Spheroid Array Research Articles

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.

Laser-assisted 3D bioprinting of exocrine pancreas spheroid models for cancer initiation study

Pancreatic ductal adenocarcinoma (PDAC) is the most common malignancy of the pancreas. It has shown a poor prognosis and a rising incidence in the developed world. Other pathologies associated with this tissue include pancreatitis, a risk condition for pancreatic cancer. The onset of both pancreatitis and pancreatic cancer follows a common pattern: exocrine pancreatic acinar cells undergo a transdifferentiation to duct cells that triggers a 3D restructuration of the pancreatic tissue. However, the exact mechanism underlying this process remains partially undefined. Further understanding the cellular events leading to PDAC could open new avenues in the development of novel therapeutic approaches. Since current 2D cell culture models fail to mimic the tridimensional complexity of the pancreatic tissue, new in vitro models are urgently needed. Here, we generated 3D pancreatic cell spheroid arrays using laser-assisted bioprinting and characterized their phenotypic evolution over time through image analysis and phenotypic characterization. We show that these bioprinted spheroids, composed of both acinar and ductal cells, can replicate the initial stages of PDAC development. This bioprinted miniaturized spheroid-based array model should prove useful for the study of the internal and external factors that contribute to the formation of precursor PDAC lesions and to cancer progression, and may therefore shed light on future PDAC therapy strategies.

A Cancer Spheroid Array Chip for Selecting Effective Drug.

A cancer spheroid array chip was developed by modifying a micropillar and microwell structure to improve the evaluation of drugs targeting specific mutations such as phosphor-epidermal growth factor receptor (p-EGFR). The chip encapsulated cells in alginate and allowed cancer cells to grow for over seven days to form cancer spheroids. However, reagents or media used to screen drugs in a high-density spheroid array had to be replaced very carefully, and this was a tedious task. Particularly, the immunostaining of cancer spheroids required numerous steps to replace many of the reagents used for drug evaluation. To solve this problem, we adapted a micropillar and microwell structure to a spheroid array. Thus, culturing cancer spheroids in alginate spots attached to the micropillar allowed us to replace the reagents in the microwell chip with a single fill of fresh medium, without damaging the cancer spheroids. In this study, a cancer spheroid array was made from a p-EGFR-overexpressing cell line (A549 lung cancer cell line). In a 12 by 36 column array chip (25 mm by 75 mm), the spheroid over 100 µm in diameter started to form at day seven and p-EGFR was also considerably overexpressed. The array was used for p-EGFR inhibition and cell viability measurement against seventy drugs, including ten EGFR-targeting drugs. By comparing drug response in the spheroid array (spheroid model) with that in the single-cell model, we demonstrated that the two models showed different responses and that the spheroid model might be more resistant to some drugs, thus narrowing the choice of drug candidates.

Inherent Photodegradability of Polymethacrylate Hydrogels: Straightforward Access to Biocompatible Soft Microstructures

AbstractHydrogels are important functional materials useful for 3D cell culture, tissue engineering, 3D printing, drug delivery, sensors, or soft robotics. The ability to shape hydrogels into defined 3D structures, patterns, or particles is crucial for biomedical applications. Here, the rapid photodegradability of commonly used polymethacrylate hydrogels is demonstrated without the need to incorporate additional photolabile functionalities. Hydrogel degradation depths are quantified with respect to the irradiation time, light intensity, and chemical composition. It can be shown that these parameters can be utilized to control the photodegradation behavior of polymethacrylate hydrogels. The photodegradation kinetics, the change in mechanical properties of polymethacrylate hydrogels upon UV irradiation, as well as the photodegradation products are investigated. This approach is then exploited for microstructuring and patterning of hydrogels including hydrogel gradients as well as for the formation of hydrogel particles and hydrogel arrays of well‐defined shapes. Cell repellent but biocompatible hydrogel microwells are fabricated using this method and used to form arrays of cell spheroids. As this method is based on readily available and commonly used methacrylates and can be conducted using cheap UV light sources, it has vast potential to be applied by laboratories with various backgrounds and for diverse applications.

Biofabrication of spatially organised tissues by directing the growth of cellular spheroids within 3D printed polymeric microchambers

Successful tissue engineering requires the generation of human scale implants that mimic the structure, composition and mechanical properties of native tissues. Here, we report a novel biofabrication strategy that enables the engineering of structurally organised tissues by guiding the growth of cellular spheroids within arrays of 3D printed polymeric microchambers. With the goal of engineering stratified articular cartilage, inkjet bioprinting was used to deposit defined numbers of mesenchymal stromal cells (MSCs) and chondrocytes into pre-printed microchambers. These jetted cell suspensions rapidly underwent condensation within the hydrophobic microchambers, leading to the formation of organised arrays of cellular spheroids. The microchambers were also designed to provide boundary conditions to these spheroids, guiding their growth and eventual fusion, leading to the development of stratified cartilage tissue with a depth-dependant collagen fiber architecture that mimicked the structure of native articular cartilage. Furthermore, the composition and biomechanical properties of the bioprinted cartilage was also comparable to the native tissue. Using multi-tool biofabrication, we were also able to engineer anatomically accurate, human scale, osteochondral templates by printing this microchamber system on top of a hypertrophic cartilage region designed to support endochondral bone formation and then maintaining the entire construct in long-term bioreactor culture to enhance tissue development. This bioprinting strategy provides a versatile and scalable approach to engineer structurally organised cartilage tissues for joint resurfacing applications.

Drug screening of biopsy-derived spheroids using a self-generated microfluidic concentration gradient

Performing drug screening of tissue derived from cancer patient biopsies using physiologically relevant 3D tumour models presents challenges due to the limited amount of available cell material. Here, we present a microfluidic platform that enables drug screening of cancer cell-enriched multicellular spheroids derived from tumour biopsies, allowing extensive anticancer compound screening prior to treatment. This technology was validated using cell lines and then used to screen primary human prostate cancer cells, grown in 3D as a heterogeneous culture from biopsy-derived tissue. The technology enabled the formation of repeatable drug concentration gradients across an array of spheroids without external fluid actuation, delivering simultaneously a range of drug concentrations to multiple sized spheroids, as well as replicates for each concentration. As proof-of-concept screening, spheroids were generated from two patient biopsies and a panel of standard-of-care compounds for prostate cancer were tested. Brightfield and fluorescence images were analysed to provide readouts of spheroid growth and health, as well as drug efficacy over time. Overall, this technology could prove a useful tool for personalised medicine and future drug development, with the potential to provide cost- and time-reduction in the healthcare delivery.

High-throughput culture and embedment of spheroid array using droplet contact-based spheroid transfer.

Spheroids are one of the most representative models of 3D cell culture, which can be easily formed using conventional hanging drop method. However, medium change and spheroid transferring process are the bottlenecks that reduce the throughput of the entire process in the hanging drop culture. In addition, the embedment of spheroid into hydrogel still depends on the individual pipetting process. To overcome these issues, we present poly(dimethylsiloxane) (PDMS)-based simple devices which can exploit droplet contact-based spheroid transfer using a drop array chip (DAC) having an array of well structures and peripheral rims. When the upper spheroid-containing drops were in contact with the lower liquid drops, the air-liquid interface disappeared at the merged surface and the spheroids settled down due to gravitational force. This method was applied to repetitive medium change and live/dead staining of spheroids cultured with the hanging drop method. To simultaneously embed the spheroids into the corresponding collagen hydrogel drops, a PDMS-based pillar array chip (PAC) was contacted in advance with the spheroid-containing DAC. The contacted PAC then contained the spheroids trapped in small drops of liquid reduced in volume to around 0.5 μl. Consequently, the spheroids were embedded into the collagen drops at once by contacting the spheroid-containing PAC with the collagen-loaded DAC. The embedded spheroids using the DAC-PAC contacting method showed a reliable invasion behavior compared to the embedded spheroids using conventional manual pipetting.

High-Content Monitoring of Drug Effects in a 3D Spheroid Model.

A recent decline in the discovery of novel medications challenges the widespread use of 2D monolayer cell assays in the drug discovery process. As a result, the need for more appropriate cellular models of human physiology and disease has renewed the interest in spheroid 3D culture as a pertinent model for drug screening. However, despite technological progress that has significantly simplified spheroid production and analysis, the seeming complexity of the 3D approach has delayed its adoption in many laboratories. The present report demonstrates that the use of a spheroid model may be straightforward and can provide information that is not directly available with a standard 2D approach. We describe a cost-efficient method that allows for the production of an array of uniform spheroids, their staining with vital dyes, real-time monitoring of drug effects, and an ATP-endpoint assay, all in the same 96-well U-bottom plate. To demonstrate the method performance, we analyzed the effect of the preclinical anticancer drug MLN4924 on spheroids formed by VCaP and LNCaP prostate cancer cells. The drug has different outcomes in these cell lines, varying from cell cycle arrest and protective dormancy to senescence and apoptosis. We demonstrate that by using high-content analysis of spheroid arrays, the effect of the drug can be described as a series of EC50 values that clearly dissect the cytostatic and cytotoxic drug actions. The method was further evaluated using four standard cancer chemotherapeutics with different mechanisms of action, and the effect of each drug is described as a unique multi-EC50 diagram. Once fully validated in a wider range of conditions, this method could be particularly valuable for phenotype-based drug discovery.

Spheroid arrays for high-throughput single-cell analysis of spatial patterns and biomarker expression in 3D

We describe and share a device, methodology and image analysis algorithms, which allow up to 66 spheroids to be arranged into a gel-based array directly from a culture plate for downstream processing and analysis. Compared to processing individual samples, the technique uses 11-fold less reagents, saves time and enables automated imaging. To illustrate the power of the technology, we showcase applications of the methodology for investigating 3D spheroid morphology and marker expression and for in vitro safety and efficacy screens. First, spheroid arrays of 11 cell-lines were rapidly assessed for differences in spheroid morphology. Second, highly-positive (SOX-2), moderately-positive (Ki-67) and weakly-positive (βIII-tubulin) protein targets were detected and quantified. Third, the arrays enabled screening of ten media compositions for inducing differentiation in human neurospheres. Last, the application of spheroid microarrays for spheroid-based drug screens was demonstrated by quantifying the dose-dependent drop in proliferation and increase in differentiation in etoposide-treated neurospheres.

Enhanced and controllable nonlinear effects in composite with aligned gold spheroid arrays

The linear and nonlinear optical properties of composite containing aligned prolate spheroid nanoparticles embedded in a dielectric host are studied as a function of depolarization factor. It is shown that the effective third-order nonlinear susceptibility of the composite can be significantly enhanced with respect to the linear optical properties, due to a combination of varying nanoparticle shape and interparticle space. The susceptibility enhancement is up to four orders larger compared to the spherical case. The susceptibility enhancement of the host is finally comparable to the inclusion after choosing property structure parameters. The geometry-dependent susceptibility enhancement can lead to an improved figure of merit for nonlinear absorption.

Detachably assembled microfluidic device for perfusion culture and post-culture analysis of a spheroid array.

Microfluidic devices permit perfusion culture of three-dimensional (3D) tissue, mimicking the flow of blood in vascularized 3D tissue in our body. Here, we report a microfluidic device composed of a two-part microfluidic chamber chip and multi-microwell array chip able to be disassembled at the culture endpoint. Within the microfluidic chamber, an array of 3D tissue aggregates (spheroids) can be formed and cultured under perfusion. Subsequently, detailed post-culture analysis of the spheroids collected from the disassembled device can be performed. This device facilitates uniform spheroid formation, growth analysis in a high-throughput format, controlled proliferation via perfusion flow rate, and post-culture analysis of spheroids. We used the device to culture spheroids of human hepatocellular carcinoma (HepG2) cells under two controlled perfusion flow rates. HepG2 spheroids exhibited greater cell growth at higher perfusion flow rates than at lower perfusion flow rates, and exhibited different metabolic activity and mRNA and protein expression under the different flow rate conditions. These results show the potential of perfusion culture to precisely control the culture environment in microfluidic devices. The construction of spheroid array chambers allows multiple culture conditions to be tested simultaneously, with potential applications in toxicity and drug screening.

Biomimetic Miniaturized Platform Able to Sustain Arrays of Liquid Droplets for High‐Throughput Combinatorial Tests

The development of high‐throughput and combinatorial technologies is helping to speed up research that is applicable in many areas of chemistry, engineering, and biology. A new model is proposed for flat devices for the high‐throughput screening of accelerated evaluations of multiplexed processes and reactions taking place in aqueous‐based environments. Superhydrophobic (SH) biomimetic surfaces based on the so‐called lotus effect are produced, onto which arrays of micro‐indentations allow the fixing of liquid droplets, based on the rose‐petal effect. The developed platforms are able to sustain arrays of quasi‐spherical microdroplets, allowing the isolation and confinement of different combinations of substances and living cells. Distinct compartmentalized physical, chemical, and biological processes may take place and be monitored in each droplet. The devices permit the addition/removal of liquid and mechanical stirring by adding magnetic microparticles into each droplet. By facing the chip downward, it is possible to produce arrays of cell spheroids developed by gravity in the suspended droplets, with the potential to be used as microtissues in drug screening tests.

713 ラベルフリー細胞アセンブリ法によるスフェロイドアレイの形成と融合(GS-2 生体材料)

713 ラベルフリー細胞アセンブリ法によるスフェロイドアレイの形成と融合(GS-2 生体材料)

Encapsulation of Adipose Stromal Vascular Fraction Cells in Alginate Hydrogel Spheroids Using a Direct-Write Three-Dimensional Printing System

The study of tissue function in vitro has been aided by the development of three-dimensional culture systems that more accurately duplicate the complex cell components of tissues and organs. Bioprinting of cells provides a rapid tissue fabrication technique that can be used to evaluate normal and pathologic conditions in vitro as well as to construct complex three-dimensional tissue structures for implantation in regenerative medicine therapies. Studies were performed using a direct write three-dimensional bioprinting system to fabricate adipose-derived stromal vascular fraction cell spheroids. Human fat–derived stromal vascular fraction cells were mixed in 1.5% (w/v) alginate solutions, and fabrication conditions were varied to produce an array of spheroids. The spheroids were placed in spinner culture, and spheroid integrity and encapsulated cell viability were assessed for 16 days. Results establish the ability to tightly control adipose SVF spheroids in the range of 800–1500 μm. Fabrication conditions were used to control spheroid size, and the results illustrate the ability to construct spheroids of precise size and shape. The adipose SVF cell population remains viable and the spheroid integrity was maintained for 16 days in suspension culture. The direct-write printing of adipose stromal vascular fraction cell containing spheroids provides a rapid fabrication technology to support in vitro microphysiologic system studies.

Magnetic Field Intensity Gradient Method on the Detection of a Remote Target

Based on the spheroid and magnetic dipole mixed array model, the magnetic field intensity gradient of ship is measured by using buoys and optically pumped magnetometers in order to achieve the purpose of eliminating the interference of the background magnetic field, thereby effectively improving magnetic detection of remote targets.

Multiarray Formation of CHO Spheroids Cocultured with Feeder Cells for Highly Efficient Protein Production in Serum-Free Medium

Functional proteins like antibody, cytokine and growth factor have been widely used for basic biological research, diagnosis and cancer therapy. Particularly, antibody drugs as attractive biopharmaceuticals will be expected to create an enormous new market. Chinese hamster ovay (CHO) cells are being increasingly used in industry for the production of recombinant therapeutic proteins including antibody drugs. Although three-dimensional culture is preferred to two-dimensional monolayer culture for the efficient large scale culture of CHO cells and subsequent mass production of recombinant proteins, it has the limitation of low protein production. Therefore, a new cell culture em essentially required for an efficient protein production. Here we report on a new three-dimensional cell culture system as a spheroid cell culture on the micropattern array for efficient production of protein in CHO cells. Furthermore, cocultivation of CHO spheroids with feeder cells including bovine aortic endothelial cells (BAEC) and NIH 3T3 was essential to more increase a protein production. The results indicated that CHO heterospheroids cocultured with BAECs were much superior to either CHO monolayers or CHO homospheroids in protein production. Significantly, the above cocultured spheroids in the serum-free medium drastically enhanced protein expression level up to 3-fold compared with CHO spheroids in serum medium, suggesting that a coculture of spheroid system with feeder layer cells is a promising method to enhance protein production under serum-free condition. The spheroid array constructed here is highly usuful as a platform of biopharmaceutical manufacturing as well as tissue and cell based biosensors to detect a wide variety of clinically active compounds through a cellular physiological response.

Magnetic force-based cell patterning for evaluation of the effect of stromal fibroblasts on invasive capacity in 3Dcultures

Biomimetic cell culture systems that recreate tumor microenvironments are necessary in understanding the progression of cancer cells in cell-to-cell interaction and in cell-to-extracellular matrix interaction. We have developed a three-dimensional spheroid array embedded in collagen for evaluation of the effect of stromal fibroblasts associated with cancer cells. When the breast epithelial cancer cell model MCF10A/myr-Akt1 was magnetically labeled and aligned in the array by an external magnetic force using a pin-holder device and a magnet, a stellate configuration was observed. Changes in MCF10A/myr-Akt1 cell behavior were only slight when normal human dermal fibroblasts (NHDF) cells coexisted in collagen (indirect-interaction array). In contrast, when NHDF were magnetically labeled and patterned together with MCF10A/myr-Akt1 (direct-interaction array), spreading and progression were observed along with NHDF. Cell image analysis indicated that the length and area were statistically significantly increased in the direct-interaction array compared to the MCF10A/myr-Akt1 alone or to the indirect-interaction array. A cell susceptibility assay was undertaken with breast cancer MDA-MB-231 associated with NHDF in the indirect-interaction array. Interestingly, although distinct suppression of cell movement and proliferation was observed with 100μM of collagenase inhibitor, formation of invadepodia significantly increased with coexistent NHDF. Since cancer progression is influenced by its microenvironment, this magnetic cell-patterning method which clarifies direct and indirect effects of stromal cells on invasion and proliferation, is well suited for evaluation and design of more efficient approaches in cancer prevention and treatment.

Long-term survival and functional maintenance of hepatocytes by using a microfabricated cell array

We developed a microfabricated cell array of hepatocyte spheroids that showed long-term viability and retained the properties of the parent hepatocytes. Fresh hepatocytes harvested from 8-week-old Wistar rats were cocultured with feeder cells to rapidly form hepatocyte spheroids; these cells retained the spheroidal formation for 42 days. We also evaluated the cellular functions of the hepatocytes such as albumin secretion and metabolic activity of cytochrome P450 (CYP). In spheroids in which hepatocytes were cocultured with feeder cells, these cellular functions were retained even after 42 days. Therefore, this novel coculture will be very useful not only for research on the mechanism and treatment of liver diseases but also for early prediction of hepatocyte toxicity in the pre-clinical phase of drug development.

Hepatocyte spheroid arrays inside microwells connected with microchannels

Spheroid culture is a preferable cell culture approach for some cell types, including hepatocytes, as this type of culture often allows maintenance of organ-specific functions. In this study, we describe a spheroid microarray chip (SM chip) that allows stable immobilization of hepatocyte spheroids in microwells and that can be used to evaluate drug metabolism with high efficiency. The SM chip consists of 300-μm-diameter cylindrical wells with chemically modified bottom faces that form a 100-μm-diameter cell adhesion region surrounded by a nonadhesion region. Primary hepatocytes seeded onto this chip spontaneously formed spheroids of uniform diameter on the cell adhesion region in each microwell and these could be used for cytochrome P-450 fluorescence assays. A row of microwells could also be connected to a microchannel for simultaneous detection of different cytochrome P-450 enzyme activities on a single chip. The miniaturized features of this SM chip reduce the numbers of cells and the amounts of reagents required for assays. The detection of four cytochrome P-450 enzyme activities was demonstrated following induction by 3-methylcholantlene, with a sensitivity significantly higher than that in conventional monolayer culture. This microfabricated chip could therefore serve as a novel culture platform for various cell-based assays, including those used in drug screening, basic biological studies, and tissue engineering applications.

Abstract 1540: Targeting PI3K/mTOR inhibitor induced survival programs abrogates resistance to cell death in ECM-attached cancer cells

Abstract Alterations in the PI3-kinase pathway are common in breast, endometrial, and ovarian cancer and are thought to serve as major drivers of proliferation and survival. Multiple compounds targeting PI3-kinase pathway are currently in clinical trials. We have examined the sensitivity of ovarian and breast tumor cell lines to dual PI3K/mTOR inhibitors in 3D tumor spheroid assays. Interestingly, tumor cells in the center of the spheroids that are not attached to matrix are generally highly sensitive to NVP-BEZ235 and undergo apoptosis, whereas those in contact with matrix are highly resistant. In order to understand the basis for the differential sensitivity to NVP-BEZ235 and to identify candidate targets to abrogate this resistance, we performed reverse phase protein array of tumor spheroids treated with NVP-BEZ235. While NVP-BEZ235 inhibited known downstream targets of PI3K/mTOR, there was significant up-regulation of multiple proteins known to regulate cell survival including IGF1R, p-EGFR and several Bcl-2 family members. This response raised the question whether combined inhibition of PI3K/mTOR and any of the induced proteins would sensitize the matrix-attached cells. Interestingly, inhibition of Bcl-2 by ABT737 caused widespread apoptosis in the 3D structures, but only in combination with NVP-BEZ235. In addition, matrix-resistance was abrogated by combined treatment with NVP-BEZ235 and inhibition of EGFR or knockdown IGF1R. These results indicate that inhibition of PI3K/mTOR leads to induction of several protective pathways and that suppression of these pathways can significantly enhance sensitivity to PI3K/mTOR targeted therapies. In addition, these studies suggest a rational approach to the development of combination therapies – that is, through identification of drug-induced survival pathways and targeting these pathways in combination. Our current efforts center on investigating the mechanisms of PI3K/mTOR inhibition resistance and addressing the efficacy of the dual treatment in preclinical models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1540. doi:10.1158/1538-7445.AM2011-1540