Simplified low-cost methodology to establish,histologically process and analyze three-dimensional cancer cell spheroid arrays

Differently of two-dimensional cell culture, three-dimensional (3D) multicellular spheroid model allows cells to establish cell-cell/cell-matrix interactions over the entire cell surface, more closely mimicking tumor microenvironments and cellular subpopulations with specific standards of morphology, differentiation and gene expression. Thenceforth several methodologies involving or the 3D cell aggregates generation or its histological processing and analysis have emerged, but in general they are laborious, expensive and complex to set up as a routine technique. Thus, we developed a complete methodology, detailing a simple, accessible and low-cost step by step, including 1) the 3D cell aggregate generation using hanging drop technique; 2) providing a simple way to assess morphological parameters of generated spheroids; followed by 3) a multiple and organized histological processing, keeping several individual spheroids inside an agarose apparatus, maintaining a known order and position of each ones, similar to tissue microarray principle; 4) until the last step, where it is allowed a simultaneous histological composition analysis of several spheroid slices, organized side by side, in a same block section, through conventional stainings or 5) immunostaining against different molecular markers. Therefore, the present methodology aims to popularize 3D cell culture, allowing to make this a regular technique in basic cell biology research, once all steps are performed without using onerous reagents, materials or equipment. In addition to bring the agarose apparatus as a simple low cost novelty, allowing high-throughput analysis of several spheroids simultaneously in an organized manner.     

Colony,hanging drop,and methylcellulose three dimensional hypoxic growth optimization of renal cell carcinoma cell lines

Renal cell carcinoma (RCC) is the most lethal of the common urologic malignancies, comprising 3% of all human neoplasms, and the incidence of kidney cancer is rising annually. We need new approaches to target tumor cells that are resistant to current therapies and that give rise to recurrence and treatment failure. In this study, we focused on low oxygen tension and three-dimensional (3D) cell culture incorporation to develop a new RCC growth model. We used the hanging drop and colony formation methods, which are common in 3D culture, as well as a unique methylcellulose (MC) method. For the experiments, we used human primary RCC cell lines, metastatic RCC cell lines, human kidney cancer stem cells, and human healthy epithelial cells. In the hanging drop assay, we verified the potential of various cell lines to create solid aggregates in hypoxic and normoxic conditions. With the semi-soft agar method, we also determined the ability of various cell lines to create colonies under different oxygen conditions. Different cell behavior observed in the MC method versus the hanging drop and colony formation assays suggests that these three assays may be useful to test various cell properties. However, MC seems to be a particularly valuable alternative for 3D cell culture, as its higher efficiency of aggregate formation and serum independency are of interest in different areas of cancer biology.     

Multiplexing Spheroid Volume,Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.     

The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells

Background aimsThe 3-dimensional (3-D) culture of various cell types reflects the in vivo situation more precisely than 2-dimensional (2-D) cell culture techniques. Spheroids as 3-D cell constructs have been used in tumor research for a long time. They have also been used to study angiogenic mechanisms, which are essential for the success of many tissue-engineering approaches. Several methods of forming spheroids are known, but there is a lack of systematic studies evaluating the performance of these techniques.MethodsWe evaluated the performance of the hanging drop technique, carboxymethyl cellulose technique and liquid overlay technique to form both mono- and co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. The performance of the three techniques was evaluated in terms of rate of yield and reproducibility. The size of the generated spheroids was determined systematically.ResultsThe liquid overlay technique was the most suitable for generating spheroids reproducibly. The rate of yield for this technique was between 60% and 100% for monoculture spheroids and 100% for co-culture spheroids. The size of the spheroids could be adjusted easily and precisely by varying the number of seeded cells organized in one spheroid. The formation of co-culture spheroids consisting of three different cell types was possible.ConclusionsOur results show that the most suitable technique for forming spheroids can vary from the chosen cell type, especially if primary cells are used. Co-culture spheroids consisting of three different cell types will be used to study angiogenic phenomena in further studies.     

3D Spheroids’ Sensitivity to Electric Field Pulses Depends on Their Size

Dramatic differences of cells behavior exist between cells cultured under classical 2D monolayers and 3D models, the latter being closer to in vivo responses. Thus, many 3D cell culture models have been developed. Among them, multicellular tumor spheroid appears as a nice and easy-to-handle 3D model based on cell adhesion properties. It is composed of one or several cell types and is widely used to address carcinogenesis, or drugs screening. A few and recent publications report the use of spheroids to investigate electropermeabilization process. We studied the response of spheroids to electrical field pulses (EP) in terms of their age, diameter or formation technique. We found that small human HCT-116 colorectal spheroids are more sensitive to electric field pulses than larger ones. Indeed, the growth of spheroids with a diameter of 300 μm decreased by a factor 2 over 4 days when submitted to EP (8 pulses, lasting 100 μs at a 1,300 V/cm field intensity). Under those electrical conditions, 650 μm spheroids were not affected. These data were the same whatever the formation method (i.e. hanging drop and nonadherent techniques). These observations point out the fact that characteristics of 3D cell models have to be taken into account to avoid biased conclusions of experimental data.     

Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types

Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue.     

3D spheroid cultures improve the metabolic gene expression profiles of HepaRG cells

3D (three-dimensional) cultures are considered to be an effective method for toxicological studies; however, little evidence has been reported whether 3D cultures have an impact on hepatocellular physiology regarding lipid or glucose metabolism. In the present study, we conducted physiological characterization of hepatoma cell lines HepG2 and HepaRG cells cultured in 3D conditions using a hanging drop method to verify the effect of culture environment on cellular responses. Apo (Apolipoprotein)B as well as albumin secretion was augmented by 3D cultures. Expression of genes related to not only drug, but also glucose and lipid metabolism were significantly enhanced in 3D cultured HepaRG spheroids. Furthermore, mRNA levels of CYP (cytochrome P450) enzymes following exposure to corresponding inducers increased under the 3D condition. These data suggest that this simple 3D culture system without any special biomaterials can improve liver-specific characteristics including lipid metabolism. Considering that the system enables high-throughput assay, it may become a powerful tool for compound screening concerning hepatocellular responses in order to identify potential drugs.     

Scaffold-free 3D culturing enhance pluripotency,immunomodulatory factors,and differentiation potential of Wharton’s jelly-mesenchymal stem cells

Mesenchymal stem cells (MSCs) show a decline in pluripotency and differentiation with increased cell culture passages in 2D cultures. The 2D monolayer culture fails to correctly imitate the architecture and microenvironments of in-vivo cell models. Alternatively, 3D culture may improve the simulations of in-vivo cell microenvironments with wide applications in cell culture and drug discovery. In the present study, we compared various 3D culturing techniques such as 3D micro-well (3D-S), hanging drop (HD), and ultra-low attachment (ULA) plate-based spheroid culture to study their effect on morphology, viability, pluripotency, cell surface markers, immunomodulatory factors, and differentiation capabilities of Wharton’s jelly-mesenchymal stem cells (WJ-MSCs). The cell morphology, viability, and senescence of 3D cultured WJ-MSCs were comparable to cells in 2D culture. The expression of pluripotency markers (OCT4, SOX2, and NANOG) was enhanced upto 2–8 fold in 3D cultured WJ-MSCs when compared to 2D culture. Moreover, the immunomodulatory factors (IDO, IL-10, LIF, ANG1, and VEGF) were significantly elevated in ULA based 3D cultured WJ-MSCs. Furthermore, significant enhancement in the differentiation potential of WJ-MSCs towards adipocyte (ADP and C/EBP-α), osteocyte (OPN and RUNX2), and definitive endodermal (SOX17, FOXA2, and CXCR4) lineages in 3D culture conditions were observed. Additionally, the osteogenic and adipogenic differentiation potential of WJ-MSCs over the time points 7 days, 14 days, and 28 days was also significantly increased in 3D culture groups. Our study demonstrates that stemness properties of WJ-MSCs were significantly enhanced in 3D cultures and ULA-based culture outperformed other methods with high pluripotency gene expression and enhanced differentiation potential. This study indicates the efficacy of 3D cultures to bridge the gap between 2D cell culture and animal models in regenerative medicine.     

Simultaneous quantitative measurement of luciferase reporter activity and cell number in two- and three-dimensional cultures of hepatitis C virus replicons

Hepatitis C virus (HCV) is a global health problem and an important human pathogen. The development of cell culture models for HCV infection has been difficult to accomplish, primarily because HCV is very sensitive to the host cell state. Future models will require the use of three-dimensional (3D) cultures that model the host cell state and environment more accurately. Higher information content screens for anti-HCV therapeutics will also involve 3D cell cultures. Here we report a method for screening cell models for HCV replication that involves normalizing luciferase reporter activity based on cell number in two-dimensional (2D) and 3D HCV replicon cultures. Human hepatoma cells stably replicating luciferase-containing HCV replicons were cultured in 2D monolayer culture and 3D spheroid culture. Optimization of cell lysis was performed so that cell lysates could be used to quantify both luciferase activity and cellular DNA content. Cellular DNA content was quantified using Hoechst 33258 dye and was converted to cell number. The method is straightforward, reproducible, and sensitive down to 5000 cells. This method enables low-throughput but high-information content screening of HCV replicons, with the potential for high-throughput screening in a variety of 3D cultures and cocultures.     

PDMS well platform for culturing millimeter‐size tumor spheroids

Multicellular tumor spheroids are widely used as in vitro models for testing of anticancer drugs. The advantage of this approach is that it can predict the outcome of a drug treatment on human cancer cells in their natural three‐dimensional environment without putting actual patients at risk. Several methods were utilized in the past to grow submillimeter‐size tumor spheroids. However, these small models are not very useful for preclinical studies of tumor ablation where the goal is the complete destruction of tumors that can reach several centimeters in diameter in the human body. Here, we propose a PDMS well method for large tumor spheroid culture. Our experiments with HepG2 hepatic cancer cells show that three‐dimensional aggregates of tumor cells with a volume as large as 44 mm³ can be grown in cylindrical PDMS wells after the initial culture of tumor cells by the hanging drop method. This is a 350 times more than the maximum volume of tumor spheroids formed inside hanging drops (0.125 mm³). © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1265–1269, 2013     

Optimization of the formation of embedded multicellular spheroids of MCF-7 cells: How to reliably produce a biomimetic 3D model

To obtain a multicellular MCF-7 spheroid model to mimic the three-dimensional (3D) of tumors, the microwell liquid overlay (A) and hanging-drop/agar (B) methods were first compared for their technical parameters. Then a method for embedding spheroids within collagen was optimized. For method A, centrifugation assisted cells form irregular aggregates but not spheroids. For method B, an extended sedimentation period of over 24 h for cell suspensions and increased viscosity of the culture medium using methylcellulose were necessary to harvest a dense and regular cell spheroid. When the number was less than 5000 cells/drop, embedded spheroids showed no tight cores and higher viability than the unembedded. However, above 5000 cells/drop, cellular viability of embedded spheroids was not significantly different from unembedded spheroids and cells invading through the collagen were in a sun-burst pattern with tight cores. Propidium Iodide staining indicated that spheroids had necrotic cores. The doxorubicin cytotoxicity demonstrated that spheroids were less susceptible to DOX than their monolayer cells. A reliable and reproducible method for embedding spheroids using the hanging-drop/agarose method within collagen is described herein. The cell culture model can be used to guide experimental manipulation of 3D cell cultures and to evaluate anticancer drug efficacy.     

Ultra High Content Image Analysis and Phenotype Profiling of 3D Cultured Micro-Tissues

In many situations, 3D cell cultures mimic the natural organization of tissues more closely than 2D cultures. Conventional methods for phenotyping such 3D cultures use either single or multiple simple parameters based on morphology and fluorescence staining intensity. However, due to their simplicity many details are not taken into account which limits system-level study of phenotype characteristics. Here, we have developed a new image analysis platform to automatically profile 3D cell phenotypes with 598 parameters including morphology, topology, and texture parameters such as wavelet and image moments. As proof of concept, we analyzed mouse breast cancer cells (4T1 cells) in a 384-well plate format following exposure to a diverse set of compounds at different concentrations. The result showed concentration dependent phenotypic trajectories for different biologically active compounds that could be used to classify compounds based on their biological target. To demonstrate the wider applicability of our method, we analyzed the phenotypes of a collection of 44 human breast cancer cell lines cultured in 3D and showed that our method correctly distinguished basal-A, basal-B, luminal and ERBB2+ cell lines in a supervised nearest neighbor classification method.     

Application of hanging drop technique for stem cell differentiation and cytotoxicity studies

The aim of our study is to explore the possibility of using an ancient method of culture technique- the hanging drop technique for stem cell differentiation and cytotoxicity testing. We demonstrate here a variety of novel applications of this age old technique not only to harness the differentiation potential of stem cells into specific lineages but also for cytotoxicity studies. Here we have prepared hanging drop cultures by placing 20 μl micro-drops of nutrient media and 10% Fetal Calf Serum (FCS) containing cells of interest on the lids of 60 mm dishes. Bottom plates of the dishes were filled with sterile Phosphate Buffer Saline (PBS) to avoid desiccation of samples. Lids were then placed on the bottom plates to achieve hanging drop cultures. We utilized this technique for cultivation of ciliated epithelia to study cytotoxicity and differentiation of bone marrow stromal cells. Most importantly the modified culture technique presented here is simple, economical and cost effective in terms of the time taken and the reagents required and are amenable to goal specific modification such as cytotoxicity testing. It is advantageous over the existing system in terms of retention of viability and functionality for longer duration and for providing three dimensional growth micro-environment making it useful for organotypic cultures and in vivo simulation.     

Multi-Contrast Imaging and Digital Refocusing on a Mobile Microscope with a Domed LED Array

We demonstrate the design and application of an add-on device for improving the diagnostic and research capabilities of CellScope—a low-cost, smartphone-based point-of-care microscope. We replace the single LED illumination of the original CellScope with a programmable domed LED array. By leveraging recent advances in computational illumination, this new device enables simultaneous multi-contrast imaging with brightfield, darkfield, and phase imaging modes. Further, we scan through illumination angles to capture lightfield datasets, which can be used to recover 3D intensity and phase images without any hardware changes. This digital refocusing procedure can be used for either 3D imaging or software-only focus correction, reducing the need for precise mechanical focusing during field experiments. All acquisition and processing is performed on the mobile phone and controlled through a smartphone application, making the computational microscope compact and portable. Using multiple samples and different objective magnifications, we demonstrate that the performance of our device is comparable to that of a commercial microscope. This unique device platform extends the field imaging capabilities of CellScope, opening up new clinical and research possibilities.     

Fluorinert, an oxygen carrier, improves cell culture performance in deep square 96-well plates by facilitating oxygen transfer

In bioprocess development, the 96-well plate format has been widely used for high-throughput screening of production cell line or culture conditions. However, suspension cell cultures in conventional 96-well plates often fail to reach high cell density under normal agitation presumably due to constraints in oxygen transfer. Although more vigorous agitation can improve gas transfer in 96-well plate format, it often requires specialized instruments. In this report, we employed Fluorinert, a biologically inert perfluorocarbon, to improve oxygen transfer in 96-well plate and to enable the growth of a Chinese Hamster Ovary cell line expressing a recombinant monoclonal antibody. When different amounts of Fluorinert were added to the cell culture medium, a dose-dependent improvement in cell growth was observed in both conventional and deep square 96-well plates. When sufficient Fluorinert was present in the culture, the cell growth rate, the peak cell density, and recombinant protein production levels achieved in deep square 96-wells were comparable to cultures in ventilated shake flasks. Although Fluorinert is known to dissolve gases such as oxygen and CO(2), it does not dissolve nor extract medium components, such as glucose, lactate, or amino acids. We conclude that mixing Fluorinert with culture media is a suitable model for miniaturization of cell line development and process optimization. Proper cell growth and cellular productivity can be obtained with a standard shaker without the need for any additional aeration or vigorous agitation.     

Array-based ELISAs for high-throughput analysis of human cytokines

In this report, we describe the development of a mini-array system suitable for high-throughput quantification of proteins. This mini-array is a multiplexed, sandwich-type ELISA that measures the concentration of seven different human cytokines--TNF-alpha, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-6, and IL-10--from a single sample in each well of a 96-well plate. The mini-array is produced by spotting monoclonal antibodies (mAbs) in a 3 x 3 pattern in the bottom of the wells of 96-well polystyrene plates. Cytokines that are captured by the arrayed mAbs are detected by using biotinylated mAbs, followed by the addition of a streptavidin-horseradish peroxidase (HRP) conjugate and a chemiluminescent substrate. The light produced from the HRP-catalyzed oxidation of the substrate is measured at each spot in the array by imaging the entire plate with a commercially available CCD camera. Here, we demonstrate that these 96-well-plate format mini-arrays have performance characteristics that make them suitable for the high-throughput screening of anti-inflammatory compounds.     

Identification of the Cancer Cell Proliferation and Survival Functions of proHB-EGF by Using an Anti-HB-EGF Antibody

Purpose

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family. The membrane-bound proHB-EGF is known to be a precursor of the soluble form of HB-EGF (sHB-EGF), which promotes cell proliferation and survival. While the functions of sHB-EGF have been extensively studied, it is not yet fully understood if proHB-EGF is also involved in cellular signaling events. In this study, we utilized the anti-HB-EGF monoclonal antibodies Y-142 and Y-073, which have differential specificities toward proHB-EGF, in order to elucidate proHB-EGF functions in cancer cells.

Experimental Design

The biological activities of proHB-EGF were assessed in cell proliferation, caspase activation, and juxtacrine activity assays by using a 3D spheroid culture of NUGC-3 cells.

Results

Y-142 and Y-073 exhibited similar binding and neutralizing activities for sHB-EGF. However, only Y-142 bound to proHB-EGF. We could detect the function of endogenously expressed proHB-EGF in a 3D spheroid culture. Blocking proHB-EGF with Y-142 reduced spheroid formation, suppressed cell proliferation, and increased caspase activation in the 3D spheroid culture of NUGC-3 cells.

Conclusions

Our results show that proHB-EGF acts as a cell proliferation and cell survival factor in cancer cells. The results suggest that proHB-EGF may play an important role in tumor progression.