroids diameter, the drug resistance (for the two tested 5‐FU concentrations) decreases. This phenomenon was observed both through cells metabolism analysis, as well as changes in spheroids sizes. In our research, we have shown that the lower 5‐FU (0.5 mM) concentration causes higher decrease in HepG2 spheroids viability. Moreover, due to the microsystem construction, we observe the drug resistance effect (10th day of culture) regardless of the initial size of the created spheroids and the drug concentration.     

Systematic Research and Evaluation Models of Nanomotors for Cancer Combined Therapy

Limited tumor permeability of therapeutic agents is a great challenge faced by current cancer therapy methods. Herein, a kind of near infrared light (NIR)‐driven nanomotor with autonomous movement, targeted ability, hierarchical porous structure, multi‐drugs for cancer chemo/photothermal therapy is designed, prepared and characterized. Further, we establish a method to study the interaction between nanomotors and cells, along with their tumor permeability mechanism, including 2D cellular models, 3D multicellular tumor spheroids and in vivo models. In vivo tumor elimination results verify that the movement behaviour of the nanomotors can greatly facilitate them to eliminate tumor through multiple therapeutic methods. This work tries to establish systematic research and evaluation models, providing strategies to understand the relationship between motion behaviour and tumor permeation efficiency of nanomotors in depth.     

Drug cytotoxicity and signaling pathway analysis with three-dimensional tumor spheroids in a microwell-based microfluidic chip for drug screening

Currently, there has been a growing need for developing in vitro models to better reflect organism response to chemotherapy at tissue level. For this reason, a microfluidic platform was developed for mimicking physiological microenvironment of solid tumor with multicellular tumor spheroids (MTS) for anticancer drug screening. Importantly, the power of this system over traditional systems is that it is simple to operate and high integration in a more physiologically relevant context. As a proof of concept, long-term MTS cultures with uniform structure were realized on the microfluidic based platform. The response of doxorubicin and paclitaxel on different types of spheroids were simultaneously performed by in situ Live/Dead fluorescence stain to provide spatial distribution of dead cells as well as cytotoxicity information. In addition, the established platform combined with microplate reader was capable to determine the cytotoxicity of different sized MTS, showing a more powerful tool than cell staining examination at the end-point of assay. The HCT116 spheroids were then lysed on chip followed by signaling transduction pathway analysis. To our knowledge, the on chip drug screening study is the first to address the drug susceptibility testing and the offline detailed drug signaling pathway analysis combination on one system. Thus, this novel microfluidic platform provides a useful tool for drug screening with tumor spheroids, which is crucial for drug discovery and development.     

Spheroid cultivation of HT-29 carcinoma cell line in liquid marbles

The ability to simulate the 3D structure of a human body is essential to increase the efficiency of drug development. In vivo conditions are significantly different in comparison to in vitro conditions. A standardly used cell monolayer on tissue culture plastic (2D cell culture) is not sufficient to simulate the transfer phenomena occurring in living organisms, therefore, cell growth in a 3D space is desired. Drug absorption, distribution, metabolism, excretion and toxicity could be tested on 3D cell aggregates called spheroids, decrease the use of animal models and accelerate the drug development. In this work, the formation of spheroids from HT-29 human colorectal adenocarcinoma cells was successfully achieved by means of the so-called liquid marbles, which are liquid droplets encapsulated by a hydrophobic powder. During the cultivation in the medium inside the liquid marbles, cells spontaneously formed spherical agglomerates (spheroids) without the need of any supporting scaffold. The study focused on the influence of different parameters—namely liquid marble volume, seeding cell density and time of cultivation—on the final yield and quality of spheroids. This work has shown that using liquid marbles as microbioreactors is a suitable method for the cultivation of HT-29 cells in the form of spheroids.     

nurP28, a New-to-Nature Zein-Derived Peptide,Enhances the Therapeutic Effect of Docetaxel in Breast Cancer Monolayers and Spheroids

The development of novel cancer therapeutic strategies has garnered increasing interest in cancer research. Among the therapeutic choices, chemosensitizers have shown exciting prospects. Peptides are an attractive alternative among the molecules that may be used as chemosensitizers. We rationally designed a new-to-nature peptide, nurP28, derived from the 22-kDa α-zein protein sequence (entry Q00919_MAIZE). The resultant sequence of the nurP28 peptide after the addition of arginine residues was LALLALLRLRRRATTAFIIP, and we added acetyl and amide groups at the N- and C-terminus, respectively, for capping. We evaluated the cytotoxicity of the nurP28 peptide alone and in combination with docetaxel in fibroblast monolayers and breast cancer monolayers and spheroids. Our results indicated that nurP28 is not cytotoxic to human fibroblasts or cancer cells. Nevertheless, when combined with 1 µM docetaxel, 3 ng/mL nurP28 induced equivalent (in MCF7 monolayers) and higher (in MCF7 spheroids) cytotoxic effects than 10-fold higher doses of docetaxel alone. These findings suggest that nurP28 may act as a chemosensitizer in breast cancer treatment. This study describes the enhancing “anti-cancer” effects of nurP28 in breast cancer 2D and 3D cultures treated with docetaxel. Further studies should explore the mechanisms underlying these effects and assess the clinical potential of our findings using animal models.     

GROWTH DELAY STUDIES OF THE RESPONSE OF V-79 MULTICELL SPHEROIDS EXPOSED TO DHE and RED LIGHT

Abstract Multicell tumour spheroids (MTS) of V-79 Chinese hamster cells have been used to study the role of a number of treatment and microenvironmental parameters in the modification of tumour response to Photodynamic Therapy (PDT) using visible light in combination with the photosensitizing compound dihematoporphyrin ether (DHE). The kinetics of DHE uptake into MTS, determined by fluorimetry of extracted porphyrins, indicate that after extended incubation (i.e. 24 h) the mean cellular DHE content in larger (˜300 μ.m and 400 u.m) MTS is significantly less than that for smaller (˜200 μm) MTS, consistent with a hypothesis that DHE uptake into the internal regions of spheroids is diffusion-limited. The response of spheroids to PDT, as assessed by the endpoint of growth delay, indicates that the kinetics of spheroid volume alteration and cell loss, as well as the potential for regfrrwth, are markedly dependent on both the drug and light exposure levels used. The oxygen dependence of this response has been investigated after light irradiation of spheroid cultures equilibrated with either 21% O₂ (i.e. air) or 0% 0₂ (i.e. N₂). While treatment in air results in significant growth delay, the growth kinetics of DHE-treated spheroids irradiated under N₂ were essentially unchanged from those of untreated spheroids. These observations clearly demonstrate an important role for oxygen, at the time of irradiation, in determining the response of spheroids to PDT.     

Comparison of protein expression profiles between monolayer and spheroid cell culture of HT-29 cells revealed fragmentation of CK18 in three-dimensional cell culture

The use of three-dimensional cell culture models, so-called multicellular tumor spheroids, is a special approach in experimental cancer research, because spheroids are similar to in vivo tumors in structural as well as functional sense. Cells grown in spheroids exhibit alterations of cell cycle regulation, induction of apoptosis and differentiation and can acquire multidrug resistance. In this study we investigated the protein expression in human colorectal cancer cells grown in monolayer and in spheroid cultures using proteomics. Evaluation by computer-assisted image analysis revealed overexpression of three cytokeratin 18 fragments that were generated in vivo. Cytokeratin 18 has previously been described as a target for caspase-mediated cleavage during apoptosis and our results indicate that apoptosis may take place in spheroids. Other proteins upregulated in spheroids include calreticulin precursor, a rho GDP dissociation inhibitor variant, several cytokeratins and peroxiredoxin 4. Some of these proteins have already been linked to chemoresistance and apoptotic phenomena.     

Biohybrid microarrays – Impedimetric biosensors with 3D in vitro tissues for toxicological and biomedical screening

To investigate the effectiveness of potential anti-cancer therapeutics or therapies, efficient screening methods are required. On the one hand, multicellular 3D aggregates (spheroids) are a powerful in vitro model for simulating the in vivo situation and on the other hand, planar electrode structures are generally highly suitable for automation and parallel testing. Here, the detection of the effect of active substances on spheroids positioned on electrodes of substrate integrated electrode arrays is exemplarily investigated. As a 3D tissue model a reaggregation system of T47D clone 11 tumor cells is used. The effect of cytotoxins (DMSO, Triton X-100) on spheroids can be detected by recording the effective impedance of planar electrodes covered by spheroids. The equivalent circuit model parameter of electrodes covered by cytotoxin treated spheroids are determined from recorded impedance spectra and compared to the parameter of electrodes covered by control spheroids as well as not covered electrodes. Spheroids on electrodes mainly influence the electrode impedance in the frequency range of 10 kHz to 1 MHz. The results are discussed in view of an optimal electrode/spheroid-interface for sensing the effects of therapeutics with high sensitivity.     

Biohybrid microarrays--impedimetric biosensors with 3D in vitro tissues for toxicological and biomedical screening

To investigate the effectiveness of potential anticancer therapeutics or therapies, efficient screening methods are required. On the one hand, multicellular 3D aggregates (spheroids) are a powerful in vitro model for simulating the in vivo situation and on the other hand, planar electrode structures are generally highly suitable for automation and parallel testing. Here, the detection of the effect of active substances on spheroids positioned on electrodes of substrate integrated electrode arrays is exemplarily investigated. As a 3D tissue model a reaggregation system of T47D clone 11 tumor cells is used. The effect of cytotoxins (DMSO, Triton X-100) on spheroids can be detected by recording the effective impedance of planar electrodes covered by spheroids. The equivalent circuit model parameter of electrodes covered by cytotoxin treated spheroids are determined from recorded impedance spectra and compared to the parameter of electrodes covered by control spheroids as well as not covered electrodes. Spheroids on electrodes mainly influence the electrode impedance in the frequency range of 10 kHz to 1 MHz. The results are discussed in view of an optimal electrode/spheroid-interface for sensing the effects of therapeutics with high sensitivity.     

Mass spectrometry imaging of endogenous metabolites in response to doxorubicin in a novel 3D osteosarcoma cell culture model

Three‐dimensional (3D) cell culture is a rapidly emerging field, which mimics some of the physiological conditions of human tissues. In cancer biology, it is considered a useful tool in predicting in vivo chemotherapy responses, compared with conventional two‐dimensional (2D) cell culture. We have developed a novel 3D cell culture model of osteosarcoma composed of aggregated proliferative tumour spheroids, which shows regions of tumour heterogeneity formed by aggregated spheroids of polyclonal tumour cells. Aggregated spheroids show local necrotic and apoptotic regions and have sizes suitable for the study of spatial distribution of metabolites by mass spectrometry imaging (MSI). We have used this model to perform a proof‐of‐principle study showing a heterogeneous distribution of endogenous metabolites that colocalise with the necrotic core and apoptotic regions in this model. Cytotoxic chemotherapy (doxorubicin) responses were significantly attenuated in our 3D cell culture model compared with those of standard cell culture, as determined by resazurin assay, despite sufficient doxorubicin diffusion demonstrated by localisation throughout the 3D constructs. Finally, changes to the distribution of endogenous metabolites in response to doxorubicin were readily detected by MSI. Principal component analysis identified 50 metabolites which differed most in their abundance between treatment groups, and of these, 10 were identified by both in‐software t test and mixed‐effects analysis of variance (ANOVA). Subsequent independent MSIs of identified species were consistent with principle component analysis findings. This proof‐of‐principle study shows for the first time that chemotherapy‐induced changes in metabolite abundance and distribution may be determined in 3D cell culture by MSI, highlighting this method as a potentially useful tool in the elucidation of chemotherapy responses as an alternative to in vivo testing.     

Microfluidic platform for studying the anti-cancer effect of ursolic acid on tumor spheroid

At present, the probability that a new anti-tumor drug will eventually succeed in clinical trials is extremely low. In order to make up for this shortcoming, the use of a three-dimensional (3D) cell culture model for secondary screening is often necessary. Cell spheroid is the easiest 3D model tool for drug screening. In this study, the microfluidic chip with a microwell array was manufactured, which could allow the formation of tumor spheroids with uniform size and easily retrieve cell spheroids from the chip. Cell spheroids were successfully cultured for over 15 days and the survival rate was as high as 80%. Subsequently, cellular response to the ursolic acid (UA) was observed on the chip. Compared to the monolayer culture cells in vitro, the tumor spheroids showed minor levels of epithelial-mesenchymal transition fluctuation after drug treatment. The mechanism of cell spheroid resistance to UA was further verified by detecting the expression level of upstream pathway proteins. But the invasive ability of tumor spheroids was attenuated when the duration of action of UA extended. The anti-cancer effect of UA was innovatively evaluated on breast cancer by using the microfluidic device, which could provide a basis and direction for future preclinical research on UA.     

Novel Photodynamic Therapy Using Water-dispersed TiO2–Polyethylene Glycol Compound: Evaluation of Antitumor Effect on Glioma Cells and Spheroids In Vitro

Titanium dioxide (TiO₂) is thought to be a photocatalytic agent excited by UV light. Our aim was to investigate the photocatalytic antitumor effect of water-dispersed TiO₂ nanoparticles on C6 rat glioma cells and to evaluate the treatment responses by the spheroid models. Water-dispersed TiO₂ nanoparticles were constructed by the adsorption of chemical modified polyethylene glycol (PEG) on the TiO₂ surface (TiO₂/PEG). Each monolayer and spheroid of C6 cells was coincubated with various concentrations of TiO₂/PEG and subsequently irradiated with UV light. Damage of the cells and spheroids was evaluated sequentially by staining with the fluorescent dyes. The cytotoxic effect was correlated with the concentration of TiO₂/PEG and the energy dose of UV irradiation. More than 90% of cells were killed after 13.5 J cm⁻² of UV irradiation in the presence of 500 μg mL⁻¹ TiO₂/PEG. The irradiated spheroids in the presence of TiO₂/PEG showed growth suppression compared with control groups. In TiO₂/PEG-treated spheroids, the number of Annexin V-FITC-stained cells gradually increased during the first 6 h, and subsequently propidium iodide-stained cells appeared. The results of this study suggest that newly developed photoexcited TiO₂/PEG have antitumoral activity. Photodynamic therapy utilizing this material can be a clue to a novel therapeutic strategy for glioma.