Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis.
Andrea Ivascu,Manfred Kubbies +1 more
TLDR
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.read more
Citations
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Journal ArticleDOI
A novel three‐dimensional model system for keloid study: Organotypic multicellular scar model
TL;DR: The three‐dimensional organotypic multicellular spheroid keloid culture will allow investigators to study keloids pathogenesis and test potential keloidal therapeutic agents.
Journal ArticleDOI
In vitro treatment of melanoma brain metastasis by simultaneously targeting the MAPK and PI3K signaling pathways.
Inderjit Daphu,Sindre Horn,Daniel Stieber,Jobin K. Varughese,Endy Spriet,Hege Avsnes Dale,Kai Ove Skaftnesmo,Rolf Bjerkvig,Frits Thorsen +8 more
TL;DR: It is demonstrated for the first time that combined therapy with vemurafenib and temsirolimus is effective on melanoma brain metastasis cells in vitro, and global gene analysis indicated that functions related to cell survival and invasion were influenced by combined treatment.
Journal ArticleDOI
Dissociation of mono- and co-culture spheroids into single cells for subsequent flow cytometric analysis.
TL;DR: Spheroids can be successfully dissociated into singular cells for subsequent flow cytometric analysis and the observed decrease in cell size in spheroids over time might be responsible for the well-known time-dependent decrease in spheroid size.
Journal ArticleDOI
Remote Control in Formation of 3D Multicellular Assemblies Using Magnetic Forces
TL;DR: The application of magnetic fields to impart forces on the cells enhanced the fusion of these spheroids, which could be used to produce larger and more complicated structures for future tissue engineering applications.
Patent
Method for obtaining a multicellular spheroid
TL;DR: In this paper, a method for producing a multicellular spheroid comprising injecting a cell suspension into a gel is described, and a method of assessing the effect of an agent on the property of a cell selected from any of survival, growth, proliferation, differentiation, migration, morphology, signalling, metabolic activity, gene expression and cell-cell interaction is presented.
References
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Journal ArticleDOI
Cell and environment interactions in tumor microregions: the multicell spheroid model
TL;DR: The special cellular microecology of tumors influences responsiveness to therapeutic agents and has implications for future directions in cancer research.
Journal ArticleDOI
Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types.
Jens M. Kelm,Jens M. Kelm,Nicholas E. Timmins,Catherine Brown,Martin Fussenegger,Lars K. Nielsen +5 more
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.
Journal ArticleDOI
The use of 3-D cultures for high-throughput screening: the multicellular spheroid model.
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.
Journal ArticleDOI
Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane
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.
Journal ArticleDOI
Rotation-mediated histogenetic aggregation of dissociated cells. A quantifiable approach to cell interactions in vitro.
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.
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