Generation of Multicellular Tumor Spheroids by the Hanging-Drop Method

Timmins, Nicholas; Nielsen, Lars K.

doi:10.1007/978-1-59745-443-8_8

Cited by 198 publications

(171 citation statements)

References 7 publications

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“…Commonly used culture techniques to create tumor spheroids include the hanging drop method, 17,18 the liquid-overlay technique, 19 and the gyratory rotation technique. 20 In the hanging drop method, spheroids are grown in droplets placed on an inverted microplate.…”

Section: Introductionmentioning

confidence: 99%

Continuously perfused microbubble array for 3D tumor spheroid model

et al. 2011

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Multi-cellular tumor spheroids ͑MCTSs͒ have been established as a 3D physiologically relevant tumor model for drug testing in cancer research. However, it is difficult to control the MCTS testing parameters and the entire process is timeconsuming and expensive. To overcome these limitations, we developed a simple microfluidic system using polydimethylsiloxane ͑PDMS͒ microbubbles to culture tumor spheroids under physiological flow. The flow characteristics such as streamline directions, shear stress profile, and velocity profile inside the microfluidic system were first examined computationally using a COMSOL simulation. Colo205 tumor spheroids were created by a modified hanging drop method and maintained inside PDMS microbubble cavities in perfusion culture. Cell viability inside the microbubbles was examined by live cell staining and confocal imaging. E-selectin mediated cell sorting of Colo205 and MDA-MB-231 cell lines on functionalized microbubble and PDMS surfaces was achieved. Finally, to validate this microfluidic system for drug screening purposes, the toxicity of the anti-cancer drug, doxorubicin, on Colo205 cells in spheroids was tested and compared to cells in 2D culture. Colo205 spheroids cultured in flow showed a threefold increase in resistance to doxorubicin compared to Colo205 monolayer cells cultured under static conditions, consistent with the resistance observed previously in other MCTS models. The advantages presented by our microfluidic system, such as the ability to control the size uniformity of the spheroids and to perform real-time imaging on cells in the growth platform, show potential for high throughput drug screening development.

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Section: Introductionmentioning

confidence: 99%

Continuously perfused microbubble array for 3D tumor spheroid model

et al. 2011

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“…Friedrich et al developed a promising protocol for spheroid-based high throughput drug screening in which they coated the bottom surface of the wells of multiwell plates with agarose [48]. PEG (polyethylene glycol) [49] and polystyrene plastic [50] materials are also used as a non-adherent surface for spheroid formation.…”

Section: Liquid Overlay Methodsmentioning

confidence: 99%

Inventions and Innovations in Preclinical Platforms for Cancer Research

2018

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Three-dimensional (3D) cell culture systems can be regarded as suitable platforms to bridge the huge gap between animal studies and two-dimensional (2D) monolayer cell culture to study chronic diseases such as cancer. In particular, the preclinical platforms for multicellular spheroid formation and culture can be regarded as ideal in vitro tumour models. The complex tumour microenvironment such as hypoxic region and necrotic core can be recapitulated in 3D spheroid configuration. Cells aggregated in spheroid structures can better illustrate the performance of anti-cancer drugs as well. Various methods have been proposed so far to create such 3D spheroid aggregations. Both conventional techniques and microfluidic methods can be used for generation of multicellular spheroids. In this review paper, we first discuss various spheroid formation phases. Then, the conventional spheroid formation techniques such as bioreactor flasks, liquid overlay and hanging droplet technique are explained. Next, a particular topic of the hydrogel in spheroid formation and culture is explored. This topic has received less attention in the literature. Hydrogels entail some advantages to the spheroid formation and culture such as size uniformity, the formation of porous spheroids or hetero-spheroids as well as chemosensitivity and invasion assays and protecting from shear stress. Finally, microfluidic methods for spheroid formation and culture are briefly reviewed.

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“…In the following protocol, Protocol 1 pertains to the initial preparation of tumor spheroids which is described here using overlay on agarose but could be substituted with a variety of other methods such as hanging drop 40 , or rotary culture 41 techniques. Protocol 2 describes the process of embedding spheroids in a collagen scaffold though alternatively, in vitro 3D tumors could be grown by encapsulation or embedding of resuspended cells in ECM 12,15 , rather than single pre-formed non-adherent spheroids.…”

Section: Introductionmentioning

confidence: 99%

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

Jones

Hanna

El-Hamidi

2014

JoVE

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The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position-and timedependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(ω). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment. Video LinkThe video component of this article can be found at

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Generation of Multicellular Tumor Spheroids by the Hanging-Drop Method

Cited by 198 publications

References 7 publications

Continuously perfused microbubble array for 3D tumor spheroid model

Continuously perfused microbubble array for 3D tumor spheroid model

Inventions and Innovations in Preclinical Platforms for Cancer Research

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

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