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Journal of Visualized Experiments : JoVE Mar 2017Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D)...
Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.
Topics: Cell Aggregation; Cell Culture Techniques; Cell Line, Tumor; Humans; Methylcellulose; Serum; Spheroids, Cellular; Time Factors
PubMed: 28448014
DOI: 10.3791/55544 -
Scientific Reports Oct 2021In this study we demonstrated that exposure of Escherichia coli (E. coli) to terahertz (THz) radiation resulted in a change in the activities of the tdcABCDEFGR and...
In this study we demonstrated that exposure of Escherichia coli (E. coli) to terahertz (THz) radiation resulted in a change in the activities of the tdcABCDEFGR and matA-F genes (signs of cell aggregation), gene yjjQ (signs of suppression of cell motility), dicABCF, FtsZ, and minCDE genes (signs of suppression of cell division), sfmACDHF genes (signs of adhesin synthesis), yjbEFGH and gfcA genes (signs of cell envelope stabilization). Moreover, THz radiation induced E. coli csg operon genes of amyloid biosynthesis. Electron microscopy revealed that the irradiated bacteria underwent increased aggregation; 20% of them formed bundle-like structures consisting of two to four pili clumped together. This could be the result of changes in the adhesive properties of the pili. We also found aberrations in cell wall structure in the middle part of the bacterial cell; these aberrations impaired the cell at the initial stages of division and resulted in accumulation of long rod-like cells. Overall, THz radiation was shown to have adverse effects on bacterial populations resulting in cells with abnormal morphology.
Topics: Cell Aggregation; Cell Division; Cell Wall; Escherichia coli; Gene Expression Regulation, Bacterial; Microscopy, Electron; Operon; Terahertz Radiation
PubMed: 34650158
DOI: 10.1038/s41598-021-99665-3 -
ELife Aug 2023Pancreatic islets are three-dimensional cell aggregates consisting of unique cellular composition, cell-to-cell contacts, and interactions with blood vessels. Cell...
Pancreatic islets are three-dimensional cell aggregates consisting of unique cellular composition, cell-to-cell contacts, and interactions with blood vessels. Cell aggregation is essential for islet endocrine function; however, it remains unclear how developing islets establish aggregation. By combining genetic animal models, imaging tools, and gene expression profiling, we demonstrate that islet aggregation is regulated by extracellular matrix signaling and cell-cell adhesion. Islet endocrine cell-specific inactivation of extracellular matrix receptor integrin β1 disrupted blood vessel interactions but promoted cell-cell adhesion and the formation of larger islets. In contrast, ablation of cell-cell adhesion molecule α-catenin promoted blood vessel interactions yet compromised islet clustering. Simultaneous removal of integrin β1 and α-catenin disrupts islet aggregation and the endocrine cell maturation process, demonstrating that establishment of islet aggregates is essential for functional maturation. Our study provides new insights into understanding the fundamental self-organizing mechanism for islet aggregation, architecture, and functional maturation.
Topics: Animals; Cell Adhesion; Integrin beta1; alpha Catenin; Cell Aggregation; Extracellular Matrix
PubMed: 37610090
DOI: 10.7554/eLife.90006 -
Current Opinion in Biotechnology Oct 2011Recapitulating the elegant structures formed during development is an extreme synthetic and biological challenge. Great progress has been made in developing materials to... (Review)
Review
Recapitulating the elegant structures formed during development is an extreme synthetic and biological challenge. Great progress has been made in developing materials to support transplanted cells, yet the complexity of tissues is far beyond that found in even the most advanced scaffolds. Self-assembly is a motif used in development and a route for the production of complex materials. Self-assembly of peptides, proteins and other molecules at the nanoscale is promising, but in addition, intriguing ideas are emerging for self-assembly of micron-scale structures. In this brief review, very recent advances in the assembly of micron-scale cell aggregates and microgels will be described and discussed.
Topics: Animals; Cell Aggregation; Humans; Hydrogels; Tissue Engineering
PubMed: 21524904
DOI: 10.1016/j.copbio.2011.04.001 -
Stem Cells Translational Medicine Aug 2021Human mesenchymal stromal cells (MSCs) are promising candidates for cell therapy due to their ease of isolation and expansion and their ability to secrete antiapoptotic,...
Human mesenchymal stromal cells (MSCs) are promising candidates for cell therapy due to their ease of isolation and expansion and their ability to secrete antiapoptotic, pro-angiogenic, and immunomodulatory factors. Three-dimensional (3D) aggregation "self-activates" MSCs to augment their pro-angiogenic and immunomodulatory potential, but the microenvironmental features and culture parameters that promote optimal MSC immunomodulatory function in 3D aggregates are poorly understood. Here, we generated MSC aggregates via three distinct methods and compared them with regard to their (a) aggregate structure and (b) immunomodulatory phenotype under resting conditions and in response to inflammatory stimulus. Methods associated with fast aggregation kinetics formed aggregates with higher cell packing density and reduced extracellular matrix (ECM) synthesis compared to those with slow aggregation kinetics. While all three methods of 3D aggregation enhanced MSC expression of immunomodulatory factors compared to two-dimensional culture, different aggregation methods modulated cells' temporal expression of these factors. A Design of Experiments approach, in which aggregate size and aggregation kinetics were systematically covaried, identified a significant effect of both parameters on MSCs' ability to regulate immune cells. Compared to small aggregates formed with fast kinetics, large aggregates with slow assembly kinetics were more effective at T-cell suppression and macrophage polarization toward anti-inflammatory phenotypes. Thus, culture parameters including aggregation method, kinetics, and aggregate size influence both the structural properties of aggregates and their paracrine immunomodulatory function. These findings underscore the utility of engineering strategies to control properties of 3D MSC aggregates, which may identify new avenues for optimizing the immunomodulatory function of MSC-based cell therapies.
Topics: Cell Aggregation; Cell Proliferation; Cells, Cultured; Extracellular Matrix; Immunomodulation; Mesenchymal Stem Cells; T-Lymphocytes
PubMed: 33818906
DOI: 10.1002/sctm.19-0414 -
Developmental Cell Dec 2018Inhibition of metastatic cancer cell colonization and outgrowth is arguably one of the greatest therapeutic challenges. Reporting in Cancer Discovery, Liu et al. (2018)...
Inhibition of metastatic cancer cell colonization and outgrowth is arguably one of the greatest therapeutic challenges. Reporting in Cancer Discovery, Liu et al. (2018) describe how homophilic interactions of CD44, a classical breast cancer stem cell marker, drive tumor cell aggregation outside the primary tumor to augment their metastatic potential.
Topics: Breast Neoplasms; Cell Aggregation; Humans; Hyaluronan Receptors; Neoplastic Stem Cells
PubMed: 30562512
DOI: 10.1016/j.devcel.2018.11.038 -
Science (New York, N.Y.) Apr 2009Topobiology posits that morphogenesis is driven by differential adhesive interactions among heterogeneous cell populations. This paradigm has been revised to include... (Review)
Review
Topobiology posits that morphogenesis is driven by differential adhesive interactions among heterogeneous cell populations. This paradigm has been revised to include force-dependent molecular switches, cell and tissue tension, and reciprocal interactions with the microenvironment. It is now appreciated that tissue development is executed through conserved decision-making modules that operate on multiple length scales from the molecular and subcellular level through to the cell and tissue level and that these regulatory mechanisms specify cell and tissue fate by modifying the context of cellular signaling and gene expression. Here, we discuss the origin of these decision-making modules and illustrate how emergent properties of adhesion-directed multicellular structures sculpt the tissue, promote its functionality, and maintain its homeostasis through spatial segregation and organization of anchored proteins and secreted factors and through emergent properties of tissues, including tension fields and energy optimization.
Topics: Animals; Biological Evolution; Cell Adhesion; Cell Aggregation; Cell Communication; Extracellular Matrix; Genotype; Homeostasis; Morphogenesis; Phenotype; Proteins; Signal Transduction
PubMed: 19359578
DOI: 10.1126/science.1170107 -
Trends in Cell Biology Jan 2009Asymmetric cell division (ACD) is a fundamental process used to generate cell diversity during metazoan development that occurs when a cell divides to generate daughter... (Review)
Review
Asymmetric cell division (ACD) is a fundamental process used to generate cell diversity during metazoan development that occurs when a cell divides to generate daughter cells adopting distinct fates. Stem cell divisions, for example, are a type of ACD and provide a source of new cells during development and in adult animals. Some ACDs produce a daughter cell that dies. In many cases, the reason why a cell divides to generate a dying daughter remains elusive. It was shown recently that denatured proteins are segregated asymmetrically during cell division. Here, we review data that provide interesting insights into how apoptosis is regulated during ACD and speculate on potential connections between ACD-induced cell death and partitioning of denatured proteins.
Topics: Animals; Apoptosis; Cell Aggregation; Cell Division; Cell Lineage; Humans; Protein Folding; Signal Transduction
PubMed: 19091567
DOI: 10.1016/j.tcb.2008.10.004 -
PloS One 2016The crawling of biological cell is a complex phenomenon involving various biochemical and mechanical processes. Some of these processes are intrinsic to individual...
The crawling of biological cell is a complex phenomenon involving various biochemical and mechanical processes. Some of these processes are intrinsic to individual cells, while others pertain to cell-to-cell interactions and to their responses to extrinsically imposed cues. Here, we report an interesting aggregation dynamics of mathematical model cells, when they perform chemotaxis in response to an externally imposed global chemical gradient while they influence each other through a haptotaxis-mediated social interaction, which confers intriguing trail patterns. In the absence of the cell-to-cell interaction, the equilibrium population density profile fits well to that of a simple Keller-Segal population dynamic model, in which a chemotactic current density [Formula: see text] competes with a normal diffusive current density [Formula: see text], where p and ρ refer to the concentration of chemoattractant and population density, respectively. We find that the cell-to-cell interaction confers a far more compact aggregation resulting in a much higher peak equilibrium cell density. The mathematical model system is applicable to many biological systems such as swarming microglia and neutrophils or accumulating ants towards a localized food source.
Topics: Animals; Cell Aggregation; Cell Communication; Cell Movement; Chemotactic Factors; Chemotaxis; Microglia; Models, Biological; Models, Neurological; Rats
PubMed: 27128310
DOI: 10.1371/journal.pone.0154717 -
PloS One 2014Stirred-suspension bioreactors are a promising modality for large-scale culture of 3D aggregates of pluripotent stem cells and their progeny. Yet, cells within these...
Stirred-suspension bioreactors are a promising modality for large-scale culture of 3D aggregates of pluripotent stem cells and their progeny. Yet, cells within these clusters experience limitations in the transfer of factors and particularly O2 which is characterized by low solubility in aqueous media. Cultured stem cells under different O2 levels may exhibit significantly different proliferation, viability and differentiation potential. Here, a transient diffusion-reaction model was built encompassing the size distribution and ultrastructural characteristics of embryonic stem cell (ESC) aggregates. The model was coupled to experimental data from bioreactor and static cultures for extracting the effective diffusivity and kinetics of consumption of O2 within mouse (mESC) and human ESC (hESC) clusters. Under agitation, mESC aggregates exhibited a higher maximum consumption rate than hESC aggregates. Moreover, the reaction-diffusion model was integrated with a population balance equation (PBE) for the temporal distribution of ESC clusters changing due to aggregation and cell proliferation. Hypoxia was found to be negligible for ESCs with a smaller radius than 100 µm but became appreciable for aggregates larger than 300 µm. The integrated model not only captured the O2 profile both in the bioreactor bulk and inside ESC aggregates but also led to the calculation of the duration that fractions of cells experience a certain range of O2 concentrations. The approach described in this study can be employed for gaining a deeper understanding of the effects of O2 on the physiology of stem cells organized in 3D structures. Such frameworks can be extended to encompass the spatial and temporal availability of nutrients and differentiation factors and facilitate the design and control of relevant bioprocesses for the production of stem cell therapeutics.
Topics: Animals; Biological Transport; Bioreactors; Cell Aggregation; Cell Culture Techniques; Cell Hypoxia; Cell Proliferation; Cells, Cultured; Human Embryonic Stem Cells; Humans; Mice; Mouse Embryonic Stem Cells; Oxygen; Oxygen Consumption
PubMed: 25032842
DOI: 10.1371/journal.pone.0102486