Preservation of the 3D Phenotype Upon Dispersal of Cultured Cell Spheroids Into Monolayer Cultures

Abstract

In functional cytometric studies, cultured cells are exposed to effectors (e.g., drugs), and the heterogeneity of cell responses are studied using cytometry techniques (e.g., image cytometry). Such studies are difficult to perform on 3D cell cultures. A solution is to disperse 3D clusters and transfer the cells to the 2D state before applying effectors and using cytometry. This approach requires that the lifetime of the 3D phenotype be longer than the duration of the experiment. Here we studied the dynamics of phenotype transformation from 3D to 2D and searched for means of slowing this transformation down in dispersed spheroids of MCF7 cells. We found three functional biomarkers of the 3D phenotype in MCF7 cell spheroids that are absent in the 2D cell culture: (i) the presence of a subpopulation with an elevated drug-expelling capacity; (ii) the presence of a subpopulation with an elevated cytoprotective capacity; and (iii) the accumulation of cells in the G1 phase of the cell cycle. Monitoring these biomarkers in cells transferred from the 3D state to the 2D state revealed their gradual extinction. We found that the combined application of an elevated cell density and thiol-containing medium supplements increased the lifetime of the 3D phenotype by several fold to as long as 96 h. Our results suggest that extending the lifetime of the 3D phenotype in the cells transferred from the 3D state to the 2D state can facilitate detailed functional cytometric studies, such as measurements of population heterogeneity of cytotoxicity, chemosensitivity, and radiosensitivity. J. Cell. Biochem. 118: 154-162, 2017. © 2016 Wiley Periodicals, Inc.

Establishment of 2D Cell Cultures Derived From 3D MCF‐7 Spheroids Displaying a Doxorubicin Resistant Profile

“…[2,6] For this purpose, the performance of a number of drugs has been analyzed using 2D in vitro models due to their simplicity, reproducibility, and lowcost. [7][8][9][10][11] Still, these 2D in vitro models can only be used to investigate the direct effects of the drugs in the cells and do not take into consideration the role of the physiological 3D environment of the cells in drugs resistance. Therefore, the exclusive use of 2D in vitro models can lead to poor predictive results concerning drug's effectiveness.…”

“…[24,25,27] On the other hand, researchers are developing drug resistant 2D cell cultures. [7,[33][34][35][36][37][38][39][40] These models show a drug resistance profile more alike to that found in in vivo tumors and can be analyzed through standardized assays and equipment used for conventional 2D cell cultures. Recently, Koshkin et al demonstrated that 3Dderived MCF-7 cells (cells obtained from 3D spheroids disaggregation) are able to preserve their 3D-phenotype when cultured in 2D.…”

“…Recently, Koshkin et al demonstrated that 3Dderived MCF-7 cells (cells obtained from 3D spheroids disaggregation) are able to preserve their 3D-phenotype when cultured in 2D. [7] Additionally, this phenotype was maintained for longer periods (up to 96 h) by culturing the 3D-derived cells in medium supplemented with 5 mM of glutathione (GSH; reducing and antioxidant agent found in high levels in various cancer [e.g., breast cancer] and an influencer of cancer cells resistance). [7,[41][42][43][44][45][46][47][48][49] However, so far, the applicability of the 3D-derived cells for evaluating a drug response has not been investigated.…”

“…[7] Additionally, this phenotype was maintained for longer periods (up to 96 h) by culturing the 3D-derived cells in medium supplemented with 5 mM of glutathione (GSH; reducing and antioxidant agent found in high levels in various cancer [e.g., breast cancer] and an influencer of cancer cells resistance). [7,[41][42][43][44][45][46][47][48][49] However, so far, the applicability of the 3D-derived cells for evaluating a drug response has not been investigated. Furthermore, the effect of drugs in 3D-derived cells in 2D has not yet been compared to that occurring in conventional 2D models and 3D spheroids.…”

FGF2 and EGF Are Required for Self-Renewal and Organoid Formation of Canine Normal and Tumor Breast Stem Cells

“…Since SCs and CSCs have the unique in vitro capacities to survive and proliferate in an anchorage‐independent manner and generate mammospheres (MMs) in suspension culture conditions [Dontu et al, ; Medina et al, ], we and others have utilized the property of MM formation to culture SCs/CSCs ex situ. Additionally, as SCs are characterized by a low rate of cell proliferation ex situ, SCs can be enriched during MM formation by their capacity to retain higher levels of a membrane‐labeling dye compared to the progeny cells generated from the SCs [Dontu et al, ; Zucchi et al, , ; Pece et al, ; Tosoni et al, ; Piscitelli et al, ; Koshkin et al, ].…”