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Journal Article•DOI•

Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis.

01 Dec 2006-Journal of Biomolecular Screening (SAGE Publications)-Vol. 11, Iss: 8, pp 922-932
TL;DR: The authors present a rapid method to generate single spheroids in suspension culture in individual wells with homogeneous sizes, morphologies, and stratification of proliferating cells in the rim and dying Cells in the core region in a true suspension culture.
Abstract: Spheroids are widely used in biology because they provide an in vitro 3-dimensional (3D) model to study proliferation, cell death, differentiation, and metabolism of cells in tumors and the response of tumors to radiotherapy and chemotherapy. The methods of generating spheroids are limited by size heterogeneity, long cultivation time, or mechanical accessibility for higher throughput fashion. The authors present a rapid method to generate single spheroids in suspension culture in individual wells. A defined number of cells ranging from 1000 to 20,000 were seeded into wells of poly-HEMA-coated, 96-well, round-or conical-bottom plates in standard medium and centrifuged for 10 min at 1000 g. This procedure generates single spheroids in each well within a 24-h culture time with homogeneous sizes, morphologies, and stratification of proliferating cells in the rim and dying cells in the core region. Because a large number of tumor cell lines form only loose aggregates when cultured in 3D, the authors also performed a screen for medium additives to achieve a switch from aggregate to spheroid morphology. Small quantities of the basement membrane extract Matrigel, added to the culture medium prior to centrifugation, most effectively induced compact spheroid formation. The compact spheroid morphology is evident as early as 24 h after centrifugation in a true suspension culture. Twenty tumor cell lines of different lineages have been used to successfully generate compact, single spheroids with homogenous size in 96-well plates and are easily accessible for subsequent functional analysis.
Citations
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Journal Article•DOI•
TL;DR: It is believed that 3D cultures will have a strong impact on drug screening and will also decrease the use of laboratory animals, for example, in the context of toxicity assays.
Abstract: Cell monolayers have serious limitations for cell biological investigations and for cell-based assays in drug screening and toxicity studies. However, the establishment of three-dimensional cultures as a mainstream approach requires the development of reliable protocols, new cell lines and suitable imaging techniques.

2,413 citations


Cites background from "Rapid generation of single-tumor sp..."

  • ...and are straightforward to apply in high-throughput screen...

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Journal Article•DOI•
TL;DR: The rationale, potential and flexibility of tumor spheroid mono- and cocultures for implementation into state of the art anti-cancer therapy test platforms are highlighted and the relevance of the cancer stem cell hypothesis for cancer cure is highlighted.

1,430 citations

Journal Article•DOI•
TL;DR: This work describes a standardized setup for reproducible, easy-handling culture, treatment and routine analysis of multicellular spheroids, the classical 3D culture system resembling many aspects of the pathophysiological situation in human tumor tissue and provides a list of human carcinoma cell lines that produce treatable sp Heroids under identical culture conditions.
Abstract: Although used in academic research for several decades, 3D culture models have long been regarded expensive, cumbersome and unnecessary in drug development processes. Technical advances, coupled with recent observations showing that gene expression in 3D is much closer to clinical expression profiles than those seen in 2D, have renewed attention and generated hope in the feasibility of maturing organotypic 3D systems to therapy test platforms with greater power to predict clinical efficacies. Here we describe a standardized setup for reproducible, easy-handling culture, treatment and routine analysis of multicellular spheroids, the classical 3D culture system resembling many aspects of the pathophysiological situation in human tumor tissue. We discuss essential conceptual and practical considerations for an adequate establishment and use of spheroid-based drug screening platforms and also provide a list of human carcinoma cell lines, partly on the basis of the NCI-DTP 60-cell line screen, that produce treatable spheroids under identical culture conditions. In contrast to many other settings with which to achieve similar results, the protocol is particularly useful to be integrated into standardized large-scale drug test routines as it requires a minimum number of defined spheroids and a limited amount of drug. The estimated time to run the complete screening protocol described herein--including spheroid initiation, drug treatment and determination of the analytical end points (spheroid integrity, and cell survival through the acid phosphatase assay)--is about 170 h. Monitoring of spheroid growth kinetics to determine growth delay and regrowth, respectively, after drug treatment requires long-term culturing (> or =14 d).

1,365 citations

Journal Article•DOI•
TL;DR: 2D and 3D culture approaches are reviewed and the strengths and relevance of each method are considered in the context of anti-cancer drug screening.

1,005 citations

Journal Article•DOI•
TL;DR: The suitability of spheroids as an in vitro platform for testing drug delivery systems is examined and the assay techniques required for the characterization of drug delivery and efficacy in sp Heroids are discussed.

936 citations

References
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Journal Article•DOI•
08 Apr 1988-Science
TL;DR: The special cellular microecology of tumors influences responsiveness to therapeutic agents and has implications for future directions in cancer research.
Abstract: Abnormal vascularization of malignant tumors is associated with the development of microregions of heterogeneous cells and environments. Experimental models such as multicell spheroids and a variety of new techniques are being used to determine the characteristics of these microregions and to study the interactions of the cells and microenvironments. The special cellular microecology of tumors influences responsiveness to therapeutic agents and has implications for future directions in cancer research.

1,791 citations


"Rapid generation of single-tumor sp..." refers background in this paper

  • ...This gives rise to a more stratified composition, with the rim of spheroids consisting of proliferating cells, followed by a layer of quiescent cells in the middle and necrotic cells in the center of the spheroid.(11) A similar cellular heterogeneity is present in nonvascularized tumor regions....

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Journal Article•DOI•
TL;DR: A mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate, which has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue.
Abstract: 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.

850 citations

Journal Article•DOI•
TL;DR: 3-D in vitro systems for drug development, with a focus on screening for novel antitumor drugs, are addressed, and the advantages and limitations of these systems of intermediate complexity are discussed.
Abstract: Over the past few years, establishment and adaptation of cell-based assays for drug development and testing has become an important topic in high-throughput screening (HTS). Most new assays are designed to rapidly detect specific cellular effects reflecting action at various targets. However, although more complex than cell-free biochemical test systems, HTS assays using monolayer or suspension cultures still reflect a highly artificial cellular environment and may thus have limited predictive value for the clinical efficacy of a compound. Today's strategies for drug discovery and development, be they hypothesis free or mechanism based, require facile, HTS-amenable test systems that mimic the human tissue environment with increasing accuracy in order to optimize preclinical and preanimal selection of the most active molecules from a large pool of potential effectors, for example, against solid tumors. Indeed, it is recognized that 3-dimensional cell culture systems better reflect the in vivo behavior of most cell types. However, these 3-D test systems have not yet been incorporated into mainstream drug development operations. This article addresses the relevance and potential of 3-D in vitro systems for drug development, with a focus on screening for novel antitumor drugs. Examples of 3-D cell models used in cancer research are given, and the advantages and limitations of these systems of intermediate complexity are discussed in comparison with both 2-D culture and in vivo models. The most commonly used 3-D cell culture systems, multicellular spheroids, are emphasized due to their advantages and potential for rapid development as HTS systems. Thus, multicellular tumor spheroids are an ideal basis for the next step in creating HTS assays, which are predictive of in vivo antitumor efficacy.

753 citations


"Rapid generation of single-tumor sp..." refers background or methods in this paper

  • ...In addition, in suspension cultures, many tumor cell lines grow poorly in 3D as compact spheroids.(1,20) They form loose aggregates of cells only, an architecture that is significantly different from the in vivo tumor morphology....

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  • ...A current limitation of the spheroid application in vitro is that numerous cancer cell lines do not form compact round spheroids in 3D.(1,20) For example, approximately half of the breast cancer cell lines tested in this study grew as aggregates of cells in 3D culture (Fig....

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  • ...Because monolayer-based high-throughput (HT) systems have only a limited predictive value for the in vivo efficacy of antineoplastic compounds, the implementation of the spheroid model in the drug discovery process may improve the predicted activity of new cancer drugs in cell-based screening assays.(20) Several approaches have been developed to enable drug screening using spheroids: confocal microscopy to measure drug or antibody permeation into the spheroid,(20,21) electrochemical microscopy to measure the respiratory activity,(22) image cytometry to quantify lactate dehydrogenase activity as a marker for Roche Pharmaceutical Research Oncology, Penzberg, Germany....

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Journal Article•DOI•
TL;DR: It is reported that tissue-specific vectorial secretion coincides with the formation of functional alveoli-like structures by primary mammary epithelial cells cultured on a reconstituted basement membrane matrix (derived from Engelbreth-Holm-Swarm murine tumour), which reproduce the dual role of mammaries to secrete vectorially and to sequester milk proteins.
Abstract: An essential feature of mammary gland differentiation during pregnancy is the formation of alveoli composed of polarized epithelial cells, which, under the influence of lactogenic hormones, secrete vectorially and sequester milk proteins. Previous culture studies have described either organization of cells polarized towards lumina containing little or no demonstrable tissue-specific protein, or establishment of functional secretory cells exhibiting little or no glandular architecture. In this paper, we report that tissue-specific vectorial secretion coincides with the formation of functional alveoli-like structures by primary mammary epithelial cells cultured on a reconstituted basement membrane matrix (derived from Engelbreth-Holm-Swarm murine tumour). Morphogenesis of these unique three-dimensional structures was initiated by cell-directed remodelling of the exogenous matrix leading to reorganization of cells into matrix-ensheathed aggregates by 24 h after plating. The aggregates subsequently cavitated, so that by day 6 the cells were organized into hollow spheres in which apical cell surfaces faced lumina sealed by tight junctions and basal surfaces were surrounded by a distinct basal lamina. The profiles of proteins secreted into the apical (luminal) and basal (medium) compartments indicated that these alveoli-like structures were capable of an appreciable amount of vectorial secretion. Immunoprecipitation with a broad spectrum milk antiserum showed that more than 80% of caseins were secreted into the lumina, whereas iron-binding proteins (both lactoferrin and transferrin) were present in comparable amounts in each compartment. Thus, these mammary cells established protein targeting pathways directing milk-specific proteins to the luminal compartment. A time course monitoring secretory activity demonstrated that establishment of tissue-specific vectorial secretion and increased total and milk protein secretion coincided with functional alveolar-like multicellular architecture. This culture system is unique among models of epithelial cell polarity in that it demonstrates several aspects of epithelial cell polarization: vectorial secretion, apical junctions, a sequestered compartment and formation of a basal lamina. These lumina-containing structures therefore reproduce the dual role of mammary epithelia to secrete vectorially and to sequester milk proteins. Thus, in addition to maintaining tissue-specific cytodifferentiation and function, a basement membrane promotes the expression of tissue-like morphogenesis.

724 citations

Journal Article•DOI•
A.A. Moscona1•
TL;DR: A cell aggregation procedure based on readily standardizable manipulations is described, whereby cells dissociated enzymatically from embryonic tissues may be aggregated in various combinations and concentrations into developmentally effective multicellular structures; it is suitable for analyzing mutual reactions of cells and their responses to diverse environmental conditions.

720 citations