Formation, Growth and Morphology of Multicellular Tumor Spheroids from a Human Colon Carcinoma Cell Line (LoVo)

Soranzo, Carla; Torre, Gabriella Della; Ingrosso, Antonella

doi:10.1177/030089168607200502

Cited by 14 publications

(10 citation statements)

References 8 publications

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“…Unfortunately, only some types of cells form cellular aggregates, and because the extracellular matrix of the aggregate is secreted by the component cells, little control over which matrix components are present is possible. Cellular aggregates also tend to agglomerate, and cells may die in regions where nutrients can no longer reach the cells by diffusion [17] .…”

Section: Introductionmentioning

confidence: 99%

Cell Encapsulation in Sub-mm Sized Gel Modules Using Replica Molding

et al. 2008

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For many types of cells, behavior in two-dimensional (2D) culture differs from that in three-dimensional (3D) culture. Among biologists, 2D culture on treated plastic surfaces is currently the most popular method for cell culture. In 3D, no analogous standard method—one that is similarly convenient, flexible, and reproducible—exists. This paper describes a soft-lithographic method to encapsulate cells in 3D gel objects (modules) in a variety of simple shapes (cylinders, crosses, rectangular prisms) with lateral dimensions between 40 and 1000 μm, cell densities of 105 – 108 cells/cm3, and total volumes between 1×10−7 and 8×10−4 cm3. By varying (i) the initial density of cells at seeding, and (ii) the dimensions of the modules, the number of cells per module ranged from 1 to 2500 cells. Modules were formed from a range of standard biopolymers, including collagen, Matrigel™, and agarose, without the complex equipment often used in encapsulation. The small dimensions of the modules allowed rapid transport of nutrients by diffusion to cells at any location in the module, and therefore allowed generation of modules with cell densities near to those of dense tissues (108 – 109 cells/cm3). This modular method is based on soft lithography and requires little special equipment; the method is therefore accessible, flexible, and well suited to (i) understanding the behavior of cells in 3D environments at high densities of cells, as in dense tissues, and (ii) developing applications in tissue engineering.

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Section: Introductionmentioning

confidence: 99%

Cell Encapsulation in Sub-mm Sized Gel Modules Using Replica Molding

et al. 2008

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“…The liquid overlay and spinner flask techniques and other 1 g methods provide specific environments for 3D formation [25]. Unfortunately, after two weeks the spheroids revealed a necrotic center [26].…”

Section: Introductionmentioning

confidence: 99%

Simulated Microgravity Influences VEGF, MAPK, and PAM Signaling in Prostate Cancer Cells

Hybel

Dietrichs

Sahana

et al. 2020

IJMS

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Prostate cancer is one of the leading causes of cancer mortality in men worldwide. An unusual but unique environment for studying tumor cell processes is provided by microgravity, either in space or simulated by ground-based devices like a random positioning machine (RPM). In this study, prostate adenocarcinoma-derived PC-3 cells were cultivated on an RPM for time periods of 3 and 5 days. We investigated the genes associated with the cytoskeleton, focal adhesions, extracellular matrix, growth, survival, angiogenesis, and metastasis. The gene expression of signaling factors of the vascular endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK), and PI3K/AKT/mTOR (PAM) pathways was investigated using qPCR. We performed immunofluorescence to study the cytoskeleton, histological staining to examine the morphology, and a time-resolved immunofluorometric assay to analyze the cell culture supernatants. When PC-3 cells were exposed to simulated microgravity (s-µg), some cells remained growing as adherent cells (AD), while most cells detached from the cell culture flask bottom and formed multicellular spheroids (MCS). After 3-day RPM exposure, PC-3 cells revealed significant downregulation of the VEGF, SRC1, AKT, MTOR, and COL1A1 gene expression in MCS, whereas FLT1, RAF1, MEK1, ERK1, FAK1, RICTOR, ACTB, TUBB, and TLN1 mRNAs were not significantly changed. ERK2 and TLN1 were elevated in AD, and FLK1, LAMA3, COL4A5, FN1, VCL, CDH1, and NGAL mRNAs were significantly upregulated in AD and MCS after 3 days. After a 5-day culture in s-µg, the PC-3 cells showed significant downregulations of VEGF mRNA in AD and MCS, and FN1, CDH1, and LAMA3 in AD and SCR1 in MCS. In addition, we measured significant upregulations in FLT1, AKT, ERK1, ERK2, LCN2, COL1A1, TUBB, and VCL mRNAs in AD and MCS, and increases in FLK1, FN1, and COL4A5 in MCS as well as LAMB2, CDH1, RAF1, MEK1, SRC1, and MTOR mRNAs in AD. FAK1 and RICTOR were not altered by s-µg. In parallel, the secretion rate of VEGFA and NGAL proteins decreased. Cytoskeletal alterations (F-actin) were visible, as well as a deposition of collagen in the MCS. In conclusion, RPM-exposure of PC-3 cells induced changes in their morphology, cytoskeleton, and extracellular matrix protein synthesis, as well as in their focal adhesion complex and growth behavior. The significant upregulation of genes belonging to the PAM pathway indicated their involvement in the cellular changes occurring in microgravity.

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“…The observation of intercellular cavities within the spheroids together with the scarce scattered pattern of necrosis formation are similar to the findings of Grümmer et al (1990) and White et al, although in the latter study JAr cells and not BeWo cells were used. Development of central necrosis has been shown to vary considerably in spheroids of different cell lines although in most spheroid systems a central necrotic core begins to develop at diameters greater than 250 µm (Soranzo et al, 1986;White et al). For example, V79 spheroids of more than 250 µm in diameter develop a necrotic core (Grümmer et al, 1990) while spheroids of other cell lines such as spheroids of human bladder carcinoma origin could reach diameters of about 500 µm or even 700 µm before central necrosis occurred in them (Erlichman & Tannock, 1986;Hermes et al).…”

Section: Discussionmentioning

confidence: 99%

A Quantitative Assessment of the Morphological Characteristics of BeWo Cells as an in vitro Model of Human Trophoblast Cells

et al. 2010

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In order to study the detailed morphology of trophoblast cells during human implantation, BeWo cells were cultured as spheroids in suspension culture. These cultures were then processed for light and electron microscopical examination. The present study showed that the BeWo spheroids consist of two cell types which are cytotrophoblast-like and syncytiotrophoblast-like. The cells with larger nuclear diameter made up only about 1% of the cell population and appear to be those of syncytiotrophoblast. Therefore the predominant cell type of the BeWo spheroids appeared to be relatively undifferentiated and cytotrophoblast-like. About 10% of the BeWo cells in the present study were mitotic, indicating a highly proliferative population. Total cell number increased about 12 times during the culture period from 107 ± 9 on day 1 to 1211 ± 145 on day 7 whereas the volume per cell increased about 2 times, from 1300 µm 3 on day 1 to 2400 µm 3 on day 7. Therefore overall growth of BeWo spheroids is due to both hyperplasia and hypertrophy. However, it appears that cell proliferation outstrips volumetric growth. These quantitative data show that BeWo cells grow mainly by hyperplasia and provide baseline values for further studies. In addition, the results show that BeWo cell morphology has marked similarities to that reported for human trophoblast, making it a useful model for subsequent in vitro studies.

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Formation, Growth and Morphology of Multicellular Tumor Spheroids from a Human Colon Carcinoma Cell Line (LoVo)

Cited by 14 publications

References 8 publications

Cell Encapsulation in Sub-mm Sized Gel Modules Using Replica Molding

Cell Encapsulation in Sub-mm Sized Gel Modules Using Replica Molding

Simulated Microgravity Influences VEGF, MAPK, and PAM Signaling in Prostate Cancer Cells

A Quantitative Assessment of the Morphological Characteristics of BeWo Cells as an in vitro Model of Human Trophoblast Cells

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